CHROMOSOME RESEARCH

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Contents

Volume 12 Supplement 1 September 2004

ICC XV,15th International Chromosome Conference 5^10 September 2004 Brunel University,West London, UK

Edited by: Joanna M. Bridger Darren K. Griffin

Editorial v Conference Programme xi Plenary Session: Lectures L01^L07 1 Animal Chromosomes Symposium: Lectures L09^L17; Posters PO:06:01^PO:06:15 3 Centromeres Symposium: Lectures L18^L28; Posters PO:06:16^PO:06:22 14 Genome Organisation Symposium: Lectures L29^L46; Posters PO:06:23^PO:06:32 21 Reproduction Symposium: Lectures L47^L54; Posters PO:06:33^PO:06:36 34 Comparative Genomics/Chromosome Evolution Symposium: Lectures L55^L65; Posters PO:06:37^PO:06:55 40 Telomeres Symposium: Lectures L66^L70; Posters PO:06:56^PO:06:64 55 Epigenetics Symposium: Lectures L71^L78; Posters PO:06:65^PO:06:71 61 Plant Chromosomes Symposium: Lectures L79^L90; Posters PO:06:72^PO:06:94 67 Prokaryote Chromosomes Symposium: Lectures L91^L95; Posters PO:08:01^PO:08:02 84 Sex Chromosomes Symposium: Lectures L96^L107; Posters PO:08:03^PO:08:14 87 Chromosome Transfer Symposium: Lectures L108^L111 Posters PO:08:15^PO:08:16 98 Gene Therapy Symposium: Lectures L112^L119; Posters PO:08:17^PO:08:22 101 Meiosis Symposium: Lectures L120^L126; Posters PO:08:23^PO:08:36 107 Population Cytogenetics Symposium: Lectures L127^L137; Posters PO:08:37^PO:08:53 117 Solid Tumours Symposium: Lectures L138^L147; Posters PO:08:54^PO:08:64 128 Aneuploidy Symposium: Lectures L148^L155; Posters PO:08:65^PO:08:77 137 New Methodologies Symposium: Lectures L156^L165; Posters PO:08:78^PO:08:83 146 Haematological Malignancy Symposium: Lectures L166^L174; Posters PO:08:84^PO:08:94 152 Post-genomic Era Symposium: Lectures L175^L180 161 Prize Nominee Special Symposium: Lectures L181^L191 163 Author Index 171 Important Notes and Reminders for Authors inside back cover CHROMOSOME RESEARCH

Cytogenetics, Genomics, Chromatin and The Nucleus

FRONT COVER PICTURE 3D reconstruction of a mouse fibroblast (NIH3T3) nucleus after visualization of telomeres by 3D-FISH and DNA counterstaining. The border of the nucleus (red outside, grey inside) was reconstructed using TO-PRO-3 counterstain and then partly removed to show the inside with reconstructed telomere signals (green). Segmentation of the nucleoli (yellow) was performed using the counterstain labelling. Maximum intensity projections (XY, XZ, YZ) of false-colouredimagestacks are shown on the background. Laser confocal serial sections were recorded by a confocal microscope (Leica TCS SP) with a voxel size ¼ 0.05 0.05 0.250 mm. Reconstruction was performed by surface rendering using software AmiraTM (Amira 3.0, TGS Europe, Merignac, France; http://www.amiravis.com). FISH experiment and 3D reconstruction were performed in the laboratory of Prof. T. Cremer by A. Brero, Department of Biology II, University of Munich, Germany.

Chromosome Research is covered in Biochemistry & Biophysics Citation Index, Biological Abstracts, BIOSIS Previews, Biotechnology Citation Index, Chemical Abstracts, Current Contents/Life Sciences, Elsevier BIOBASE/Current Awareness in Biological Sciences, EMBASE/Excerpta Medica, IBIDS, Index Medicus/MEDLINE, The ISI Alerting Services, Science Citation Index, Science Citation Index Expanded, and in CD-ROM via Adonis.

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# 2004 Kluwer Academic Publishers. Printed in the Netherlands ICC XV 15th International Chromosome Conference September 5^10, 2004; Brunel University,London, UK

CONFERENCE ORGANIZING COMMITTEES

Local Organizers Hosts: Joanna Bridger and Darren Griffin (Cell and Chromosome Biology Group, Brunel University) Zac Peake (Cambridge Publications) Jackie Storey (Cambridge Publications) Charmaine Grimes (Brunel Conferencing Services) Shiela Killen (Brunel Conferencing Services)

Standing Committee of the International Chromosome Conferences Hans de Jong (Wageningen,The Netherlands) Peter Moens (Toronto, Canada) Ettore Olmo (Ancona, Italy) Maria Puertas (Madrid, Spain) Jeremy Searle (York, UK) Adrian Sumner (North Berwick, UK) ^ Chair DwayneWise (Mississippi, USA)

International Advisory Board Martin Bentz (Ulm, Germany) Harold Klinger (NewYork, USA) Adrian Bird (Edinburgh, UK) Peter Lichter (Heidelberg, Germany) Nigel Carter (Cambridge, UK) Richard Losick (Harvard, USA) Titia De-Lange (NewYork, USA) Jennifer Marshall-Graves (Canberra, Australia) Joy Delhanty (London, UK) Rob Newbold (Brunel University,UK) Bill Earnshaw (Edinburgh, UK) Michael Schmid (Wuerzburg, Germany) Malcolm Ferguson-Smith (Cambridge, UK) Jeremy Searle (York, UK) Terry Hassold (Cleveland, USA) Kevin Sullivan (La Jolla, USA) Pat Heslop-Harrison (Leicester, UK) HansTanke (Leiden, Netherlands) Pat Hunt (Cleveland, USA) AndrewWright (Boston, USA) Clare Huxley (London, UK)

Chromosome Research 12 (Suppl 1): v–x, 2004. v # 2004 Kluwer Academic Publishers. Printed in the Netherlands

Editorial

ICC XV, 15th International Chromosome We were also overwhelmed by the response of our Conference would-be presenters at the conference; around 5^10 September 2004, Brunel University 250 new abstracts were received (in addition to those of our invited speakers) and approximately This issue of Chromosome Research contains the 100 of these have been selected as oral abstracts of ICC XV, the 15th International presentations. In addition there will be two pos- Chromosome Conference held at Brunel ter sessions, collectively comprising around 150 University on 5–10 September 2004. The Inter- posters accompanied by our commercial exhibi- national Chromosome Conferences began in tion space. We hope to have over 700 delegates, 1964, 1967 and 1970 as the Oxford Chromosome each contributing to the unique nature of ICC. Conferences. They became ‘‘International’’ at Our intention is to create an atmosphere of the the fourth conference in Jerusalem in 1972 and a highest quality science and the opportunity for tri-annual format has continued more or less delegates to discuss new projects and ideas in a thereafter with conferences in Leiden (1974), relaxed environment. It is our belief that the best Helsinki (1977), Oxford (1980), Lubeck (1983), science is not always done in the lecture rooms Marseille (1986), Uppsala (1989), Edinburgh but in the bars (i.e. The Chromosome Arms, spe- (1992), Madrid (1995), Ancona (1998) and Wurz- cially renamed for the conference) and dining burg (2001). The year 2004 sees a return to the rooms of conference centres. The International UK and a first chance for Brunel University to Chromosome Conferences have always promoted host the ICC. The decision to hold ICC XV at the social side of conferencing as well as the sci- Brunel was initiated in a Franconian wine cellar ence and ICC XV will be no exception. At ICC at The Residence in Wurzburg and we’d like to XV there will be visits to the West End, Windsor think that we were awarded it because of our Castle, Virginia Water, the Tate Modern Gallery conference facilities and proximity to Heathrow and the London Eye. Events on-campus will Airport. Delegates will benefit from being only include real ale tasting, a FISH and chip supper, 15–20 minutes away from their arrival and ¢lm shows and a musical recital and ^ for the con- departure gates and Brunel will house the major- ference dinner ^ a medieval banquet, with tradi- ity of delegates on campus for the duration of tional dancing. We would like to express our the conference. Brunel University is known appreciation for the e¡orts of senior management locally for its landscaped gardens and ‘‘brutalist’’ at Brunel for listening to our pleas when the cam- architecture; we have over 850 academic staff pus building work threatened to impede our pro- and over 12,000 students. Delegates from Great gress, to Brunel Conferencing Services for Britain will be aware of Isambard Kingdom Bru- arranging the room bookings and catering and, nel, the great Victorian engineer after whom the especially to Cambridge Publications for their University is named, leading the ‘‘greatest organisational and management skills. Briton’’ poll for the majority of the long-running Each day will follow a similar theme with ses- saga before being usurped by Winston Churchill sions starting about 9 a.m. We will favour the at last minute. ‘‘continental style’’ of lunch break with sessions We were delighted that, following our initial winding up around noon and re-starting about approaches, our ‘‘wish-list’’ of speakers was largely 2.30 p.m. The reason for this is to allow time for ful¢lled. We have attracted large contingents all delegates to have a decent lunch and to allow from the US, Asia and Australasia and there are adequate time for the viewing of posters and around 120 invited speakers. This volume con- exhibitions. We would also encourage as much tainsmostoftheabstractsoftheirpresentations. social interaction and scienti¢c discussions and vi 15th ICC: Editorial are willing to provide rooms for that purpose if burst on the scene and silenced the doubters. required. Prof. Ward will inform the conference about the advancements in FISH and how these have helped move cytogenetics into the 21st century. Day 1 We would like to take the opportunity of ICC XV to propose that we are now entering the Monday 6 December will see the whole con- fourth era of chromosome research, namely that ference gathered together for the keynote and of chromosome engineering. The Brunellian over- plenary lectures. Our keynote speaker will be tones have not escaped us but we would argue Professor John Burn of the University of that they are well founded given the extensive Newcastle. Professor Burn is the Public Orator interest in gene therapy, gene function, genome for Newcastle University and is well known for his organization and chromosome transfer, all of television appearances and futuristic vision. We which derive their origins from chromosome look forward with great enthusiasm to his aptly research. Therefore our second plenary session entitled presentation ‘‘The Future of Genetics.’’ will be themed on chromosome engineering. We Chromosome research is fortunate to have had are delighted to have attracted some of the world three eras that have laid down fundamentals for leaders in the ¢eld of chromosome engineering biological and biomedical studies; our ¢rst plen- interested in the development and utility of ary session will celebrate this. The ¢rst, the classi- human and mammalian arti¢cial chromosomes. cal era, began with the discovery of the correct They are Professor Andy Choo of Melbourne number of human chromosomes and the correla- Australia, Dr Christine Farr of the University of tions between numerical aberrations and disease Cambridge UK and Professor Huntington F. (such as Down syndrome and Klinefelter syn- Willard of Duke University USA. Dr Farr and drome). Our plenary speaker to represent this era, Professor Willard are pioneers of the ‘‘top down’’ Professor Pat Jacobs, was involved in the dis- and ‘‘bottom up’’ construction strategies for mam- covery of the genetic basis of what we now know malian arti¢cial chromosomes, respectively. In as sex chromosome abnormalities and her activ- addition, Professor Choo’s laboratory were the ities continue to this day. Prof. Jacobs will not ¢rst to prove the existence of human neocen- only discuss numerical abnormalities of the karyo- tromeres, regions of chromosomes that form func- type but other chromosome abnormalities such as tional centromeres without alpha satellite DNA fragile X that led to the discovery of trinucleotide and use them to create arti¢cial chromosomes. repeat expansions in disease. The ability to band Prof. Willard will inform the conference about human chromosomes brought chromosome behavioural and functional properties of de novo research into a second era, enabling the detection constructed human arti¢cial chromosomes. The of subtle rearrangements and heralding the properties of ‘‘top-down’’ constructed mamma- advent of prenatal diagnosis and cancer cytoge- lian arti¢cial chromosomes will be discussed by netics. The name that is most synonymous with Dr Farr, with respect to what is required of a mini- chromosome banding is Dr Adrian Sumner who chromosome-based vector system in vertebrate also happens to be chair of the standing commit- cells and aspects of centromere structure and func- tee for ICC. Dr Sumner will discuss chromosome tion in them. Prof. Choo will talk about many banding pre- and post-1968 and inform the dele- aspects of neocentromeres, from why they may gates on the usefulness of banding for chromo- exist to revealing centromeric fundamentals, the some identi¢cation, referencing the position of extent of matrix attachment regions in them and particular sequences and in diagnostics. Finally, their protein binding properties. the decision about whom to represent the renais- The day will conclude with a reception spon- sance of chromosome research, namely the FISH sored by Chromosome Research then a tradi- era, led us to the name of Professor David Ward. tional English FISH and chip supper with British In a time when molecular biologists were largely real ale tasting. All will be accompanied by live writing the obituaries for cytogenetics, the techni- traditional jazz. All subsequent days will begin que of £uorescence in situ hybridization (FISH) with the opportunities for young scientists (PhD 15th ICC: Editorial vii students and post-docs) to meet, and breakfast Bridger will present her data on genome and with, their scienti¢c heroes at ‘‘Mentor Break- health, specifically in diseases of the nucleus and fasts.’’ in differentiating adult stem cells. Also included on this day will be the sessions on centromeres and telomeres led by Bill Day 2 Earnshaw and Titia De-Lange, respectively (both of whom are ¢gureheads in their respective ¢elds); Tuesday 7 September will see the beginning of no self-respecting chromosome conference could the concurrent sessions. The largest lecture room do without sessions on these most fundamental of is devoted to genome organization which structures. Speakers in the centromere session are describes how the chromosomes are positioned Iain Cheeseman who will tell us about new pro- and function in their main environment, the cell teins discovered at the C. elegans kinetochore, nucleus. This is a fast-growing and complex area Stephen Taylor who will demonstrate that Bub1 of genome/chromosome biology and it is becom- and Aurora act together to bind the anaphase pro- ing apparent how important creating the right moting complex in human cells, Tatsou Fuka- micro-environment for particular sequences of gawa will describe how the centromere/ DNA could be in terms of epigenetics, control of kinetochore is assembled and functions by using transcription, repair and health. This session is DT40 cells to knockout speci¢c genes, Beth Sulli- chaired by Kevin Sullivan, who has done some van will describe the unique organization of his- elegant work using GFP to study elements of tone modi¢cation found at centromeres, Poala genome movement and organization in real-time. Vagnerilli will discuss aspects of centromere func- Speaking in this session is Wendy Bickmore, tion and Alison Pidoux will speak from Robin All- whose research has been instrumental in deci- shire’s laboratory on centromere/kinetochore phering how the genome is arranged in cell function in ¢ssion yeast. Bill Earnshaw, as chair, nuclei. She will talk about her recent research on will impart the discovery, using proteomics, of a distribution and organization of open and con- new chromosomal sca¡old protein. The telomere densed regions of chromatin throughout the gen- session contains the following speakers Julie ome. Susan Gasser, will inform delegates about Cooper who will tell us about aspects of telomere her latest work defining a higher-order compac- function in ¢ssion yeast, Predrag Slijepcevic will tion of chromatin within yeast nuclei and by discuss his studies on DNA damage response on using sophisticated live imaging techniques how telomere maintenance, Woodring Wright will her group have mapped the organization of yeast show us how the shortest telomeres co-localize interphase chromosomes in 3 dimensions. with DNA damage and reveal temporal issues Thomas Cremer, one of the world leaders in concerning the replication timing of speci¢c telo- terms of understanding the 3-dimensional organi- meric ends and the negative regulation of telomer- sation of chromosomes and nuclei, will present asebyPOT1willbedemonstrated,inadditionto data supporting the view that chromosomes are quanti¢cation of telomere length at the nucleotide organized in the interphase nucleus according to level in yeast by Joachim Lingner. their size. He will also discuss his gene relocation The day also includes sessions on animal chro- hypothesis, i.e. when genes become silenced dur- mosomes and comparative genomics (led by ing differentiation they can translocate to new Michael Schmid and Malcolm Ferguson-Smith regions of the nucleus (this promises to be a respectively) as well as afternoon sessions from most colourful talk). Tom Misteli will tell us our selected abstracts in the topics of plant chro- about his exciting new data on how genome mosomes and new methodologies. In animal organization is dissimilar in different tissue types chromosomes we will hear about the chicken and how this may be linked to the prevalence of karyotype from Darren Gri⁄n, a genetic map for specific tissue-related translocations. Andrew rabbit from Helene Hayes, Ross Jones will Belmont will discuss the exquisite molecular tools enlighten us about hybrid zone analysis corre- he uses to decipher how chromatin is folded and lated with geographical distribution in shrews and organized in the interphase nucleus. Joanna Helen Foster will describe nuclear organization of viii 15th ICC: Editorial the pig genome in di¡erent cell types. The com- strate chromosome segregation in Bacillus sub- parative genomic session is also related to chromo- tilis, Remus Dame will speak on DNA some evolution and is chaired by Malcolm compaction in E. coli and Dave Sherrat will talk Ferguson-Smith, and he will be discussing the about bacterial chromosome segregation. The role of cross-species chromosome painting in com- ICC has always been a bastion for researchers in parative and evolutionary genomics. The talks plant chromosomes and this year a large con- will be given by Stefan Mueller on primate chro- tingent of scientists studying plant chromosomes mosomes, and Dave Burt will discuss the resource will be present. The chair of this session, Pat chickNET containing large amounts of sequence Heslop-Harrison is well known for his pioneering information on the chicken genome and how this work on the cytogenetics of plant chromosomes can be used in evolution studies. We are also look- which has been recognized all over the world. In ing forward to hearing from Stephen O’Brien this session, Andreas Houben will discuss histone about his recent advances in comparative geno- H3 phosphorylation in plants, Jiming Jiang will mics. The Reproduction session is headed by Joy tell us about aspects of centromeres in rice, Delhanty, an expert on reproductive cytogenetics, whereas Minoru Murata will tell us about cen- who will discuss her latest data and use of technol- tromeres in Arabidopsis, Paul Fransz will talk ogy in using FISH and comparative genome hybri- about the consequences of a paracentric inver- dization to look for aneuploidies in oocytes and sion in Arabidopsis. This session will also com- polar bodies. Leeanda Wilton will inform us on plement that of the previous day where the her e¡orts to use single cell CGH in aneuploidy selected abstracts from the plant chromosome screens in IVF embryos and Maj Hulte¤ n will dis- session will be given the opportunity to speak. cuss aspects of male infertility related meiotic Chromosome transfer is a technique that was abnormalities. Meiotic segregation of Robert- initially used to isolate individual chromosomes sonian and reciprocal translocations in sperm and from the rest of the karyotype and now has a pre-implantation embryos will be presented by wide range of applications. The chromosome Elvire van Assche. Finally Helen Tempest will dis- transfer session will concentrate on the diverse cuss the prospects for the e⁄cacy of alternative way that chromosome transfer has been incorpo- herbal therapy in the treatment of high levels of rated into chromosome biology to study genome sperm aneuploidy in infertile men. organization (Karen Meaburn), for chromosome The day will conclude with a classical music engineering (Mitsou Oshimura) and to study recital and wine reception. tumour suppressor genes (Christopher Parris and Harold Klinger). Epigenetics is an extremely important topic in Day 3 chromosome biology, since it concerns the mod- i¢cations to and in£uences on DNA that a¡ect Wednesday 8 September will be half devoted to chromosome function, that are not directly to do science and half to social events. The topics of with DNA sequence. The epigenetics session, sex chromosomes, plant chromosomes, prokaryo- chaired by Adrian Bird, a world-leader in this tic chromosomes, chromosome transfer and epi- topic, will contain talks on the e¡ect of histone genetics will all grace the lecture halls. Invited modi¢cations in disease such as cancer (Tony chairs are Jennifer Marshall-Graves, Pat Heslop- Kouzarides), the ¢ne mapping of epigenetic Harrison, Andrew Wright, Harold Klinger and domains in an individual gene cluster (Douglas Adrian Bird. We are particularly excited to Higgs), silencing of chromosomal regions in introduce a session on structure and organization plants (Alan Herr) and aspects of epigenetic of bacterial chromosomes which shows the rapid alterations in nuclear transfer embryos (Petra development of this field in recent years. The Hajkova). We are delighted to welcome some of chair, Andrew Wright, will show us how fluores- the world leaders in sex chromosome research led cence-based methods can be used to study bac- by Jenny Graves. Evolution, X-inactivation, Y terial DNA and how F plasmid segregation is chromosome deletions, ectopic homologous facilitated in E. coli, Jeff Errington will demon- recombination event, genome organization and 15th ICC: Editorial ix theavianZchromosomewillallbecovered.Paul 3D genome organization, and Andreas Ladurner: Burgoyne, Carolyn Brown, Heather McQueen, with the intriguingly titled ‘‘ChIP on Chip’’ pre- Julian Lange, Horst Hameister, Dimitri Filatov sentation. This is in addition, to the session from and Mike Mitchell, among others, will all be the selected abstracts on Monday afternoon. giving presentations. In the afternoon delegates Another newly introduced session will be one will have a choice of a journey: East into the centre on population cytogenetics, chaired by Jeremy of London to visit the Tate Modern Gallery, the Searle. Jeremy will enlighten us on the use of chro- London Eye and the Theatre District; or West to mosomal variation as a marker in natural popula- Virginia Water and Windsor. tions. Johannes Vogel will present his work on identifying areas where plants have survived gla- ciers using chromosome studies of the fern Asple- Day 4 nium. Natural selection is studied by Antonio Fontdevila by assessing the role of the tempera- Our final day of concurrent sessions (Thursday ture in latitudinal chromosomal clines in Droso- 9 September) will consist of presentations on gene phila. Ira Greenbaum is presenting data on therapy, post genomic era, new methodologies studies relating to the identi¢cation, variation and and population cytogenetics. The gene therapy evolution of common fragile sites in human and session, chaired by Clare Huxley, will comprise deer mice. Dave Rowell travels here to speak to some more talks on the engineering of mamma- us about the causes and consequences of Robert- lian artificial chromosomes from Brenda Grimes, sonian translocations using di¡erent organisms William Brown, Zoia Larin-Monaco and Hiroshi that have repeated Robertsonian translocation Masumoto. George Dickson will describe his occurrence or are stable for this event. Kevin outstanding recent work on using gene therapy Livingstone will discuss his theories on the role of to add or correct genes in vivo. This session is chromosome rearrangements in speciation. Juan full of abstracts on chromosome engineering so Camacho will inform us about supernumerary the talks selected from the abstracts should be chromosomal elements in population dynamics. especially exciting. The post-genomic era session An all-day session on meiosis and aneuploidy is chaired by Nigel Carter who will present will also be a feature of Thursday with Terry advances in DNA microarray used to understand Hassold and Pat Hunt teaming up to head the structure and function within the human genome. foremost proponents of the ¢elds. Christer Celia May will demonstrate the use of SNP-class H˛˛g, Sharon Bickel, Stephanie Sherman, Wendy DNA markers to understand meiotic recombina- Robinson, Kim Nasmyth, Paula Cohen, Rol¡ tion and Eric Green will talk about studying Jessberger and Antoine Peters will cover areas human genome function via multi-species com- such as age-related non-disjunction in oocytes, parisons. Bryan Young will speak about his meiotic-speci¢c proteins, the role of recombina- research on looking for novel therapies for can- tion in aneuploidy, meiosis in fetal oocytes, and cer and David Vetrie will present his recent work synaptonemal complex length. Meiotic errors in on using DNA microarray in improvement of the form of aneuploidy are amongst the most com- health. We are particularly pleased to welcome mon genetic abnormalities known to mankind Peter Lichter who is well known as a pioneer of leading to pregnancy loss, stillbirth, birth defects molecular cytogenetics. In recognition of his con- and mental retardation. Despite this we know tribution to the field, we have given the all dele- relatively little about their causes and thus are gates the opportunity to hear his presentation by some way from e¡ective treatments. We look starting the new methodologies session half an forward to hearing about the latest research in hour early at 2 p.m. Peter has selected a number this most crucial of areas. of leaders in devising new technology for our Cytogenetics has done tremendous amounts for field and so we look forward to the contributions the cancer biology ¢eld in terms of diagnosis and in this session from Guido Sauter on tissue spe- cause. Thus we have two sessions on tumour cyto- cific DNA microarrays, Roland Eils discussing genetics with a number of world-leaders speaking elaborate methods for visualizing and analysing about how they use chromosome biology in their x 15th ICC: Editorial cancer research. Tumour cytogenetics is divided (LNA) probes for FISH experimentation in place into two sessions covering solid tumours and hae- of DNA or PNA probes, Uchiyama et al. will mocytological malignancy. Speakers in the solid show the use of 2D electrophoresis to analyse the tumour session will present data on automation protein composition of human chromosomes, to detect malignancy associated changes (Calum Laner et al. will demonstrate the use of a EGFP MacAulay), advances in microarray technology in transfection efficiencies of human artificial combined with comparative genome hybridiza- chromosome generated from PAC constructs tion (Donna Albertson), breast cancer (Jorma containing artificial centromeres and telomeres Isola), premature condensation of chromosomes and Bailey et al. present their work on the appli- to aid in chromosome analysis in slow growing cations of strand-specific CO-FISH. Lightfoot tumours (Karoly Szuhai) and assessing genomic and Ho¨o¨g present a study of embryos from a imbalances in neuroblastoma employing a variety model ‘‘Scyp-3-null’’ mouse that produces aneu- of techniques. Hans Tanke, the chair of this ses- ploid oocytes and subsequent embryos after ferti- sion, will give an overview on modern day technol- lisation with normal sperm. Rotkov et al. will ogies used to study the genome in solid tumours. present an analysis of telomeres in Asparagales In the haemotological malignancy session, while Herron et al., in studying grasshopper B chaired by Martin Bentz, an expert in lymphopro- chromosomes, propose that they persist in evolu- li¢c disorders, the diseases that will be covered tion via a metaphase checkpoint alteration that, include mantle cell lymphoma (Martin Dyer), in turn, was more forgiving of misaligned chro- acute lymphoblastic leukaemia and non-random mosomes. The evolutionary theme is continued aneuploidy associated with it (Oskar Haas) and by Ferreri et al. with a study of the centromere adult acute myeloid leukaemia and gene expres- in marsupials drawing general conclusions about sion pro¢les that allow sub-grouping of patients the role of this vital structure in chromosome (Jonathan Pollack). evolution. We look forward to hearing all We are particularly delighted to be able to pre- their presentations on the final day of the con- sent our conference dinner which will take the ference and to the choosing of the prize-winners form of an old English Medieval banquet com- by the International Advisory Panel. Finally, we plete with traditional music and entertainment invite all our delegates to consider their most from the renowned Dragons¢re (http://www. recent research for our late-breaking news dragons¢re.btinternet.co.uk/). This will be fol- session. The purpose of this session is to keep lowed by traditional medieval dancing also led by delegates as up-to-date as possible with the most Dragons¢re. Bring an empty stomach with you recent developments in the field of chromosome and leave your table manners behind! research. So, in conclusion, at Brunel we are gathering some of the world’s best scientists to celebrate the Day 5 successes of chromosome research and look to its future achievements. We plan outstanding scien- The morning of our final day (Friday 10 ti¢c and social programmes and even a plenary September) will see the whole conference reuni- session on chromosome engineering. Isambard ted for prize giving and late-breaking news. Kingdom Brunel, we are sure, would have liked Nominees for the prizes were chosen by the local to attend! organizing committee from those abstracts who made the early submission deadline and con- tributed a manuscript to a special issue of Joanna M. Bridger and Darren K. Gri⁄n Cytogenetic and Genome Research. They include Cell and Chromosome Biology Group multicolour meiotic preps of Codina-Pascual and Dept of Biological Sciences colleagues, atomic force microscopy studies of Brunel University Takeyasu et al. in prokaryotes and Hoshi et al. West London in human chromosomes. Silahtaroroglu et al. will UB8 3PH demonstrate the use of Locked Nucleic Acid UK Conference Program

Sunday 5th September Monday 6th September

7:30 8:00 Conference hall opens 8:30 Start of Conference 9:00 Keynote address: 9:30 Prof. John Burn - "The Future of Genetics" 10:00 Break 10:30 Plenary Session 1: Chair - Dr Darren Griffin 11:00 The 3 eras of chromosome research 11:30 Prof. Pat Jacobs, Dr Adrian Sumner, Prof. David Ward 12:00 Registration desk opens Lunch 12:30 Start of Poster Session 1 13:00 13:30 14:00 14:30 Plenary Session 2: Chair Dr Joanna Bridger 15:00 Meeting of Int. Advisory Committee Chromosome Engineering 15:30 Prof. Hunt Willard, Dr Christine Farr 16:00 Break 16:30 Prof. Andy Choo 17:00 Poster viewing 17:30 18:00 18:30 19:00 Welcome reception Chromosome Research reception 19:30 20:00 FISH and chip supper, real ale, trad jazz Film shows

xi Tuesday 7th September Wednesday 8th September

7:30 Mentor breakfasts Mentor breakfasts 8:00 8:30 9:00 Morning concurrent sessions start: Morning concurrent sessions start: 9:30 Reproduction, Animal Chromosomes, Centromeres, Sex Chromosomes, Plant Chromosomes, Prokaryotes, 10:00 Genome Organisation Chromosome Transfer, Epigenetics 10:30 11:00 11:30 Dining room opens for lunch Dining room opens for lunch 12:00 Start of Poster Session 2 12:30 13:00 13:30 Trip A: Trip B: 14:00 Virginia Water Tate Modern 14:30 Afternoon concurrent sessions start and Windsor London Eye 15:00 Genome Organisation (cont), Comparative Genomics, and West End 15:30 Centromeres (cont.), Plant Chromosomes, Telomeres, 16:00 New methodologies 16:30 17:00 Poster viewing 17:30 18:00 18:30 19:00 19:30 20:00 Classical music recital Film shows Film shows

xii Thursday 9th September Friday 10th September

7:30 Mentor breakfasts Mentor breakfasts 8:00 8:30 9:00 Morning concurrent sessions start: Presentations by prize nominees 9:30 Post-Genomic Era, Meiosis, Population Cytogenetics, 10:00 Tumour Cytogenetics (solid), Gene Therapy Break 10:30 11:00 11:30 Dining room opens for lunch 12:00 Lunch 12:30 13:00 13:30 14:00 Late breaking news session 14:30 Afternoon concurrent sessions start 15:00 New methodologies, Aneuploidy, Sex Chromosomes (cont.) 15:30 Tumour Cytogenetics (haematological) Closing remarks, prize giving 16:00 End of conference 16:30 17:00 Poster viewing 17:30 18:00 18:30 19:00 19:30 20:00 Conference dinner: Medieval banquet with traditional entertainment and dancing

xiii

Chromosome Research 12: 1–169, 2004. 1 # 2004 Kluwer Academic Publishers. Printed in the Netherlands have proved so important in our understanding a Plenary Session wide range of neurological diseases. The advances made in the 15 years of the classi- cal era are indeed astonishing. Research under- L01 taken in an attempt to understand the causes of some of the abnormalities recognised during this The future of genetic research period and the way this has in£uenced our think- John Burn ing about human genetics will be described. Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne, L03 NE1 3BZ, UK Chromosomes ^ the banding era Keynote talk for the conference. Adrian Sumner1 17 Smileyknowes Court, North Berwick EH39 L02 4RG, UK The classical era 1 Chromosome banding – the differential staining Pat Jacobs of parts of chromosomes – began no later than 1Wessex Regional Genetics Laboratory, Salisbury the 1890s, only some 20 years after the study of District Hospital, Salisbury, SP2 8BJ and Division chromosomes began, and is today still extensively of Human Genetics, University of Southampton used. Thus there is really no distinctive banding School of Medicine, Southampton, SO16 6YD era: since the early days cytogeneticists have tried to differentiate different regions of chromosomes, The classical era of human cytogenetics dates and today banding is an essential part of the from the publication by Tjio and Levan in 1956 study of chromosomes, to which all other obser- of the correct chromosome number of our spe- vations refer. Nevertheless, banding is often held cies. Their observation was confirmed by Ford to have started in 1968 when Caspersson and his and Hamerton and it is from that time that colleagues first stained chromosomes with quina- the resurgence of human cytogenetics and the crine mustard and obtained longitudinal differ- contribution of chromosome abnormalities to entiation of plant and mammalian chromosomes. disease can be dated. Since that date a wide variety of techniques have The main autosomal trisomies, the numerical been developed for studying heterochromatic and abnormalities of the sex chromosomes and both euchromatic banding, the centromeres, and the balanced and unbalanced structural rearrange- nucleolus organisers. The important difference ments of the chromosomes were all discovered between pre- and post-1968 banding techniques without the bene¢t of banding techniques. Fur- is that the latter are of general application and thermore the amazingly high frequency of chro- can be applied to routine chromosome prepara- mosome abnormalities among spontaneous tions, and are applicable to all organisms with abortions was described and showed just how little modification. common chromosome abnormalities are in our Chromosome banding is now mature technol- species. The ¢rst recognition of an acquired ogy, used routinely for chromosome identi¢ca- abnormality, the Philadelphia chromosome, ush- tion, as a reference for localising FISH and ered in a new approach to the study and under- immuno£uorescence signals on chromosomes, for standing of malignancies, in which chromosome evolutionary studies, and for studying chromoso- status not only de¢ned the disease, but also the mal changes in cancer. Curiously, in spite of type of treatment and its e⁄cacy. It was a chromo- much e¡ort, the biological basis of chromosome some abnormality, the fragile X, which gave the banding is still poorly understood, as is its phylo- ¢rst clue to a whole new class of mutations, genetic distribution. While chromosome banding namely trinucleotide repeat expansions, which will continue to be a vital practical tool for the 2 15th ICC: Abstracts foreseeable future, investigations of its structural L07 and functional signi¢cance will be important for our understanding of chromosome organisation. What price neocentromere? Andy Choo1 L04 1Murdoch Childrens Research Institute, Plenary Talk ^ The FISH era Melbourne, Australia

David Ward Neocentromeres are fully functional centromeres Molecular Biophysics and Biochemistry that are formed ectopically at non-centromeric Department, Yale University, 333 Cedar Street, locations of the genome. During neocentromere New Haven, CT 06520, USA formation, unknown epigenetic mechanisms underlie the transformation of non-centromeric, No abstract was submitted for this talk. often gene-encoding genomic regions into cen- tromeric chromatin. The principal trigger for L05 neocentromere formation is through chromoso- mal rearrangements that result in the loss of the Chromonomics: The human genome endogenous functional centromere on a chromo- from the chromosome’s point of view some or chromosomal fragment. In humans such Huntington Willard chromosomal rearrangements often cause kar- yotype imbalance that leads to genetic abnormal- Department of Molecular Genetics and ities and even cancer. Given the general Microbiology, Duke University, Box 3382, detrimental effects of neocentromere formation Durham, North Carolina 27710, USA to the individual, what is the biological advan- tage of having a neocentromere forming mechan- No abstract was submitted for this talk ism? One model is to provide a potentially powerful driving force for karyotype evolution L06 and speciation through a process of centromere Chromosome engineering: its repositioning. We discuss recent evidence in humans, lower primates, and plant in support of application in vector development such a model. and in the functional dissection of Fully sequenced human neocentromeres also vertebrate centromeres provide unique practical advantages as a tool for 1 the dissection of the complex properties of the Christine Farr mammalian centromeres. Use of neocentromeres 1Department of Genetics, University of has allowed us to de¢ne the extent of enhanced Cambridge, Downing St, Cambridge CB2 chromosomal sca¡old/matrix binding at the 3EH UK human centromere and to show the permissibility of transcription within the enhanced sca¡old/ We have used the targeted manipulation of a matrix-binding domain. It has also allowed us to human chromosome, maintained in a somatic begin the construction of a linear organisational cell hybrid, to generate an extensive series of lin- map showing distinct and non-overlapping cen- ear minichromosomes. These are being used to tromere protein-binding domains, and the malle- (i) investigate the requirements of a chromosome- ability of these domains in response to based vector system for vertebrate cells; and (ii) chromosomal truncation or treatment with his- to functionally dissect the centromere domain. tone or chromosomal sca¡old modi¢cation Our progress in both these areas will be discussed. agents. 15th ICC: Abstracts 3

Animal Chromosomes Symposium et Etude du Ge´nome, UMR INRA CEA, 78352 Jouy-en-Josas Cedex, France; 3Station d’Amelioration Genetique des Animaux, INRA, L09 BP27, 31326 Castanet-Tolosan Cedex, France Molecular cytogenetic de¢nition of the Rabbits are bred both for the production of fur chicken genome: The ¢rst complete and meat and for biomedical research. It is still a avian karyotype map-poor species and thus our long-term objec- tive is to provide mapping data and tools, which 1 1 2 D. Griffin , J. Masabanda , D. Burt , will help increase the likelihood of finding genes 3 4 5 P. O’Brien , M. Groenen , A. Vignal , associated with complex multifactorial traits. R. Crooijmans4, V. Fillon5, Since mapping data is very limited in rabbit, we M. Ferguson-Smith3 and J. Smith2 have decided to construct an integrated genetic and cytogenetic map using a direct strategy. 1Cell and Chromosome Biology Group, Brunel First, we have chosen 350 genes regularly dis- University, UK; 2Roslin Institute, UK; 3University of Cambridge, UK; 4Waageningen tributed over the human genome approximately University, Germany; 5INRA, Toulouse, France every 10 cM, hypothesizing that they will be equally regularly distributed in rabbit. A three- Chicken genome mapping is important for a genome equivalent rabbit BAC library has been range of scientific disciplines and the ability to screened for each of these genes and we isolated distinguish chromosomes of chicken and other clones for 302 genes. On the one hand, these birds is thus a priority. Here we describe the clones are being localized on rabbit chromo- molecular cytogenetic characterisation of every somes by fluorescent in situ hybridization (FISH) chicken chromosome using chromosome painting and on the other hand, they are being used to and mapping of individual clones by fluorescence isolate microsatellite markers. Eight reference in-situ hybridization FISH. Where possible we rabbit families will be genotyped with these mar- have assigned the chromosomes to known linkage kers to construct a genetic map. Thus, the origi- groups. We propose, on the basis of size, that the nal feature of this strategy is the direct NOR chromosome is around the size of chromo- construction of an integrated map, the cytoge- some 22; however we suggest its original assign- netic results providing accurate comparative ment of 16 should be retained to avoid confusion. mapping data with man. Indeed, in bovine and We suggest the definitive chromosome classifica- porcine species, cytogenetic and genetic maps tion system and propose that the probes devel- were built separately and then integrated. To oped here will find wide utility in the fields of date, 180 gene-containing BAC clones have been developmental biology, DT40 studies, agriculture, FISH-mapped, which represents a four-fold molecular ecology, vertebrate genome organisa- enrichment of the rabbit gene map and 161 tion and comparative mapping of avian species. microsatellite markers have been isolated. We shall present an updated version of the L10 cytogenetic gene map and the first integrated genetic map in rabbit. Use of FISH-mapped genes to construct an integrated genetic map L11 in rabbit 1 2 3 Genome and nuclear architecture H. Hayes , C. Chantry-Darmon , D. Allain , organisation during development and B. Pena3, C. Urien2, M. Bertaud1, H. De Rochambeau3 and C. Rogel-Gaillard differentiation using the pig as a model 1 organism Laboratoire de Genetique biochimique et 1 2 2 2 Cytogenetique, INRA, 78352 Jouy-en-Josas H. Foster ,R.Sturmey,P.Stokes, H. Leese , Cedex, France; 2Laboratoire de Radiobiologie L. Abeydeera3, D. Griffin1 and J. Bridger1 4 15th ICC: Abstracts

1Cell and Chromosome Biology Group, Biological L12 Sciences Department, Brunel University, Uxbridge, Middlesex, UB8 3PH, UK.; Geographical analysis of a 2Department of Biology, The University of York, chromosomal hybrid zone in 3 PO Box 373, York, YO10 5YW, UK.; Sygen the common shrew International, P. O. Box 348, 3033 Nashville Road, Franklin, KY 42135, USA. R. Jones1 1Department of Biology, University of York, The precise organisation of the genome and its PO Box 373, York YO10 5YW, UK environment, the cell nucleus, is critical for correct development and differentiation. Thus, The common shrew (Sorex araneus) is subdivided we are performing studies to analyse genome into numerous races distinguished by Robertso- organisation and nuclear architecture at impor- nian fusions and whole-arm reciprocal transloca- tant stages in early development and differ- tions. Two of these races, the Oxford and entiation. Hermitage, make contact in Britain and form a We have analysed the nuclear position of the zone of hybridisation. Hybrid zones are of inter- majority of porcine chromosomes in a number of est because they may be sites of speciation, i.e. in vitro cultured cell-types: embryonic ¢broblasts, where gene flow is halted. In fact the Oxford- adult ¢broblasts, lymphocytes and mesenchymal Hermitage hybrid zone has a structure that is stem cells. Apart from one chromosome all chro- unlikely to promote speciation; instead natural mosomes are positioned in similar places in the selection has led to a structure where gene flow is nucleus. maximised. The zone was characterised along a To study nuclear and genome organisation in small part of its length in the 1980s. I have now di¡erentiated cells we have established in vitro dif- mapped nearly the whole length of the zone ferentiation systems as models for cell di¡erentia- between the Thames, Severn and Mersey estu- tion during embryogenesis by di¡erentiating aries and the structure is the same along this porcine mesenchymal stem cells into myocytes total length, consistent with powerful selective and adipocytes. To compare in vitro with in vivo forces favouring that structure. The detailed growth conditions we are studying the same mapping of such a long hybrid zone provides a aspects of nuclear and genome organisation in valuable opportunity to relate hybrid zone posi- frozen tissue sections. tion and width to geographic features and I will For developmental studies we have assessed present an analysis of this in which Geographic chromosome positioning in sperm from three dif- Information Systems and population genetic ferent breeds of pig. These chromosomes are non- mapping techniques have been applied. randomly organised both radially and long- itudinally. Chromosome positioning in sperm is also totally di¡erent to somatic cells and thus, L13 may have a role in controlling early paternal gene Avian lampbrush chromosomes: expression in the new embryo. We are also comparing aspects of genome and molecular composition and variety nuclear organisation in in vivo and in vitro con- of lateral loops and chromosome structed porcine embryos. This will reveal any det- associated structures rimental alterations common in IVF embryos and E. Gaginskaya1, S. Derjusheva1, may lead to improved selection criteria for IVF 1 1 embryos. A. Krasikova , T. Kulikova , A. Kurganova 1 These studies presently make the pig the organ- and A. Saifitdinova ism that has the most chromosomes mapped in 1Biological Research Institute of Saint-Petersburg the most cell types. University, 198504, Russia 15th ICC: Abstracts 5

Meiotic diplotene chromosomes in germinal 1Department of Biotechnology, Graduate School vesicles (GVs) of avian oocytes transform to of Engineering, Osaka University; 2Institute for typical lampbrush chromosomes (LBCs). Microbiological Diseases, Osaka University; 3 Numerous simple lateral loops are characterized Nara Advanced Institute of Science and by intensive transcription and rather uniform Technology, Japan morphology when stained with antibodies against phosphorylated form of RNA poly- Despite more than a century of research on chro- merase II (pol-II) or probed with chromosome mosome structure, it still remains poorly under- paints according to DNA/RNA hybridization stood. One of the reasons for this situation has protocol. The variety of simple loops clarifies been the difficulty in the biochemical study of itself under immunocytochemical investigation metaphase chromosomes. of bound proteins. Lateral loops, specifically In the present study, isolated human metaphase enriched with either mRNA processing protein chromosomes, which were prepared from the syn- K, or dsRNA-editing enzyme ADAR, or a chronized human cell line by a centrifugation protein sharing the epitope with RPC39 subunit method by two representative solutions for chromo- of RNA pol-III, etc., have been revealed. This some isolation (polyamine bu¡er, PAB and citric intriguing feature appears to be a general func- acid solution, CAS), were examined by both scan- tional characteristic of pol-II lateral loops on ning electron microscopy (SEM) and one-dimen- LBCs. Earlier, such a peculiarity of individual sional SDS-PAGE. SEM observation of isolated lateral loops was also described for amphibian chromosomes in PAB and CAS revealed the two LBCs. Lateral loops of complex morphology, types of surface structures which have repeatedly in particular telomeric giant loops (TGLs) on been reported to date. The surface of the human avian LBCs, appear not to be transcribed by chromosomes in PAB was relatively £at structure pol-II. TGLs accumulate splicing snRNPs covered irregularly with scales di¡erent in shapes and SR protein SC35 being considered as a and sizes, while the surface of the chromosome in kind of SC35 domains in avian GVs. Chromo- CAS had the dense ¢brous structure with uniform some associated structures of various morphol- diameter of 50^70 nm. Analyses of protein composi- ogy (protein bodies, spaghetti marker and tions of these two chromosomes by one-dimen- some others) do not either reveal any transcrip- sional SDS-PAGE showed the marked di¡erence tional activity and are mainly composed of from each other where linker histone, H1, was proteins of special nature. The protein bodies removed from the chromosomes isolated with CAS. may reflect a specific function of centromeric These results suggested that the removal of chromo- DNA repeats. An attempt to systematize the somal proteins including the linker histone might be types of lateral loops according to their mole- responsible for the speci¢c morphology of the chro- cular composition and suggested function has mosome surface structure. Identi¢cation of chromo- been made. somal proteins by biochemical analysis combined with the morphological analysis would provide the The research is supported by Russian Foundation for Basic essential data for the elucidation of higher order Research (project 02-04-49116), and partly by Russian Minis- structure of metaphase chromosomes. try of Education and Science (projects PD02-1.4-291 and 2655.2003.04). L15 Differential gene expression in L14 yellow-neckedmiceApodemus£avicollis Changes in chromosomal surface with and without B chromosomes structure are directly related to protein N. Tanic1, M. Vujosevic1, N. Dedovic-tanic2 composition and B. Dimitrijevic2 1 1 2 3 K. Fukui , S. Uchiyama , T. Sone , M. Iwano 1Institute for Biological Research ‘Sinisa and S. Matsunaga1 Stankovic’ 2Institute for Nuclear Sciences ‘Vinca’ 6 15th ICC: Abstracts

Most B chromosomes are heavily heterochro- On the other hand, the progress of scienti¢c stu- matic promoting the general idea that they are dies on the molecular level including the genome genetically inert. B chromosomes of Apodemus project requires chromosome identi¢cation. The flavicollis are euchromatic and show high degree success of FISH using B. mori BAC probes of homology with the A chromosomes. Euchro- (Sahara et al. 2003) opens the gate of BAC-FISH matic nature of Bs in A. flavicollis suggests that chromosome identi¢cation. A library consisting of they may carry active genes and have transcrip- 36,864 BACs constructed from two strains (Wu et tional activity. We applied Differential Display al., 1999) and, physical map based on RAPDs and Reverse Transcription – Polymerase Chain Reac- mutants (Yasukochi 1998; Fujii et al. 1998) are the tion (DD RT-PCR) in order to analyze and com- basis of this approach. pare gene expression in animals possessing Bs Here we identify all 28-chromosome bivalents and animals without B chromosomes. After sec- including the WZ by using BAC-FISH in B. mori. ond and third round of amplification three Positions of hybridization signals from BAC cDNA fragments were differentially expressed in probes correspond roughly to physical map +B mice compared to 0B animals. These cDNAs position. Moreover, we accomplish B. mori karyo- were: chaperonin containing TCP-1, subunit 6b typing using 2-color combination of 60 BAC (zeta) (Cct6b), Fragile histidine triad gene (Fhit) probes. We expect the BAC-FISH method is uni- and hypothetical gene XP transcript. Differential versally applicable for karyotyping of species in expression pattern was confirmed by Real Time which chromosome identi¢cation is problematic. RT-PCR. Nature of Bs and significance of these effects are discussed in the context of presented data. L17 A tandemly repetitive centromeric L16 DNA sequence from the ¢sh Hoplias BAC-FISH karyotyping in Bombyx malabaricus is derived from the 5S mori rDNA K. Sahara1, A. Yoshido1 and Y. Yasukochi2 C. Martins1, I. Ferreira1, C. Oliveira1, 1 2 1Graduate School of Agriculture, Hokkaido F. Foresti and P. Galetti Jr. 2 University, Japan; Genome Research 1Departamento de Morfologia, Instituto de Department, National Institute of Agrobiological Biociencias, Universidade Estadual Paulista, CEP Sciences, Japan 18618-000, Botucatu, SP, Brazil. 2Departamento de Gene´tica e Evolucio, Universidade Federal de Chromosome identification is fundamentally Sao Carlos, CEP 13565-905 Sao Carlos, SP, Brazil important for detection of gene mutations such as point mutations, insertions, deletions, inver- A substantial fraction of the eukaryotic genome, sions or translocations. Cytogentic mapping and in some instances greater than 90%, consists of the generation of BAC libraries are inevitable repetitive DNA sequences that include satellite, requirements of whole genome shotgun sequen- minisatellite, microsatellite, and transposable ele- cing projects. ments. Although extensively studied for the past In Lepidoptera, chromosomes are generally four decades, the molecular forces that propagate small, and uniform in shape and size without and maintain repetitive DNAs in the genome are primary constrictions in mitotic metaphase. Since still not well understood. To further the under- an applicable banding technique has not yet been standing on the dynamics and evolution of repe- found, chromosome identi¢cation and cytogenetic titive DNAs in vertebrate genome, we have mapping were thought to be not feasible. The silk- searched repetitive sequences in the genome of worm, Bombyx mori (2n ¼ 56 chromosomes), has the fish Hoplias malabaricus, a model species for been one of the most problematic lepidopteran genetic studies. We have discovered a tandemly species to be employed in chromosome research. repetitive DNA family, named Hmal1, that has 15th ICC: Abstracts 7 derived from repeat units of the 5S rDNA by detected satellite III on the Tragelaphini (Tragela- insertions/deletions and base substitutions. The phus euryceros). This ¢nding could be revealing of 5S rRNA bona fide gene repeats are clustered in an earlier appearance or formation of 1.706 satel- the interstitial position of two chromosome pairs lite III DNA, prior to the Bovini and Tragela- and its 5S variant type is clustered in the cen- phini divergence. tromeric region of nine chromosome pairs. The Although the existence of satellite III in water satellite sequence Hmal1 could has arisen by bu¡alo (Bubalus bubalis) had been reported, it transposition from the bona fide 5S gene repeats was noticed to only one autosomal pair. In the pre- and spread out in the centromeric region of sev- sent work we were able to observe signal hybridi- eral chromosomes due to the action of concerted sation in the majority of the autosomes, and evolution events. Such results are evidencing that remarkably, in the sex chromosome X, in contrast the Hmal1 family escaped from the selective with the absence of satIII in Bos chromosome X. pressure that maintain the structure and organi- These observations are revealing of a di¡erent zation of the 5S rDNA and is free to disperse in evolutionary mechanism for the sex chromosome the genome and has been maintained in the cen- X of this species. tromeric regions because of any structural advan- tage conferred by a possible centromeric function in the chromosomes. PO:06:02 Karyological comparative study of PO:06:01 the genus Leiolepis (Reptilia, New insights into Bovidae satellite III Uromastycidae) from Thailand 1 2 evolutionary history V. Aranyavalai , W. Chulalaksananukul and K. Thirakhupt3 F. Adega1, R. Chaves1, J. Heslop-Harrison2, 1 J. Wienberg3 and H. Guedes-Pinto1 Biological Science Program, Faculty of Science, Chulalongkorn University, Bangkok 1033, 1Department of Genetics and Biotechnology, Thailand; 2Genetic Program, Department of Centre of Genetics and Biotechnology, Vila Real, Botany, Faculty of Science, Chulalongkorn Portugal; 2Department of Biology, University of University. Bangkok 10330, Thailand; Leicester, UK; 3Institute of Human Genetics, 3Department of Biology, Faculty of Science, University of Munich, Munich, Germany Chulalongkorn University, Bangkok 10330, Thailand Repeats like satellite DNA sequences constitutes a rich palaeontological record, holding crucial The karyotype of four species of butterfly lizards clues about evolutionary events. As in the others collected in Thailand, Leiolepis belliana belliana, mammalian orders, the Bovidae centromere is L. belliana ocellata, L. reevesii rubritaeniata, were greatly composed of satellite DNA families. described in detailed for the first time. All have Satellite 1.706 (satIII) is a long unit of 2350 bp 2n ¼ 36 chromosomes and the same karyotypic composed of complex and altered repetitions of formulae were 10m þ 2sm þ 24mc (12 macro- two related and homogeneous 23-mer tandem chromosomes and 24 microchromosomes). The subrepeats, the Pvu and the Sau motifs. L. boehmei has 2n ¼ 34 chromosomes and its kar- The scope of this work was the isolation, yotypic formula was 10m þ 2sm þ 22mc (12 mac- sequencing and analysis of the genome organiza- rochromosomes and 22 microchromosomes). The tion and physical distribution (by Southern Blot higher microchromosome number occurred in the and Fluorescent in situ Hybridization) of these first three types of lizard which may be due to satellite sequences in several Bovidae species. the karyotypic change by centric fission in L. The procedures used showed that the presence boehmei. All the time of our exploration, L. of this satellite family was not restricted to the boehmei male was never found in southern part Bovini (Bos taurus, Bos indicus and Bubalus buba- of Thailand, indicating that this population was lis), as described in previous works; instead we a unisexual species of Leiolepis. Moreover, the 8 15th ICC: Abstracts difference in microchromosome number of L. commonality in the processes that trigger hyper- boehmei compared to the others can be explained condensation and to distinguish two propositions more clearly by Von Wolfgang Ba¨hme (1982) from each other. The first, that the conforma- about the Leiolepis triploida found in south of tional change is due to slight shifts in rotational Thailand. It was hypothesized that triploid could positioning of adjacent nucleosomes and the sec- be allotriploid hybrid clone between the diploid- ond that the conformational change is due to a parthenogenetic female and males of the normal re-arrangement of the placing of bridge-histones bisexual species. The hypothesis of L. triploida relative to the nucleosome core histones. occurrence can be proposed from our karyotypic data. The relationship of these species with the other congeneric species will be described from PO:06:04 the cytotaxonomic point of view. Cytogenetic characterization of metaphase chromosomes of alpaca Reference (Lama pacos): I-GTG/RBG-banded

Ba¨hme, W. 1982. Uber schmetterlingsagmen, Leiolepis b. karyotypes and idiograms belliana (Gray, 1827) der Malayischen halbinsel und ihre D. Di berardino1, G. Balmus2, D. Nicodemo1, parthenogenetischen linien (Sauria: Uromastycidae). Zool. 1 1 1 Jb. Syst. 109: 157–169. G. Coppola , C. Verdoliva , G. Coppola , G. Enne3, G. Bozzini4, G. Di Meo5 and L. Iannuzzi5 PO:06:03 1Department of Animal Science and Food Hypercondensation; an old change of Inspection, University of Naples, Portici, Italy; 2Faculty of Veterinary Medicine, Iasi, Romania; conformation that is still somewhat 3Italian Experimental Institute, Milan, Italy; mysterious 4Italian Alpaca Breeder Center, Grosseto, Italy; 5 1 1 National Research Council (CNR), ISPAAM, L. Burgoyne and D. Repucci Laboratory of Animal Cytogenetics and Gene 1School of Biological Sciences, Flinders Mapping, Naples, Italy University of South Australia, Bedford Park 5042, Australia The present study was undertaken to provide GTG- and RBG- banded karyotypes and repre- From the literature, there are four very broad sentative ideograms for the alpaca species (Lama levels of condensation of chromatin in order of pacos, 2n ¼ 74), to be used as a basis for future increasing condensation., (A) the tran- work on karyotype standardization and gene scriptionally-enabled (least condensed class), (B) mapping, as well as for future genetic improve- heterochromatic, (C) metaphase-level of con- ment programs within the family Camelidae. Per- densation, and (D) hypercondensed; pyknotic.- ipheral blood cultures from eight alpaca the most condensed class. Chromosomes are individuals, four males and four females, were set commonly collapsed into this latter class by up according to the standard methods. After 48 colchicine or DNA breakage or other death hours of growth, cells were synchronized with processes. The process is readily scored by phase- methotrexate; after 16 hours, the S-phase block contrast microscopy but it is difficult to study by was released by washing and the cells allowed to electron microscopy as handling and or fixatives grow for further 5–6 hours in fresh medium, sup- often induce it before embedding. This presenta- plemented either with thymidine for conventional tion is part of an in-vitro study into the nature of karyotyping and GTG-banding, or with BrdU/ pyknosis, its triggers, the events that accompany H33258 for RBG- banding, respectively. The it, and the causal relationships between the GTG- and RBG- banded karyotypes reveal a events surrounding it. Evidence will be examined large submetacentric X-chromosome, a very in an attempt to discover any underlying small metacentric Y- and 72 autosomes which 15th ICC: Abstracts 9 have been morphologically classified into 2 chromosomes were involved in the creation of a groups, as follows: group (A) includes 29 pairs, metacentric chromosome. ranging from sub-meta, sub-telo and acro- centrics; group (B) includes 7 distinct pairs of metacentrics. The GTG/RBG-banding pattern of PO:06:06 individual chromosome pairs and their ideograms The metaphase chromosome proteome are presented at 362 bands level. M. Emrick1, K. Resing1, L. Aveline-Wolf2, K. Meyer-Arendt2, K. Pierce1 and N. Ahn2 PO:06:05 1University of Colorado, Boulder, CO; 2Howard Robertsonian translocation in the Hughes Medical Institute Caspian minature horse Chromosomal condensation and separation of S. Dordari1, R. Mehran Nejhad1 and 2 sister chromatids during cell division requires S. Seyed Rassaghi coordinated assembly and function of hundreds 1Tehran Agricultural Research Center, Iran; of proteins. Malfunction of these proteins, many 2Private Veterinary Laboratory, Iran of which are unknown, can compromise genetic stability, which is critical to cell integrity, Przewalski’s horse has extensive chromosome proliferation, and survival. homology with the domestic horse. Two acro- Here, we perform a proteomic analysis of mito- centric pairs in Equus prezualskii appear to be tic chromosomes from metaphase-arrested HeLa combined in one metacentric chromosome (chro- S3 cells enriched on a sucrose gradient. Bound mosome 5) in the E. caballus karyotype. The Cas- proteins are sequentially extracted from the chro- pian horse is one of the exception that has mosomes with urea then with an acid-labile surfac- reported as translocation heterozygotes and have tant (ALS), prior to trypsin proteolysis. The not been directly implicated in fertility problems resulting peptides are separated by reverse-phase in this breed. This breed of horse has been chromatography before undergoing fragmenta- thought to be a pony type but is one of the min- tion on an LCQ mass spectrometer. The spectra iature horses today. In the recent study on 30 are analyzed using in-house search algorithms registered Caspian horses, blood samples were that rely on peptide chemical properties, as well used for metaphase chromosomes provided by as consensus between Sequest and Mascot search use of short-term culture and studied by simple programs (Resing et al., 2004). staining and Giemsa banding method. The chro- These preliminary studies identify several mosome spreads were photographed and kar- known chromatin-binding proteins, including yotyped. The results showed the diploid model four histones, several transcriptional regulators, karyotype as 2n ¼ 64 and 2n ¼ 65. Those which many chromosomal structure regulators, and a had 64 chromosomes were obtained 31 autosomal kinetochore protein. This con¢rms that our novel chromosomes that 7 pairs were metacentric and 6 extraction procedure enriches for chromosomal pairs were submetacentric and 18 pairs were telo- proteins. Extraction under these conditions enri- centric. Preparation with 65 chromosomes were ched nuclear proteins to *40% of the total pro- contained similar chromosome type except that teins identi¢ed, with an additional *30% of one extra metacentric chromosome was included. unknown localization. The X sex chromose was large metacentric and To detect lower abundance proteins, we are the Y chromosome was small telocentric as same improving this procedure with strong cation as other domestic horse species. exchange and gas-phase fractionation. In order to This karyotype with 65 chromosome in the Cas- eliminate the identi¢cation of contaminating pro- pian miniature horse is unique and this extra chro- teins, we plan to employ protein fraction pro¢ling mosome is the result of a natural centric fusion (Anderson et al., 2003). Additionally, immuno- obtained from crossing between E. przewalski £uorescence studies will be performed using exo- (2n ¼ 66) chromosomes in which two acrocentric genously expressed proteins to con¢rm mitotic 10 15th ICC: Abstracts chromosome localization of novel proteins. tilapia. The physical mapping of repetitive DNAs Studies are also being undertaken to look for to metaphase chromosomes represents a powerful post-translational modi¢cations such as acetyla- toll to the knowledge of the genome of this tion, phosphorylation, and methylation. important teleost fish.

PO:06:08 PO:06:07 Analyses of human chromosomal The role of repetitive DNA sequences proteins using monoclonal antibodies for the comprehension of Nile tilapia 1 1 1 (Oreochromis niloticus) genome T. Higashi , S. Miyakawa , H. Takata , A. Terauchi2, T. Shimizu2, M. Oda2,S. 1 1 1 I. Ferreira , R. Shimizu , C. Oliveira , Uchiyama1, S. Matsunaga1, T. Azuma2 1 1 F. Foresti and C. Martins and K. Fukui1 1 Departamento de Morfologia, Instituto de 1Department of Biotechnology, Graduate School Biociencias, Universidade Estadual Paulista, CEP of Engeneering, Osaka University, Japan; 18618-000, Botucatu, SP, Brazil 2Division of Biosignaling, Research Institute for Biological Sciences, Tokyo University of Science, The Nile tilapia (Cichlidae, Perciformes) has Japan received increasing scientific interest over the past few decades due to its enormous importance in Chromosomes consist of DNA and various kinds worldwide aquaculture and the rapid and exten- of proteins such as histone proteins, high mobi- sive evolutionary radiation undergone by the lity group proteins and topoisomerase II. How- cichlid fishes. Data on Nile tilapia genome will ever, the total image of chromosomal proteins immediately accelerate research in basic and are still remained unclearly. To identify chromo- applied biology. One unclear point in the genome somal proteins and elucidate their nature, we comprehension is the large regions that contain prepared monoclonal antibodies against human repetitive sequences. Such regions remain as gaps chromosomes which were extracted by the poly- even in most complete sequenced genomes. Stu- amine method. After screening of hybridoma dies of repetitive sequences can provide the strains based on the localization pattern of anti- understanding of these regions, contributing to a gen proteins by the indirect immunofluorescence complete physical map for the species. Concern- method, twenty-five strains were detected to pro- ing the Nile tilapia genome, studies on repetitive duce the monoclonal antibodies against chromo- sequences have been directed to satellite DNAs, somal or nuclear-localized proteins. Then the telomeres, 45S and 5S rDNAs, and the short and antigen-proteins localized at chromosomes or long interspersed nucleotide elements (SINEs and nuclei were studied. The antigen proteins were LINEs). Although several repetitive DNA purified by immunoprecipitation using HeLa cell sequences have been characterized, the presence extracts. The antigen proteins purified, which of a large number of repetitive families remains could be detected by western blotting, were iden- to be described in the genome of Nile tilapia. In tified by MALDI-TOF MS using the peptide this way, we have searched a restriction finger- mass finger printing method. Most proteins were print database (freely available at http://hcgs.un- core and linker histones. However, nuclear mito- h.edu/cichlid/#bacs.), assembling 35,000 BAC tic apparatus protein (NuMA), hnRNP and beta- clones (5x genome coverage) from the Nile tila- actin were identified as chromosomal proteins by pia. Candidate BACs were identified and dot- our method. For confirmation of chromosomal blot screened for the presence of repetitive localization of these proteins more precisely, the sequences. Several BAC clones were isolated and indirect immunofluorescence method using meta- used as probes for the chromosome mapping. phase spreads was also performed. These results These results are contributing to the construction indicate that our method is useful for identifica- of a physical chromosome map for the Nile tion of chromosomal and nuclear proteins. 15th ICC: Abstracts 11

PO:06:09 Calreticulin (CRT) is a calcium binding protein which mainly exists in an endoplasmic reticulum, SC-FISH of repeated sequences in and functions as a molecular chaperon that three species of clams and cockles assists folding of newly synthesized proteins. On N. Hurtado1, P. Moran1 and J. Pasantes1 the other hand, recently, the existence of CRT in the nuclear matrix has been reported and its 1 University of Vigo, Spain involvement in the regulation of gene expression has been discussed. Investigation of the sub- The species belonging to the Order Veneroida cellular localization of CRT through the cell (Mollusca, Bivalvia) inhabit an extensive variety of cycle will lead us to understand the function of marine ecosystems. Although many of these species this multifunctional protein. In this study, spe- are commercially important, chromosome research cific localization of calreticulin on human meta- in these organisms is still rather scarce. During the phase chromosomes was revealed biochemically last few years the combination of FISH techniques and immunohistochemically. Quantitative analy- with surface spreading of whole synaptonemal sis using western-blotting with anti-CRT anti- complexes (SC-FISH) has greatly increased the use body revealed that the amount of CRT in the of pachytene cells in chromosome research. Never- metaphase chromosomes was more than three theless, none of these methods have been applied times compared to that in the G2-nuclei. Immu- to meiotic chromosomes of bivalves. nostaining against thin sections of human mitotic Synaptonemal complexes were prepared from cells as well as isolated chromosomes showed males of three species of Veneroida, the clams Dosi- the localization of CRT on the surface of the nia exoleta and Spisula solida and the cockle metaphase chromosomes. To identify the binding Cerastoderma edule, using a surface spreading target of CRT to metaphase chromosomes, we technique. FISH was performed using all human performed the far-western experiment against telomeric probes and species speci¢c 18 þ 28S and chromosomal proteins and the binding assay 5S rDNA probes. Species speci¢c probes for using chromatin fibers purified from metaphase 18 þ 28S rDNA internal transcribed spacers (ITS) chromosomes. As the result, it was indicated that and 5S rDNA coding and spacer sequences were recombinant human CRT directly binds to core generated by PCR. The ampli¢cation products histones. Present results suggest that CRT is one were analysed by SSCPs, cloned and sequenced. of the proteins transported to daughter cells by Major ribosomal gene clusters are localised in mitotic chromosomes during the cell division. only one bivalent in the three species analysed. ITS signals appear in a loose way surrounding the PO:06:11 telomeric region of the SC. On the contrary, the telomeric signals appear tightly associated at the Heterochromatin patterns on end of the SC both on the NOR-bearing region chromosomes of beetle species and all the other telomeric regions. The number (Insecta: Coleoptera). Present stage and position of the 5S rDNA clusters is di¡erent for each species but they also appear loosely of knowledge surrounding the SCs. D. Lachowska-Cierlik1, M. Rosek1, E. Petitpierre2 and M. Holecova3 PO:06:10 1Institute of Systematics and Evolution of Animals Polish Academy of Science, Krakow, Localization of calreticulin on human 2 Poland; Laboratori de Genetica, Deparatment de metaphase chromosomes Biologia, Facultat de Ciencies, UIB, Palma de 3 S. Kobayashi1, A. Kuwae1, S. Uchiyama1, Mallorca, Spain; Department of Zoology, Comenius University, Bratislava, Slovakia S. Matsunaga1 and K. Fukui1 1Department of Biotechnology, Graduate School Data on heterochromatin patterns are known of Engineering, Osaka University, Japan only for some of beetle families. It seems that the 12 15th ICC: Abstracts main reason for the small number of data is the their high commercial value is an important limited amount of heterochromatin in the chro- resource for fishing sector and now it is carried mosomes of most beetle species. Hetero- out their experimental farm. The knowledge of chromatin patterns on the chromosomes of beetle this species is restricted to ecological, nutritional species were studied using the C-banding techni- and reproductive aspects. No genetic studies have que. Banding techniques allow a better character- been done before. ization of beetle karyotypes and selectively reveal As a step toward the genetic knowledge of P. chromosome regions consisting of constitutive adspersus in this work we describe their kar- heterochromatin. The results confirm that most yotype by means of classic and molecular cytoge- of the beetle species possess a small amount of netic techniques. Paralichtys genera is formed of heterochromatin. All chromosomes obtained 35 species and it is known the 2n in P. olivaceus from spermatogonial cells possess C-banded seg- and P. lethostigma. ments which are visible during the pachytene and In chromosomes obtained by lymphocytes cul- diplotene. When the chromosomes become more ture, the Giemsa stain and CMA3 banding was condensed during the mitotic metaphase, and used, as well as major (28S and 18S) and minor diakinesis, metaphase I or II of meiosis, short (5S) rDNA probes were located by FISH. heterochromatic segments are weakly visible or P. adspersus possesses 2n ¼ 46, with one meta- undetectable. In some families with a large centric and 22 telocentric pairs (NF 48). This kar- amount of heterochromatin, C-bands were yotype di¡ers with the reported for the other two observed in the centromeric region in all auto- members of this genera, with 48 telocentric somes during all nuclear division. Some species chromosomes. The conservation of the NF 48 also show additional heterochromatic regions in indicates the occurrence of Robertsonian rearran- the interstitial and/or telomeric regions. In the X gements in the Paralicthys genome. The NOR chromosome, C-positive segments are usually bearing pair is telocentric, but we found a third present in the centromeric region or distributed NOR bearing chromosome in some individuals. along the chromosome, while the Y chromosome Like in the most of teleosts the 28S and 5S riboso- is often completely euchromatic. Large hetero- mal genes were located in separate loci being the pycnotic parts were observed in the mealworm 5S located in a small telocentric pair. beetle, Tenebrio molitor, and other tenebrionids, The cytogenetic information obtained in this and also in species from the genera Bembidion work is a necessary step for the establishment of and Trechus (Carabidae) which show very triploid and homozygote lines in this species. conspicuous blocks of procentric C-bands in almost all chromosomes. Grant FONDEF D02I 1094

PO:06:12 PO:06:13 Chromosomal characterization and Restriction enzyme digestion physical mapping of the ribosomal chromosome banding in Crassostrea genes (5S and 28S) of Chilean £ounder and Ostrea species: Comparative (Paralichtys adspersus). karyological analysis within Ostreidae 1 2 2 N. Lam1, P. Iturra2 and A. Diaz1 A. Leitao , R. Chaves , S. Santos , H. Guedes-Pinto2 and P. Boudry3 1Universidad Andre´s Bello, Facultad de Ecologia y Recursos Naturales. Republica 252, Santiago, 1Institut National des Sciences et Technologies de Chile; 2Universidad de Chile, Facultad de la Mer, Tunisia; 2Department of Genetics and Medicina, ICBM, Independencia 1027, Santiago, Biotechnology, ICETA-UTAD, Portugal; Chile 3IFREMER, Station de La Tremblade, France

The Chilean flounder P. adspersus is distributed Reliable banding techniques are one of the major from Paita in Peru until Lota in Chile. Due to needs to develop the genetic research in oysters. 15th ICC: Abstracts 13

In this study, we have carried out the cytogene- not always contain linker histone (H1) genes. In tical characterization of four oyster species order to map the core histone genes in Mytilus gal- (family Ostreidae) using restriction endonucleases loprovincialis (Lamarck 1758) and Mytilus edulis treatments. Chromosomes were treated with platensis (d’Orbigny 1846) by FISH, chromosome three different restriction enzymes (REs), stained preparations were obtained from gill and gonadal with Giemsa, and examined for banding patterns: tissues of juvenile mussels, and from whole Crassostrea gigas (2n ¼ 20, total number of bands embryos and larvae, after hypotonic treatment with ApaI: 74, HaeIII: 61, PstI: 76), Crassostrea and ¢xation with methanol/acetic acid. Probes angulata (2n ¼ 20, total number of bands with for H2B-H2A and 18 þ 28S rDNA internal tran- ApaI:62, HaeIII:61, PstI: 55 ) (sub-family Cras- scribed spacers (ITS) were generated by PCR. sostreinae) and Ostrea edulis (2n ¼ 20, total num- Core histone gene clusters are located in two ber of bands with ApaI: 82, HaeIII: 59, PstI: 66), middle sized chromosome pairs, a metacentric Ostrea conchaphila (2n ¼ 20, total number of pair and a submeta/subtelocentric one. These bands with ApaI: 68, HaeIII: 62, PstI: 69) (sub- gene clusters are close to the centromere on the family Ostreinae). The treatment of samples with short arm of the metacentric chromosome and in ApaI, HaeIII and PstI REs produced specific the subtelomeric region of the submeta/sutelo- banding patterns, which demonstrate the poten- centric long arm. This submeta/subtelocentric tial of these enzymes for chromosome banding in chromosome pair is none of the two NOR- oysters. This is of special interest since it has bearing chromosomes of these species. been recently shown in mammal chromosomes that restriction enzyme banding is compatible PO:06:15 with fluorescent in situ hybridisation. This study therefore provides a fundamental step in genome The karyotype of the wild asses (Equus mapping of oysters, since the chromosome band- hemionus Khur), one of the two types of ing with restriction enzymes will facilitate donkeys in India physical gene mapping in these important aqua- cultured species. The analysis of the banded J. Solanki1, R. Sabapara1, S. Bheemineni2, karyotypes revealed a greater similarity within R. Uppala2 and R. Uppala2 the genera of Crassostrea and Ostrea than 1Department of Animal Genetics and Breeding, between them. Veterinary College, Gujarat Agriculture University, Anand-388001; 2Green Cross Blood PO:06:14 Bank & Genetic Research Centre, Anil Kunj, Paldi, Ahmedabad-380014, India Chromosomal mapping of core histone genes in mussels The Indian wild ass (Equus hemionous Khur) widely known by the local name: Ghurkhur is C. Perez-Garcia1, P. Moran1 and J. Pasantes1 found in the little Rann of Kutch desert on the 1University of Vigo, Spain kathiawadi peninsula of Gujarat state, India. They are living in a small population and adult The class Bivalvia includes some of the best- animals are study, medium sized, ranging in known marine invertebrate species, many of height from 110–120 cm at shoulder and about which are commercially harvested around the 200 kg of the body weight. They are light gray world. Most chromosome studies of the family to almost white in color with a dark brown Mytilidae have been performed using classical or bright yellow sandy line extending down the cytogenetic techniques and there are only a few back to the root of the tail. Other markers works using fluorescent in situ hybridisation include short ears with fawn colored patches (FISH). on the shoulder, saddle and sides up to the In the genus Mytilus the core histone genes are rump. Cytogenetic investigations were carried arranged as regular gene repeats of all four core out on two males and two female wild asses. histones (H4, H2B, H2A and H3). The repeats do Chromosomal preparations were obtained from 14 15th ICC: Abstracts peripheral heparinized blood drawn from jugular chromosomal, 8 were likely contaminants, 38 vein by standard culture techniques followed by were known, but not described in mitosis and 16 Giemsa (GTG), centromeric (CBG) banding and were uncharacterised. Of this latter group, we Ag-NOR staining techniques were performed to have tagged three with GFP and shown all of identify the chromosomes. Analysis of chromo- them to be chromosomal. Two appear to be some preparations indicates that the diploid nucleolar proteins that coat the chromosome per- chromosome number is 2n ¼ 56 and a funda- iphery in mitosis. The third tagged component mental number NF is 100. This includes 54 auto- turned out to have a distribution in cells identical somes (42 metacentric, submetacentric and 12 to the chromosomal passenger complex of acrocentric autosomes), a submetacentric X chro- INCENP, Aurora B and survivin. Studies to be mosome and a small Y chromosome, which presented indicate that the novel protein, which appears to be metacentric. In the female, both we call Borealin, is a component of the complex the X-chromosomes were homologous and did that is essential for normal mitotic progression. not show any polymorphism. It was not possible These results suggest that the chromosomal pas- to identify G-banded pattern of the Y chromo- senger complex is likely to be signi¢cantly more some on contracted preparations. To the best elaborate than has heretofore been appreciated. of our knowledge, this is the first report on the banded karyotype of this relatively rare and endangered species. L19 Molecular dissection of the Centromeres Symposium kinetochore-microtubule interface in Caenorhabditis elegans L18 I. Cheeseman1, S. Niessen2, J. Yates2, Aurora and Borealin: Not poles apart K. Oegema1 and A. Desai1 in the mitotic spindle 1Ludwig Institute for Cancer Research/UCSD; 2The Scripps Research Insitute, CA, USA R. Gassmann1, A. Carvalho1, A. Henzing1, 1 1 S. Ruchaud and W. Earnshaw Kinetochores play a crucial role in chromosome 1Wellcome Trust Centre for Cell Biology, segregation by mediating attachments between University of Edinburgh, Scotland, UK chromosomes and spindle microtubules. To iden- tify novel components of the Caenorhabditis Chromosomes lacking functional condensin lack elegans mitotic kinetochore, we have taken a chromosome scaffold fraction. One down- a biochemical approach by purifying native kine- stream consequence of this is that the structure tochore complexes from worm protein extracts. of the chromosomes is defective and they are Using this approach, we identified a 10-subunit highly sensitive to hypotonic swelling, and lack a KNL-1/3 complex that is essential for kine- structural ‘memory’. Thus non-histone proteins tochore-microtubule attachments. Functional of the chromosome scaffold fraction have an analysis of each subunit and determination of essential role in mitotic chromosome structure. their kinetochore localization requirements indi- In order to characterise chromosome sca¡old cates that the KNL-1/3 complex is primarily components in greater detail, we isolated mitotic composed of three distinct functional modules: chromosomes from human cells. Chromosome KNL consisting of KNL-1 and KNL-3, a˜MISa¨ sca¡olds prepared from this fraction were sub- consisting of MIS-12, KBP-1, and KBP2, and jected to SDS-PAGE, and 42 bands excised from a˜NDCa¨, consisting of NDC-80, Nuf2(HIM-10), the gel were subjected to MALDI-tof analysis. and Spc25(KBP-3). KNL subunits are essential This analysis identi¢ed 85 candidate proteins that for the assembly of the outer domains of the were likely to be bona fide components of the kinetochore, a˜MISa¨ subunits control the rate and chromosome sca¡old. Of these, 39 were known extent of formation of these outer domains, and 15th ICC: Abstracts 15

‘NDC’ subunits are necessary to sustain tension repressed by RNAi or when cells are exposed to at the kinetochore-microtubule interface. The Aurora inhibitors, cells arrest following spindle wide conservation of subunits from each module destruction. However, we have recently dis- implies that the functions and physical interac- covered that when Bub1 and Aurora are simulta- tions defined we have defined for the KNL-1/3 neously inhibited, the checkpoint is defective. complex will be relevant throughout the eukar- Furthermore, both Bub1 and Aurora activity are yotic kingdom. required to maintain BubR1as interaction with the APC/C. One explanation for these observa- tions is that the checkpoint is composed of two L21 arms, one dependent on Bub1, the other on Aur- Chromosome condensation without ora kinase activity, with both of these arms con- verging on BubR1, promoting its ability to bind condensin APC/C-Cdc20. We suggest that these two arms P. Vagnarelli respond to different spindle cues: while the Bub1 arm monitors kinetochore-microtubule attach- Wellcome Trust Centre for Cell Biology, ment, the Aurora arm monitors biorientation. University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, Scotland, UK This bifurcation in the signalling mechanism may help explain why aneuploid tumour cells No abstract was submitted for this talk. mount a robust checkpoint response following spindle damage despite exhibiting chromosome instability. L22 Bub1 and Aurora cooperate to prevent L23 aneuploidy by maintaining BubR1 Formation of kinetochores and mediated inhibition of the APC/C heterochromatin structures in C. Morrow1, V. Johnson1, C. Ditchfield1 vertebrate cells and S. Taylor1 T. Fukagawa1 1School of Biological Sciences, University National Institute of Genetics, Mishima, of Manchester, UK Schizuoka, Japan

The spindle checkpoint prevents aneuploidy by The centromere is a component of the chromo- inhibiting Cdc20 mediated activation of the ana- some that is required for accurate segregation of phase promoting complex/cyclosome (APC/C) chromosomes during mitosis in eukaryotes. Cen- until all the chromosomes correctly align on the tromere functions include sister chromatid cohe- microtubule spindle via their kinetochores. sion and separation, microtubule attachment, BubR1, an essential component of this check- chromosome movement, formation of hetero- point, localises to kinetochores and its kinase chromatin structures and mitotic checkpoint con- activity is regulated by the kinesin related motor trol. Because problems in chromosome protein CENP-E. BubR1 also inhibits APC/C- segregation can lead to cancer, aneuploidy, and Cdc20 in vitro, thus providing a molecular link cell death, we must understand the mechanism between kinetochore-microtubule interactions by which centromeres are formed and interact and the proteolytic machinery that regulates with the microtubules of the spindle apparatus mitotic progression. Several other protein kina- during cell division. To understand the molecular ses, including Bub1 and members of the Ipl1/ mechanism underlying centromere function, we Aurora family, also regulate anaphase onset. used hyper-recombinogenic chicken B lympho- However, in human somatic cells Bub1 and Aur- cyte cell line, DT40. The high level of homo- ora kinase activity do not appear to be essential logous recombination in DT40 cells permit for spindle checkpoint function: when Bub1 is efficient targeted modification of genes of 16 15th ICC: Abstracts interest. We have improved this system and have structural and functional foundation for the created several cell lines with conditional knock- kinetochore. We have used extended chromatin outs of several centromere proteins to investigate fibers and metaphase chromosomes from cul- the molecular mechanism of kinetochore assem- tured human cells and Drosophila cells and lar- bly and centromere function. Here we talk about val neuroblasts to study histone modifications phenotype of several mutants and discuss about within centromere regions as markers for the mechanism of kinetochore assembly. In addition chromatin state within these domains. H3 K9- to kinetochore assembly, it is important to diMe and H3 K9-triMe were not present within understand how heterochromatin structure is CEN chromatin, and only H3 K9-diMe was formed in centromere region of verterbrate cells. observed in regions immediately flanking the In this viewpoint we generated a conditional loss- CEN. Surprisingly, H3 K4-diMe staining was of-function mutant of Dicer in a chicken-human observed between CENP-A/CID subdomains in hybrid DT40 cell line that contains human chro- both humans and fly centromeres. We conclude mosome 21. We will discuss about implication of that centromeric nucleosomes contain histone RNAi machinery for formation of hetero- modifications previously associated with both chromatin in vertebrate cells. euchromatin and heterochromatin, but in a combination that is distinct from each chroma- L24 tin state individually. This modification pattern may contribute to the unique domain organiza- Centromeric chromatin demonstrates a tion and 3D structure of centromeric regions, distinct spectrum of histone and/or to epigenetic information that deter- modi¢cations mines centromere identity. B. Sullivan1, A. Lam1 and G. Karpen2 1Dept of Genetics and Genomics, Boston L25 University School of Medicine, USA; 2Dept of Genome Science, Lawrence Berkeley National Speci¢c DNA modi¢cations are Lab and Dept of Mol and Cell Biology, required for CENP-A assembly University of California, Berkeley, CA, USA S. Zeitlin1, S. Patel1 and G. Slupphaug2 Post-translational histone modifications are 1University of California, San Diego, USA; thought to regulate epigenetic switching between 2NTNU, Trondheim, Norway different chromatin states. In fact, distinct his- tone modifications have been correlated with DNA sequences in metazoan centromeres are not discrete functional chromatin domains. Acetyla- conserved. In spite of this, there is a conserved tion of histones H3 and H4, and H3 methyla- protein component of metazoan centromeres. tion at lysine 4 (K4), are predominantly The essential, centromere-specific histone H3 associated with euchromatin. H3 K4 di-methyla- variant, CENP-A, is required for kinetochore tion (H3 K4-diMe) is associated with ‘permis- formation. To determine how CENP-A is sive’ chromatin, and H3 K4 tri-methylation (H3 specifically assembled at centromeres, we develo- K4-triMe) is linked with transcriptional activity. ped a binding assay using sperm chromatin in Conversely, H3 K9 di- and tri-methylation (H3 cell-free extract derived from Xenopus eggs. We K9-diMe and H3 K9-triMe) mark constitutive show that the catalytic activities of APOBEC3G, and facultative heterochromatin, and are asso- a deoxycytidine deaminase, and UNG2, a uracil ciated with stochastic gene silencing (position DNA glycosylase, are required to create a stable effect variegation). Centromeric (CEN) chroma- binding site for CENP-A. In support of this tin is frequently located in or near hetero- model, inhibiting deoxycytidine deaminase with chromatin, and contains blocks of histone-H3 zebularine, or uracil DNA glycosylase with nucleosomes interspersed with blocks of CENP- Ugi, uracil or UTP results in a lack of DNA- A nucleosomes, the H3 variant that provides a bound CENP-A. Our data suggest that specific 15th ICC: Abstracts 17

DNA modifications are required for CENP-A L27 assembly. An increase in scaffold/matrix association is an epigentic event that L26 accompanies neocentromere formation New proteins at the metaphase H. Sumer1, J. Craig1, M. Sibson1 and mammalian centromere A. K. Choo1 1 1 1 J. Craig , P. Canham , E. Earle and 1The Murdoch Childrens Research Institute & A. K. Choo1 Dept. of Paediatrics, University of Melbourne, 1 Royal Children’s Hospital, Flemington Road, Chromosome Research Group, Murdoch Parkville, Melbourne, Victoria 3052, Australia Childrens Research Institute & Dept. of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville, Melbourne, Human neocentromeres arise at previously non- Victoria 3052, Australia centromeric loci in the human genome. They are functionally equivalent to alpha-satellite based The centromere, identifiable as the primary con- centromeres both in terms of mitotic stability striction on a metaphase chromosome, is an and centromere protein association. We have essential structure that coordinates the correct begun a systematic dissection of chromatin struc- segregation of chromosomes during cell division. ture at human neocentromeres, which have pro- Centromeres contain kinetochores, the structures ven to be valuable tools for dissecting centromere responsible for microtubule capture and spindle biology. Using a novel method which combines attachment. Centromeres also contain hetero- Scaffold/Matrix Attachment Region (S/MAR) chromatin, which has been shown to be involved DNA isolation and genomic array hybridization, * in gene silencing and chromatid cohesion. we have identified a 2.5 Mb region of densely A large number of kinetochore and hetero- packed chromatin surrounding the core neocen- chromatin-associated proteins have so far been tromere of a marker chromosome and confirmed localised to mammalian centromeres and further this by fluorescence in situ hybridization on salt- characterised using dicentric and neocentric chro- extracted metaphase chromosomes. These results mosomes. However, the role of some of these suggest that the dense S/MARs seen at normal proteins at centromeres is not understood; many centromeres are not just a consequence of the do not occur exclusively at centromeres and this tandemly repetitive nature of alpha-satellite suggests that the centromeres and certain regions DNA, but have an important role in the epige- within euchromatin share epigenetic marks and netic maintenance of centromere structure and macromolecular complexes. To help define these function. relationships, we have performed a large scale immunofluorescence analysis using antibodies L28 to proteins which, by association with known centromere proteins and with known macro- Functional mapping of an American molecular chromatin-related complexes, may be trypanosome centromere present at mitotic centromeres. We have per- 1 1 1 formed this analysis on Colcemid-arrested mito- J. Kelly , S. Obado and M. Taylor tic chromosomes from mouse and human cell 1London School of Hygiene and Tropical lines, including those containing neocentromeric Medicine, London, UK and dicentric chromosomes. A number of existing proteins were shown to be exclusively present or The trypanosomatids are an ancient family that enhanced at mammalian centromeres and fell diverged from the main eukaryotic lineage early into two broad groups of kinetochore- or hetero- in evolution. They include several species of chromatin-enhanced. The implications of these parasitic protozoa that are major public health results will be discussed. problems in many parts of the developing world. 18 15th ICC: Abstracts

Although genome sequencing is now complete, techniques made it possible to determine the chromosomal elements that mediate segregation chromosomal origin of those chromosomal mar- in these organisms have not been identified. In kers. In case 1, diagnosed prenatally, the NMC this report we will describe the functional map- originated from the short arm of chromosome 1, ping of a centromere in the American trypano- comprising two bands (1q21-q22). Autopsy, per- some, Trypanosoma cruzi, a parasite with an formed after intrauterine foetal atrophy on the unusual mechanism of the genetic exchange that 24th week of gestation, demonstrated mild intra- involves the generation of aneuploidy by nuclear uterine growth retardation while neither congen- hybridisation. Using telomere-associated chromo- ital anomalies nor malformations were detected. some fragmentation, we show that the region Case 2 was diagnosed postnatally, demonstrating required for the mitotic stability of chromosome increased postnatal growth, obesities and a few 3 encompasses a transcriptional strand-switch features if facial dysmorphism. Molecular cytoge- domain and a 16 kb GC-rich island. This region netic analysis by CGH technique revealed ampli- has been fully sequenced and contains several fication of only one chromosome band (15q22) degenerate retrotransposon-like insertions but from the distal arm of chromosome 15. The M- atypically, lacks the arrays of satellite repeats FISH technique confirmed the origin of NMC. that characterise many eukaryotic centromeres. The presented chromosome markers are among This may represent a paradigm for centromere the smaller human NMCs described in literature. organisation in trypanosomes and other primitive Additionally, case 2 is the first case of NMC, as eukaryotes. described in literature, with such chromosomal breaking points.

PO:06:16 Two new cases of neocentric marker PO:06:17 chromosomes (NMCs): molecular Fusions of GFP to the separate cytogenetic and clinical SAF-A/hnRNP U domains con¢rm characterisation computer analysis of the SAF-A M. Constantinou1, E. Zajac1, I. Plowas1 domain structure and B. Kaluzewski1 A. Kukalev1, O. Podgornaya1 and P. Percipalle2 1Department of Medical Genetics, Medical University of Lodz, Sterling str. 1/3, 1Institute of Cytology RAS, Saint-Petersburg, 91-425 Lodz, Poland Russia; 2Karolinska Institutet, Stockholm, Sweden Extra Structurally Abnormal Chromosomes (ESACs) without detectable alphoid DNA SAF-A/hnRNP U is an abundant nuclear pro- sequences represent a rare and interesting class of tein, which has been originally described as rearranged marker chromosomes. These ESACs nuclear matrix protein. SAF-A is known as pro- are predicted to have a neocentromere and have tein binding satellite DNA in centromeric (CEN) been referred to as Neocentric Marker Chromo- region, S/MARs DNA sequences and RNA, as somes (NMCs). We report molecular cytogenetic component of ribonuclear particles. Immuno- and clinical characterization of two new cases of fluorescence experiments revealed three types of NMCs (DAPI-, NOR-), one originated from the localization on methaphase plates according chromosome 1 and the other o¨ from chromo- to this activities: a) outside chromosomes in resi- some 15. Both cases were examined, using FISH dual nuclear matrix b) dots on chromosome technique with alpha-satellite DNA probes arms, c) main signal is in the CEN region. specific for chromosomes 1 and 15 and a pance- Immunoblot confirms the significant loss of SAF- tromeric probe; no alphoid DNA was detected. A in the isolated chromosomes contrary to Only the application of CGH and M-FISH CENP-B, another satellite DNA binding protein. 15th ICC: Abstracts 19

The computer analysis has been done in order to Arabidopsis plants simultaneously transformed understand how the observed complex pattern of with SKP1/CFP and H2B/YFP, showed YFP the SAF-A localization correlates with its proper- £uorescence only within the nuclei, and CFP £uor- ties and functional domains. In addition to the escence in nuclei, cell wall and cytoplasm. This known ones, a potential NTP-binding region and expression pattern might be explained by involve- coiled coil domain have been found. ment of SKP1, a part of the ubiquitin-ligase Three protein’s domains revealed by computer complex, in di¡erent cellular processes. analysis were selected for GFP-fusion proteins A number of T-DNA insertion mutants for construction and investigation of their behavior. Bub1, Bub3.1, Bub3.2 were selected from Arabi- This is: a) DNA-binding domain, 1-250 a/a; dopsis databases. The absence of the corresponding b) potential NTP-binding domain, 250-550 a/a; transcripts in homozygous lines was demon- c) RNA-binding domain, 550-823 a/a. Patterns of strated by RT-PCR. The mutants revealed no their localizations correspond to theoretically pre- clear phenotype. Bub3.1 and Bub3.2 mutants dicted. Constructs without DNA-binding domain were crossed to inactivate both isoforms. do not colocalized with heterochromatin de¢ned Several monoclonal antibodies (expressed in according to DAPI staining. C-terminal domain stabile hybridoma lines) that speci¢cally bind to with RNA-binding activity could be found in centromeric regions of isolated faba bean and bar- nucleolus. The functional role of the separate ley chromosomes have been generated. PAGE, SAF-A domains is discussed. Western blot analysis and mass spectroscopy are applied to identify the centromeric antigens recognized by these MAbs. PO:06:18 Cloning and functional PO:06:19 characterisation of plant kinetochore Conserved satellite DNA sequences in proteins (KP) the pericentromeric heterochromatin I. Lermontova1, S. Hudakova1, V. Schubert1, 1 1 1 of tenebrionid beetles (Coleoptera, R. Manteuffel , B. Schlesier , A. Tewes and Insecta) I. Schubert1 1 1 1 1 B. Mravinac , N. Mestrovic , Z. Pezer , Institute of Plant Genetics and Crop Plant 1 1 Research (IPK), D-06466 Gatersleben, Germany M. Plohl and D. Ugarkovic 1Ruder Boskovic Institute, Dept. Molecular We have cloned putative homologues of two Biology, Bijenicka 54, 10 000 Zagreb, Croatia constitutive (CBF5, one isoform of SKP1) and three transient KPs (Bub1 and two isoforms of Pericentromeric heterochromatin, located on all Bub3) of Arabidopsis thaliana. chromosomes, makes a substantial portion of up CBF5/YFP and CBF5/CFP constructs, tran- to 50% of the genome in many tenebrionid bee- siently expressed in Arabidopsis protoplasts, yiel- tles. Molecular and cytogenetical studies reveal ded £uorescent signals clustered within and tandemly repeated satellite DNAs as the major around the nucleolus. A. thaliana was trans- heterochromatic DNA components. Usually, a formed with a CBF5-RNAi construct to knock single highly abundant satellite DNA co-exists in out CBF5. To detect interacting proteins, a the heterochromatin with a number of other, low tobacco library was screened in the yeast two- copy number satellites. The same set of satellite hybrid system with CBF5 cloned into the bait vec- sequences is present in related species and the tor. For three candidate clones (presumably satellites are differentially amplified among the encoding CBF1, CENP-F and a kinesin-like pro- species forming species-specific profiles. In five tein) corresponding Arabidopsis cDNAs were species of tenebrionid genus Palorus six unrelated ampli¢ed to con¢rm interaction with CBF5 by satellite DNAs are found. Major, highly abun- yeast two-hybrid screening and in vivo by FRET. dant satellite is organized in the form of long 20 15th ICC: Abstracts arrays regularly interspersed with the short stret- mouse minor satellite (MiSat) are involved in ches of low copy number satellites. Such type of thread formation, former to the lesser extend, in organization encompasses the whole pericen- contrast to GC rich mouse satellites MS3 and tromeric, as well as centromeric regions. The MS4 (Kuznetsova et al., 2004), which were never peculiar characteristic of Palorus satellite DNAs found in interchromosome connection on con- is their extremely slow sequence evolution resulting ventional spreads, in chromosome aggregates in the absence of divergence and species from FASC or between meiotic prematurely con- diagnostic mutations. Complete sequence con- densed chromosomes; (2) telomeric sequence is servation is detected among the species within never member of the thread on the same pre- the genus as well beyond the level of genus, parations; (3) thread are synthesised in the very despite separation for 50-60 Myr. This unexpect- late G2 as shown with BrdU incorporation toge- edly high conservation might be induced by a ther with immunostaining; (4) main part of the bias in turnover mechanisms favouring the ances- connection went into midbody after mitosis; tral sequence in the process of molecular drive. (5) MiSat binding protein p68/helicase is one of Selective pressure on Palorus satellite DNA the main protein components of the interchromo- sequences can be also suggested. This could be some connection. related to their possible role in the formation of chromatin structure characteristic for pericen- PO:06:21 tromeric and centromeric regions. Histone H3 modi¢cations in the PO:06:20 centromeric regions of Arabidopsis thaliana Connective thread between 1 2 chromosomes F. Shibata and M. Murata 1 1 2 1CREST, Japan Science Technology Corporation, O. Podgornaya , I. Kuznetsova , A. Shatrova 2 2 Kawaguchi, Japan; Research Institute for and A. Dyban Bioresources, Okayama University, Kurashiki 1Institute of Cytology RAS, St. Petersburg, 710-0046, Japan Russia; 2Institute of Experimental Medicine RAMS, St. Petersburg, Russia The 180-bp family of tandem repetitive sequences is the major centromeric satellite in Arabidopsis Physical connections between the chromosomes thaliana. Our previous study showed that part of have been previously identified and shown to be the 180-bp repeats are able to assemble the cen- neither an artifact of colchicine nor of hypotonic tromere-specific proteins such as HTR12 (Arabi- treatment. It has been demonstrated that the dopsis centromeric histone H3 variant) and interchromosome connection in mammalian cells AtCENP-C (Arabidopsis CENP-C homolog). It can be completely destroyed with DNAse but was also suggested that ordinary histone H3 was when treated with RNAse or pepsin it becomes involved together with HTR12 at the centromeric more rigid. Sill interchromosome connection is regions. To investigate those histone H3 mod- not counted as well established structure. We ifications, we performed indirect fluorescence showed by Fluorescence-Activated Cell Sorting immunolabeling with antibodies against human (FASC) that (1) FASC spectrums are never free phosphorylated histone H3 at Ser10, at Ser28, of aggregates: if there is chromosomes – there is and dimethylated histone H3 at Lys9 (H3K9). aggregates; (2) the durability of connection is The H3K9 methylation was observed in nuclei comparable with durability of chromosome itself; and chromosomes all through the mitotic cycle, (3) the aggregates distribution is in agreement but limited to occur on the heterochromatic with the curve built on supposition that every regions. The immunosignals appeared on the cen- one of the chromosome is free to associate with tromeric heterochromatins were overlapped with each other. FISH and immunoFISH shows that those from HTR12 and AtCENP-C. The H3K9 (1) AT rich mouse major satellite (MaSat) and methylation signals on extend chromatin fibers 15th ICC: Abstracts 21 were linear and long stretched, whereas the site of etoposide-sensitive topo II cleavage. HTR12 and AtCENP-C signals appeared pre- Moreover, through systematic manipulation of ferentially on knobs of the 180-bp repeats. In this locus and the generation of a large series of mammals, phosphorylation of histone H3 at deletion derivatives we have shown that cen- Ser10 is known to occur all through at the tromere activity, the inner kinetochore proteins mitotic cell division, and estimated to promote CENP-C and -H and topo II cleavage co-localise chromosome condensation. In A. thaliana, how- to the same sub-domain of the DXZ1 array. ever, both phosphorylations at Ser10 and 28 Molecular characterisation of this deletion series were thought to occur for ordinary histone H3, also revealed that only part of the starting 2.1 and were observed preferentially at the cen- Mb DXZ1 array shared by all centric chromo- tromeric regions only during late prophase to some derivatives recovered is a region, of <50 metaphase. Compared with HTR12, phospho- kb, that encompasses the site of topo II cleavage histone H3 (Ser10) localized at the inner parts of in the starting array. The ability to map topo II the kinetochores, which suggests that ordinary activity at the molecular level presents exciting phospho-histone H3 has a role in chromatid opportunities for the dissection of this enzymeaˆs cohesion. centromere-specific role. Our progress in this will be presented.

PO:06:22 Genome Organisation Symposium Topoisomerase II and the vertebrate centromere L29 J. Spence1, L. Alonso-Gonzalez1, W. Mills1, A. Carpenter2, W. Earnshaw3, T. Fukagawa4, Biochemical dynamics in and around A. Porter2 and C. Farr1 the centromere 1 1 1 1Department of Genetics, University of K. Sullivan , K. Monier and A. Visser Cambridge, Downing St, Cambridge CB2 3EH 1 2 The Scripps Research Institute, La Jolla, UK; MRC Clinical Sciences Centre, CA, USA Hammersmith Campus, Du Cane Rd, London W12 ONN UK; 3Wellcome Trust Centre for Cell Heterochromatic regions of the chromosomes are Biology, Institute of Cell and molecular Biology, University of Edinburgh, King’s Buildings, distinctive sites thought to play roles in gene Mayfield Road, Edinburgh EH9 3LR UK; silencing, chromosome spatial organization and 4National Institute of Genetics and Graduate mitosis During G2 histone H3 becomes highly University for Advanced Studies, Mishima phosphorylated at serine 7. Phosphorylation initi- Shizuoka 4118540, Japan ates in pericentromeric heterochromatin. Initia- tion does not occur at random, but preferentially A number of studies have suggested that topo II, at specific chromosomes in a number of cell while being involved in a wide range of cellular types. Immunofluorescence demonstrates that functions, may also have a specific role at the this is mediated by preferential recruitment and centromere. Molecular support for this has come activation of Aurora B kinase into specific peri- from the characterisation of topo II cleavage centric heterochromatin domains exhibiting activity at the human Y and X centromeres using extensive methylation of DNA. Recruitment of etoposide, which acts by stabilising the covalent Aurora B into pericentric regions is abrogated enzyme DNA intermediate resulting in double- upon treatment with 5-azacytidine or antisense stranded DNA breaks. Where a centromere is depletion of DNMT1. We hypothesize that one molecularly well defined it is possible to use function of heterochromatin in the cell cycle is to pulsed-field gel electrophoresis to map the immo- serve as a compartment for the assembly of bilised DNA breaks. Within the human X active Aurora B kinase. A second G2 event at a-satellite array we have localised a single major centromeres is the initiation of assembly of the 22 15th ICC: Abstracts kinetochore. The centromere-specific histone H3 These nuclear envelope related disorders include homologue CENP-A is specifically expressed in Emery-Dreifuss muscular dystrophy (AD-, AR- G2. To investigate CENP-A assembly, CENP- and X-linked EDMD), Dunnigan type of partial A-GFP plasmid was microinjected into cells at familial lipodystrophy (FLPD), Charcot-Marie- different stages of the cell cycle. CENP-A was Tooth 2 (CMT2), Mandibulacral dysplasia (MAD) able to assemble promiscuously into chromatin and Hutchinson-Gilford Progeria Syndrome at any stage of the cell cycle, indicating that gen- (HGPS). We have found that genome organisa- eral chromatin assembly factors can recognize tion changes dramatically over time in culture in CENP-A. However, preferential assembly of these cells. We have a number of assays that mea- nucleosomal CENP-A at centromeres was also sure the degree of chromatin disruption in these observed throughout the cell cycle. This demon- cells. These include chromosome positioning by strates that there is a resident mechanism at cen- £uorescence in situ hybridisation, telomere bind- tromeres that can continuously exchange ing to the nuclear matrix, DNA halo prepara- available CENP-A into centromeres. Centrome- tions, replication studies and 3-dimensional res and pericentric heterochromatin are thus sites reconstruction, visualisation and analysis of chro- of regulated assembly of both resident and non- matin domains. resident macromolecular complexes during the We have also established a model system for cell cycle. di¡erentiation of porcine ex vivo stem cells to cell types a¡ected in laminopathies. Thus, we are able to determine aspects of genome organisation associated with di¡erentiation and study where L30 this process may go wrong in cells with disturbed The role of nuclear structure in nuclear structure. genomic health J. Bridger1, K. Meaburn1, H. Foster1, M. Figgitt1, G. Bonne2, N. Levy3, D. Griffin1 L31 1 and I. Kill The chromatin ¢bre architecture of 1Cell and Chromosome Biology Group, heterochromatin and euchromatin in Department of Biological Sciences, Brunel the human genome: relationship to University, Cleveland Road, Uxbridge, UB8 3PH, UK; 2INSERM UR 582, Institut de Myologie, genes and their expression Groupe Hospitalier Pitie´-Salpetriere, 47, W. Bickmore1, N. Gilbert1, S. Boyle1, boulevard de l’Hoˆpital, 75 651 Paris Cedex 13, 2 2 France; 3INSERM U491, Faculte de Medecine de H. Fiegler and N. Carter la Timone, 13385 Marseille, Cedex 05, France 1MRC Human Genetics Unit, Edinburgh, EH4 2XU, UK; 2The Wellcome Trust Sanger Institute, Chromosomes and specific types of chromatin Hinxton, Cambridge CB10 1SA, UK are positioned into specific areas in an interphase nucleus. This complex organisation is funda- Whilst much is now known about nucleosome mental for the correct functioning of the genome. structures, little is known about higher-order Chromatin positioning and reorganisation in chromatin structures. We present the first bio- particular stages of a cell’s lifespan depend physical analysis of chromatin fibre structure upon underlying structures in the nucleus that across the human genome. Compact and open influence the genome in a variety of ways, chromatin fibre structures were separated by structurally to controlling transcription. sucrose sedimentation and their genomic dis- We have investigated genome organisation in tributions analysed. We show that compact chro- a number of cell lines derived from patients matin fibres are enriched at some sites of carrying mutations in proteins associated with cytological heterochromatin (C-bands), and in nuclear structures, i.e. emerin or lamins A/C. G-bands (euchromatin). However. some regions 15th ICC: Abstracts 23 that are thought of as heterochromatic also con- an euchromatic to a heterochromatic nuclear tain open chromatin structure suggesting that zone (gene relocation hypothesis). heterochromatin is a heterogeneous structure. Bolzer, A., Kreth, G., Solovei, I., Saracoglu, K., Fauth, C., In contrast, the most open chromatin ¢bres are Mu¨ller, S., Eils, R., Cremer, C., Speicher, M. R., Cremer, T. enriched at the regions of highest density in the (2004) Complete 3D-maps of chromosome positions in human genome (T-bands), even when the genes in human male fibroblast nuclei and prometaphase rosettes these regions are not expressed. We show that demonstrate a chromosome size dependent, probabilistic many of these regions are also cytologically decon- arrangement (submitted). Cremer, M., Zinner, R., Stein, S., Albiez, H., Wagler, B., densed, An open chromatin ¢bre structure could Cremer, C., Cremer T. (2004) Three dimensional analysis of facilitate transcription and we suggest that this histone methylation patterns in normal and tumor cell may provide a evolutionary constraint to maintain nuclei. Eur. J. Histochem. 48: 15–28. clusters of genes together along chromosomes. L34 L33 Large scale chromatin structure and dynamics Chromosome territory arrangements 1 in the cell nucleus: non-random, A. Belmont probabilistic order and its implications 1University of Illinois for nuclear functions No abstract was submitted for this talk. T. Cremer1 1Department Biology II, Ludwig Maximilians L35 University, Munich, Richard-Wagner-Str. 10/I, The analysis of long-range compaction D-80333 Mu¨nchen, Germany and positioning of interphase Chromosome territory (CT) arrangements were chromatin in budding yeast by high analyzed in nuclei of postmitotic human diploid resolution live imaging fibroblasts (Bolzer et al., 2004). We demonstrate 1 1 2 size dependent, non-random distribution of S. Gasser , K. Bystricky , P. Heun , 3 1 small CTs towards the nuclear center and of J. Langowski and H. Schober large CTs towards the nuclear rim (radial CT 1University of Geneva, Frontiers in order), This pattern deviates strongly from the Genetics-NCCR, Geneva, Switzerland; gene density correlated, radial CT arrangements 2Lawrence Berkeley Laboratory, Berkeley, reported for lymphocyte nuclei and several CA USA; 3German Cancer Research Center, other cell types. Side-by-side arrangements Heidelberg, Germany (neighborhoods) of heterologous and homo- logous CTs were highly variable in all cell Using optimized in situ hypbridization and live types. We propose the following hypothesis: Cell imaging techniques we have determined point- type specific gene expression and silencing pat- to-point compaction ratios for interphase chro- terns depend on the position of transcribed matin in budding yeast. Quantitative two color genes in euchromatic nuclear zones, and of FISH data on sequences found at 14- to 100-kb silent genes in heterochromatic nuclear zones intervals along single chromatids are compared (Cremer et al., 2004). In case of a differentia- with 3D measurements of whole chromosome tion dependent switch of gene expression, e.g. arms ranging from 122 to 623 kb in length, mon- from an active to a permanently silent state, itored through the binding of GFP-repres- affected genes may either maintain their nuclear sor fusions to targeted arrays of repressor position, while the embedding chromatin sites. The resulting distance measurements are structure changes from an ‘open’ to a ‘closed’ introduced into flexible polymer modeling configuration, or affected genes may move from algorithms. The results argue that interphase 24 15th ICC: Abstracts chromatin exists in a higher order conformation that the same duplicon is present in 9p21.1, with a persistence length of 100-150 nm, or *15 9p11, 9q11, 9q21 and in 9q22.3. kb based on a mass density of 6-7 nucleosomes A growing body of evidence indicates that a per 11 nm. This degree of compaction, equiva- relationship can be established between con- lent to a folded 30 nm fiber, is remarkable stitutive rearrangements and acquired somatic given that all intervals monitored are open, aberrations. Particular rearrangement prone chro- transcriptionally competent chromatin. We pro- mosome regions show clustering of recurrent can- pose that this state unfolds transiently as trans- cer breakpoints. These data support the idea that cription occurs. duplicons play a role in driving not only meiotic, Using similar quantitative £uorescence analysis but also somatic exchanges. We found a cancer of of GFP-,YFP- and CFP-tagged chromosomal breakpoint hot spot in 9p21 and more than 100 loci we also show a spatially distinct tethering of inter-chromosomal tumor rearrangements between centromeres and telomeres in the yeast interphase 9p21 and 11q23. Sequence analysis demonstrated nucleus. By integrating unrelated bacterial opera- that band 11q23 contains a segment displaying tor arrays into unique chromosomal sites we mea- a high degree of homology to the duplicon that sure telomere-telomere and telomere-spindle pole we isolated from 9p21.1. body distances in living cells. We show that yeast In silico analysis demonstrated that segmental Chr 3, 5 and 6 loop back upon themselves. Unlike duplications showing more than 90% similarity to the ends of these chromosomes, those of Chr 14 chromosome 9 dupicons are present in 22 loci are not clustered together. Results from telomere distributed on chromosomes 1, 2, 3, 4, 5, 6, 11, swapping, analysis of subtelomeric sequences, 12, 13, 15, 16, 18, 21, 22, and Y. FISH locali- and telomere interaction in mutant strains will be zation con¢rmed the in silico data. presented. L37 L36 Large-scale chromatin organization Identi¢cation and chromosomal and gene expression distribution of a human chromosome P. Verschure1, J. Mateos-Langerak1, I. Van 9 duplicon mediating meiotic and der Kraan1, A. Belmont2 and R. Van Driel1 somatic rearrangements. 1SILS, Univ. of Amsterdam, Amsterdam, 2 1 1 1 The Netherlands; Dept. of Cell and Structural M. Paulis , D. Moralli , L. De Carli and Biology, Univ. of Illinois, Illinois, USA E. Raimondi1 1Dipartimento di Genetica e Microbiologia Research focuses on the functional organization ‘A. Buzzati Traverso’ Universita di Pavia. Via of chromosomes and chromatin in the interphase Ferrata, 1. 27100 Pavia, Italy cell nucleus. Actively transcribed loci and epigen- etically silenced genes locate at condensed chro- The origin of an accessory marker chromosome matin surfaces [1, 2, 3, 4]. Condensed chromatin derived from a chromosome 9 rearrangement has domains are accessible for large macromolecules, been elucidated. Isolation, sequencing and implicating that other mechanisms than inaccessi- sequence analysis of one of the breakpoints map- bility of heterochromatin regulate gene silencing ping in 9p21 have been performed. We found a within heterochromatin [5]. complex duplicon, containing the breakpoint, Recently we investigate the inter-relationship composed of sub-domains with a redundant between chromatin structure, gene expression and structure consisting of blocks of repetitive regulatory proteins involved (i.e. HP1) using two sequences belonging to the Alu and L1 families, strategies: (i) interference with the functionality DNA transposons and three full-length genes of HP1, expressing truncated HP1alpha or of unknown function. Database search for HP1beta lacking a functional chromodomain, paralogous segments on chromosome 9 revealed and (ii) in vivo targeting of HP1alpha or HP1beta 15th ICC: Abstracts 25 to an integrated ampli¢ed chromosome region, tissues. Consistent with the notion that spatial using a lac-operator/repressor based system. proximity contributes to translocation frequency, Our data indicate that HP1alpha and HP1beta we find a tissue-specific correlation between spa- are not the only proteins involved to maintain tial proximity and tissue-specific translocation and reassemble condensed heterochromatin prevalence. Our results demonstrate that the domains, whereas in vivo HP1 targeting causes spatial organization of genomes is tissue specific local chromatin condensation. We aim to unravel and point to a role of spatial genome organization the molecular pathways for functional organiza- in the prevalence of cancerous chromosome tion of transcriptionally active and epigenetically arrangements. silenced genes.

1. Verschure PJ, Van der Kraan I, Manders EMM, Van Driel R (1999) J. Cell Biol. 4: 13. L39 2. CMarko D, Verschure PJ, Martin TE, Dahmus ME, Krause S, Fu X-D, Van Driel R, Fakan (1999) Mol. Biol. Chromosome intermingling in the Cell. 10: 211. nucleus of human lymphocytes 3. Verschure PJ, Van der Kraan I, Enserink, JM, Mone MJ, 1 1 Manders EMM, Van Driel R (2002) J. Histochem. M. Branco and A. Pombo Cytochem. 50: 1303. 1 4. CMarko D, Verschure PJ, Otte AP, Van Driel R, Fakan F MRC Clinical Sciences Centre, UK (2003). J. Cell Science 116: 335. 5. Verschure PJ, Van der Kraan I, Manders EMM, Houts- The discreteness of chromosome territories has muller AB, Van Driel R (2003) EMBO Reports 4: 861–866. been studied by chromosome painting, known to miss repetitive sequences and less condensed L38 DNA, or through more indirect methods, like CldU/IdU labelling followed by chromosome Tissue-speci¢city of spatial genome segregation. We optimised the chromosome organization painting procedure in ultrathin cryosections * 1 2 1 ( 150 nm thick), and found that they allow the L. Parada , P. McQueen and T. Misteli use of harsher treatments while preserving the 1National Cancer Institute, NIH, Bethesda, organisation of chromatin. Painting efficiency MD 20892, USA; 2Mathematical and Statistical could therefore be improved and this led us to Laboratory, DCB, CIT, NIH, Bethesda, question whether DNA from different chromo- MD 20892, USA somes might intermingle. We co-hybridized 9 pairs of chromosomes in activated human Genomes are organized in vivo in the form of lymphocytes and found a measurable degree of chromosomes. The spatial positioning of chro- intermingling. Interestingly, the degree of inter- mosomes within the cell nucleus is non-random. mingling correlated with the frequency of Patterns of chromosome positioning have been gamma-ray-induced translocations in the same described in lymphocytes and fibroblasts. How- cell type (Cornforth et al., 2002, JCB, 159: 237). ever, it is unclear how conserved the spatial orga- Intermingling was also studied in resting and nization of genomes is amongst tissues. Using 2D alpha-amanitin-treated (activated) lymphocytes, and 3D fluorescence in situ hybridization we have aiming to assess a possible role for transcription. investigated whether spatial genome organization When compared with activated cells, resting cells is conserved amongst tissues. We show that gen- showed differences in the degree of intermingling omes are differentially organized in different tis- of different pairs of chromosomes, but there was sues. Chromosomes exhibit tissue-specific radial not a general tendency for increase or decrease. positioning with respect to the center of the Alpha-amanitin treated cells tended to have nucleus and relative to each other. Subsets of increased degrees of intermingling, which chromosomes form distinct types of spatial clus- suggests that transcriptional activity may ters in different tissues and the relative distance constrain chromosome territories. In activated between chromosome pairs varies amongst lymphocytes, intermingling is not correlated to 26 15th ICC: Abstracts chromosomal volume, probably due to differential nucleus periphery, accordingly, results in increase positioning of chromosomes within the nucleus. of chromosome spatial dissociation. At the same In contrast, intermingling in resting cells can be time, in all cases the nucleolus-forming chromo- justified by differences in chromosomal volume some 4 remained attached to the nucleus periphery. alone, suggesting that chromosome position is That is, such ratio can be speci¢c to separate random. Studies are currently being undertaken chromosome and can participate in maintenance to analyse chromosome intermingling at the elec- of chromosome order in interphase nuclei. tron microscope level and to assess preferential intermingling of specific subchromosomal regions. L41 L40 When is a chromosome is too large Chromosome subdividing to haploid to handle? sets in diploid metaphase plates of W. Rens1, P. O’Brien1, F. Yang1 and 1 some mammalian species M. Ferguson-Smith 1 T. Glazko1 Molecular Cytogenetics Laboratory, Centre for Veterinary Science, Department of Clinical 1Instituteof Agriecologyand Biotechnology, Ukraine Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, UK In investigation of metaphase plates of small- sized and large mammalian species (bone marrow The extremely long X-chromosome of the field cells of laboratory lines of mice, Microtus arvalis, vole, Microtus agrestis, appears to hamper Microtus oeconomus, Clethrionomys glareolus; proper nuclear divison. This observation was cells of peripheral blood of cattle) the space obtained using FISH with chromosome specific chromosome subdividing on two haploid sets was paints, and immuno-fluorescence with an anti- revealed. The comparative analysis of Robertso- body against the nuclear pores and an antibody nian translocations in bone marrow cells (in vivo) against INCENP (inner centromere protein). and in cell lines (in vitro) of laboratory lines of Cells, preserved in their 3D configuration, were mice had shown, that the transition to passing of investigated by Z-stack fluorescent microscopy at cells from in vivo to in vitro was accompanied by different stages of the cell cycle, from metaphase destruction of chromosome organization in hap- till furrow formation. loid sets. So, the Robertsonian translocations The DNA content of most diploid mammalian between homologous chromosomes were revealed cells is around 7 pg. However, the DNA is dis- only in cells in vitro in difference from in vivo tributed over a di¡erent number of chromosomes populations. It allows to suppose that chromo- in di¡erent species, and consequently the sizes of some haploid set is an autonomous element in these chromosomes vary considerably. The posi- organization of a genetic material, which destabi- tion of the centromere along the chromosome is lizing is connected with the appearance of some not ¢xed either, leading to chromosomes of varying cytogenetic anomaly. length. At late anaphase chromosomes are pulled The analysis of polytene chromosome positions apart by microtubules attached to kinetochores at in salivary gland cells of Chironomus thummi the centromeres, but the space in which the chro- demonstrated that the chromosome space order mosomes can move apart is limited. In plants, can be related with the dynamics of ratios arti¢cially elongated chromosomes result in between strength of their attachment to nucleus impairment of viability. We have already shown periphery and between chromosomes. The in the potoroo that long chromosomes a¡ect the decreasing of chromosome connections with a shape of the nucleus and their relative position in nucleus periphery is accompanied by the increase the nucleus (Chromosoma 2003; 112:66-76). The of interchromosome associations; the increase case of the ¢eld vole is more extreme. The long strength of the chromosome attachment to X-chromosome leads to DNA bridges between 15th ICC: Abstracts 27 nuclei, i.e. incomplete nuclei divison, and the tin essentially random or not? Does the amplifi- occurrence of micronuclei, i.e. loss of chromo- cation of heterochromatin and segmental some fragments. Apparently there is no mitotic duplications affect the position of neighbouring check- point for lagging chromosome arms. The chromatin segments? We expect these studies to signi¢cance of these ¢ndings on the natural his- contribute to the detailed analysis of preserva- tory of the ¢eld vole remains to be determined. tion/changes of higher order CT arrangements in various primate evolutionary lineages. On the other hand these experiments may help to clarify L42 whether non-random higher order chromatin arrangements affect the nature and direction of Effects of chromosome karyotype evolution. rearrangements during primate evolution on the higher order L43 chromatin architecture 1 1 1 Spatial position of satellite DNA in M. Neusser , V. Schwarz , A. Koch , respect to chromosome territories in T. Cremer1 and S. Mu¨ller1 interphase nuclei of human cells 1 Department Biology II, Human Genetics, 1 2 Ludwig-Maximilians University, Munich, N. Enukashvily , R. Donev and 1 Germany O. Podgornaya 1Cell Cultures Dept., Institute of Cytology, It is not known whether certain evolutionary Tikhoretsky, 4., St. Petersburg, 194064, Russia; chromosome rearrangements are favoured by dis- 2University of Wales College of Medicine, tinctly non-random positions of chromosome ter- Medical Biochemistry & Immunology Dept, ritories (CT). Depending on specific structural Biology of the Complement Group, Heath Park, properties of chromosome segments involved in Cardiff CF14 4XN, UK evolutionary rearrangements they may or may not lead to the dislocation of chromosomal mate- The existence of chromosome territories (CT) is a rial to different nuclear compartments with dif- well-established fact. The role of satellite DNA ferent functional environments. Considering of centromeric and pericentromeric regions in possible effects of higher order chromatin nuclear spatial organisation has been also pro- arrangements on gene activity, it is important to posed. The aim of our work was to investigate study whether different evolutionary rearrange- the spatial position of satellite DNA and some ments do affect the nuclear topology of the proteins bound to it in respect to CTs. In human respective chromosome segments. This is particu- lymphocytes, placenta parenchyma cells, fibro- larly interesting in cases, where translocations blasts, A431 and HeLa cells, centromeres brought together chromosome segments with revealed by FISH with labelled alphoid DNA high and low gene density. We performed 3D were distributed throughout the nuclear interior. FISH studies of interphase nuclei from human Centromeres of some chromosomes were posi- and a variety of other primate species in order to tioned on the surface of CTs as shown by FISH. trace the influence of chromosomal rearrange- The spatial proximity of some centromeres to ments that have occurred during evolution on interchromosomal SC35 domains was demon- nuclear organization. These studies address the strated by immunoFISH. Thus, some cen- following questions: Are gene density-correlated tromeres can be located at the CT periphery. radial arrangements of chromatin evolutionary Human pericentromeric DNA (human satellite 3, conserved in different species despite exten- HS3) of chromosome 1 but not 14 was always sive evolutionary chromosome reshuffling? Does adjacent to nuclear envelope in primary cultures. the CT size of evolutionary translocation pro- In HeLa cells we used, 3-4 signals corresponding ducts affect their nuclear localization? Is the to chromosome 1 HS3 were revealed by FISH. side-by-side arrangement of rearranged chroma- Only two of them were adjacent to nuclear envel- 28 15th ICC: Abstracts ope. The contacts between HS3 regions of chro- the 3-D localisation of centromeres, chromo- mosomes 1 and 14 were observed in all cell cul- centeres and nucleoli. In two-cell embryos, cen- tures analysed. HS3 regions of these tromeres and chromocenters cluster in half of chromosomes remained always compacted in nucleus and are localised at its periphery. An aver- these cells. In old but dividing MRC 5 fibroblasts age of seven nucleoli are dispersed within the culture (passage 30-36), HS3 was unfolded. It’s nucleus. At four-cell stage, most of the centromeres threads were invading neighbour CTs. AB and chromocenters are associated with the nucleoli against RNA helicase p68 decorated a network which average number is four. During next stages, framing CT. Thus CTs have well defined bor- nucleoli tend to cluster, as well as their associated ders, but some chromosomal regions can pene- centromeres and chromocenters, whereas the others trate into CT of neighbour chromosomes. are dispersed throughout the nuclear volume. Satellite DNA is involved in these processes. Our results are indicative of a dynamic 3-D spa- tial organisation of the genome and provide the L44 molecular morphology ground necessary for understanding the regulatory mechanisms that 3-D localisation of centromeres and control gene expression at di¡erent hierarchical chromocenters in preimplantation levels during preimplantation development. mouse embryos L45 1 1 2 V. Merico , M. Monti , M. Zuccotti , Organization and dynamics of plant C. Redi1 and S. Garagna1 chromosome territories (Arabidopsis) 1Dipartimento di Biologia Animale and Centro di A. Pecinka1, V. Schubert1, A. Meister1, Eccellenza in Biologia Applicata, University of 2 3 1 1 Pavia, Piazza Botta 10, 27100 Pavia, Italy; G. Kreth , N. Kato , M. Lysak , J. Fuchs , 2Dipartimento di Medicina Sperimentale, Sezione M. Klatte1, E. Lam3 and I. Schubert1 di Istologia ed Embriologia, Parma University, 1 Via Volturno 39, 43100 Parma, Italy Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany; 2Kirchhoff Institute Development requires a precise program of gene for Physics, University of Heidelberg, expression to be carried out. Epigenetic mechan- Im Neuenheimer Feld 227, D-69120 Heidelberg, isms such as DNA methylation, histone acetyla- Germany; 3Biotech Center, Cook College, tion, chromatin organisation and nuclear Rutgers & The State University of New Jersey, architecture are involved in modelling and 59 Dudley Rd., Foran Hall, New Brunswick, moulding genome functioning. Antral oocytes NJ 08901, USA isolated from the mouse ovary display meiotic and developmental competence which is corre- Differential painting allowed to study organiza- lated with their nuclear architecture. Oocytes tion and potential dynamics of chromosome showing a homogeneous diffused chromatin are territories (CTs) in A. thaliana nuclei. The side- incapable of developing beyond the two-cell stage by-side arrangement of heterologous chromo- whereas oocytes showing a rim of chromocenters somes and the homologous association of the and centromeres surrounding the nucleolus are symmetric chromosomes 1, 3 and 5 are in accor- capable of further development to the blastocyst dance with the random frequency predicted by stage after in vitro fertilisation. computer model simulations for differently In preimplantation embryos, the highly methy- shaped nuclei. Homologues are spatially asso- lated maternal and the paternal genome are topo- ciated on average in 35-50% of nuclei and in up logically separated up to the four-cell stage to 70% of spheric nuclei. Only the NOR-bearing (Mayer et al., Nature, 403:501, 2000). chromosome 2 and 4 homologues associate In order to follow the changes of the nuclear more frequently than at random, apparently due architecture in mouse embryos throughout to attachment of NORs to a single nucleolus. preimplantation development we have determined Punctual homologous pairing of *100 kb seg- 15th ICC: Abstracts 29 ments (in *5% of nuclei) occurs 7-10 times less of the rearrangements involved in the transloca- frequently than homologue association, not sig- tions should probably help us to better under- nificantly more often than expected at random stand the molecular events responsible for these and not simultaneously along the homologues. translocations. Here, we report the molecular Thus, CT arrangement in Arabidopsis differs study of a familial t(1;4)(q31.3;p15.32) transloca- from that in Drosophila (characterized by tion associated with primary microcephaly. We somatic pairing of homologues in 60-90% of showed that this translocation disrupts the nuclei) in spite of similar genome size, sequence ASPM gene in 1q31.3. Recently, mutations in the organization and chromosome number. Never- ASPM gene have been reported in some cases of theless, in 9.3-31.5% of investigated Arabidopsis familial microcephaly Bond et al. 2002. Nat. nuclei allelic sequences may share positions close Genet. 32:316-320). Although G-Banding karyo- enough for homologous recombination. The gramme and CGH did not reveal any imbalance average punctual pairing frequency is approxi- within the breakpoints, FISH and South- mately doubled at the integration loci in lines ernblotting have shown several rearrangements transgenic for the lacO/lacI-GFP chromatin tag- including duplication, deletion and insertion. We ging system and becomes further increased by are currently being delineate and sequence these expression of the GFP-LacI fusion protein. With rearrangements in order to propose mechanism increasing endopolyploidy level of nuclei, the for translocation. number of homologous CTs does not increase due to association of NORs and of homologous PO:06:23 pericentromeric chromocenters. However the frequency of sister chromatid cohesion becomes Characterization of two families of dramatically reduced, particularly at interstitial tandem repeated DNA sequences in chromosome arm positions. Potamogeton pectinatus L. 1 1 1 L46 M. Ceccarelli , S. Minelli , V. Sarri and M. Gelati2 Molecular characterisation of a t(1;4) 1Dipartimento di Biologia Cellulare e Molecolare translocation associated with primary della Universit, Sezione Citologia e Genetica, microcephaly Perugia, Italy; 2Dipartimento di Agrobiologia e 1 1 Agrochimica, Universit della Tuscia, Viterbo, B. Pichon , S. Vankerckhove , Italy G. Bourrouillou2, M. Abramowicz3 and 1 L. Duprez Two families of tandem repeated DNA sequen- 1Laboratoire de Cytoge´ne´tique, Hoˆpital Erasme, ces, PpeRsa1 and PpeRsa2, have been isolated Universite´ Libre de Bruxelles, Belgique; 2CHU from a partial genomic DNA library of the Purpan, Toulouse, France; 3Service de Ge´ne´tique submerged macrophyte Potamogeton pectinatus Me´dicale, Hoˆpital Erasme, Universite´ Libre de L. (fennel or sago pondweed). The consensus Bruxelles, Belgique sequences of PpeRsa1 and PpeRsa2 are 365 and 359 base pairs in length, respectively. Both are Reciprocal translocations associated with abnor- enriched in adenine þ timine (62,5% with mal phenotypes constitute powerful tools for the PpeRsa1 and 59,3% with PpeRsa2) and do not study of genotype-phenotype association and the contain internal repeats. Repeat structure within identification of gene function. The molecular the two sequence families is well conserved mechanisms responsible for these translocations because the nucleotide sequence similarity are poorly understood today. Detailed study of among the sequenced clones is 92-96% with these translocations at the molecular level PpeRsa1, and 86-99% with PpeRsa2. No obvious revealed that they are generally associated with nucleotide sequence similarity occurs between cryptic complex rearrangements in the vicinity of the two repeats, but certain sequence traits may the translocation breakpoints. The identification suggest their evolution from a common ancestor. 30 15th ICC: Abstracts

No significant nucleotide sequence identity was Analysis of satellite DNAs has demonstrated a found after comparing PpeRsa1 and PpeRsa2 fundamental similarity of the mechanisms of com- with DNA sequences in the EMBL-GenBank. As pactization of chromosomal DNA regions encod- shown by fluorescent in situ hybridization experi- ing and not encoding proteins. In the latter case, ments, the two sequence families are present in however, a more comlicated and more consider- 52 out of the 78 chromosomes of P. pectinatus. able compactization of tandem repeats of satellite PpeRsa1-related sequences have a marked pre- DNAs is observed. This is in agreement with cyto- ferential localization at the chromosome ends, logical data on a greater degree of compactization while PpeRsa2-related sequences occur also at of chromosomal parts which contain extended various intercalary chromosome regions. regions of satellite DNAs (constitutive hetero- The two sequence families proved to be useful chromatin) as compared to euchromatin. molecular markers to assess intraspeci¢c varia- tion of the genome size and organization in fennel The work was supported by an RFBR grant. pondweed. Indeed, their copy number and struc- ture vary signi¢cantly among ¢ve populations in Central Italy. PO:06:25 An Investigation on status of tylosis with oesophageal cancer (TOC) locus PO:06:24 in Iranian patients af£icted with DNA/DNA interactions may be oesophageal squamous cell carcinoma involved in chromosomal compaction M. Noori Daloii1, M. Shahabi2, E. Jahanzad3, M. Glazkov1 E. Khoshbin4, M. Taghikhani2, J. Field5, J. Langan5 and J. Risk5 1Vavilov Institute of General Genetics, Russian Academy of Sciences, Russia 1Department of medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, A possible mechanism of compactization of Tehran, Islamic Republic of Iran; 2Pasteur eukaryotic chromosomes with the involvement of Institute of Iran, Tehran, Islamic Republic of Iran; 3Cancer Institute, Imam Khomeini Hospital, triple-stranded DNAs was studied by using com- 4 puter modelling carried out for analysis of Tehran, Islamic Republic of Iran; Department of Mathematics, Faculty of Science, University of nucleotide sequences of 17 eukaryotic genes, Tehran, Tehran, Islamic Republic of Iran; satellite DNAs as well as LINEs and SINEs. 5Department of medical Genetics, Faculty of The obtained results indicate that chromoso- Medicine, Tehran University of Medical Sciences, mal regions containing genes as well as satellite Tehran, Islamic Republic of Iran; 2Molecular DNAs, LINEs and SINEs are capable of compac- Genetics and Oncology Group, Department of tization into small loops. Chromosomal domains Clinical Dental Sciences, University of Liverpool, of genes often form rosette-like structures. The Liverpool L69 3BX, UK fact that gene domains of animal and plant chro- mosomes are able to compact into ‘isolated’ series Tylosis (hereditary hyperkeratosis palmaris et of loops can serve as a structural basis of their plantaris) is an autosomal dominant disease independent expression. Rosette-like structures characterized by thickening of the skin that is have been previously found in somatic and meio- associated with a very high risk of esophageal tic chromosomes by cytological methods. It has squamous cell carcinoma (ESCC) in three pedi- also been established earlier that these structures grees investigated. Haplotype and linkage ana- are form of organization of transcription inactive lyses using microsatellite markers have previously genes. The results suggest that protein noncoding mapped the tylosis with oesophageal cancer regions of chromosomal domains of genes (£anks (TOC) locus to a 500 kb region on 17q25. LOH and introns of genes) carry information about the of markers within this region have been found in mechanisms of their compactization. sporadic cases of ESCC. Further fine mapping of 15th ICC: Abstracts 31 the TOC locus within tylotic families using addi- by homology for LMW glutenin sequences. 90 tional microsatellite and SNP markers reduced full-length LMW glutenin sequences were defined the TOC minimal region to 42.5 kb. In the pre- that clustered into 8 distinct groups representing sent study we investigated loss of heterozygosity at least 20 different LMW glutenin subunits. A (LOH) of markers spanning 17q25, three of LMW glutenin composite probe was used to which were within the TOC minimal region, in screen a hexaploid wheat BAC library by hybri- 60 Iranian ESCCs. LOH patterns ranged from dizing a set of 24 high-density filters. A subset of complete loss of 17q25 to small deletions within 536 BAC clones was selected and fingerprinted as the minimal region and to no deletion at all. a result of two rounds of hybridization. A set of Seventy three percent (44/60) of patients had 25 pairs of PCR primers was designed from full- LOH in at least one marker within the 17q25 length LMW glutenin sequences and used as mar- region and all markers tested had more than 50% kers on the BAC clones. The combined finger- LOH. One known and two putative genes lie printing and marker data was used to build the within the minimum deletion region, including physical maps using FPC. The total length of the the cytoglobin gene. We performed a mutation 90 contigs was 14.5 Mb; the average contig com- analysis on exons of the cytoglobin gene using prised three BAC clones and was 161 kb long. single strand conformational polymorphism The longest contigs comprised 14 and 11 clones (SSCP) and DNA sequencing. We found a few and spanned 403 Kb and 343 Kb respectively. mutations which suggests that this gene may be involved in the ESCC phenotype. PO:06:27 Characterization and chromosome PO:06:26 location of a repetitive DNA and a Physical mapping with mariner-like element in the ant high-throughput ¢ngerprinting for Tapinoma nigerrimum low-molecular-weight glutenin loci T. Palomeque1, J. Carrillo1, M. Mun˜oz1 (Glu-3) in hexaploid wheat (Triticum and P. Lorite1 aestivum) 1Departamento de Biologia Experimental, 1 2 N. Ozdemir and S. Cloutier Universidad de Jae´n, Spain 1Yildiz Technical University, Department of Biology, Molecular Biology, Davutpasa str. 127 Genomic DNA from T. nigerrimum digested with Esenler, Istanbul, Turkey 34210; 2Cereal Research HindIII originates a single 200-bp band (TANI), Center, Agriculture and Agri-Food Canada, which has been sequenced. Southern blotting 195 Dafoe Road, Winnipeg, Manitoba, Canada using TANI as a probe did not reveal the ladder R3T 2M9 pattern typical of tandemly repetitive DNA. Using the TANI sequence, we have designed the Glutenins are important storage proteins of primers and carried out PCR amplification. The wheat and help determine bread-making quality. PCR results show that it produced a single They contain different polypeptides, which are amplification band of 1kb (TANIPCR). Southern subdivided into high molecular weight (HMW) blotting using this sequence as a probe also pro- and low molecular weight (LMW) categories. duced the ladder pattern typical of tandemly Genes tightly linked to Glu-A3, Glu-B3, and Glu- repetitive DNA. These sequences did not show D3 on the short arms of chromosomes 1A, 1B significant similarity to sequences previously and 1D, respectively, control most of the LMW deposited in GenBank. The results of in situ glutenin subunits. Three developing seed cDNA hybridization techniques using TANIPCR libraries were constructed and a total of 5000- sequences as a probe showed pericentromeric 6000 ESTs were created from these libraries. hybridization signals in all chromosomes. Never- ESTs were assembled into contigs and searched theless, some signals in other chromosome 32 15th ICC: Abstracts regions were also observed. We also carried out a chromosomes 1, 3 and 5 is in accordance with partial genomic library using genomic DNA the model prediction for the random arrange- from T. nigerrimum. In all the clones in which we ment. In contrast, a higher than random fre- were able to amplify sequence TANIPCR, we quency of association was observed for the have also been able to amplify a mariner-like homologues of the NOR-bearing chromosomes 2 element. This fact could indicate a relationship and 4, apparently due to the frequent attachment between the repetitive DNA and these elements, of NORs to a single nucleolus in a way mediat- it has been as has been shown in other species of ing association of homologues. FISH with ants. The mariner elements cloned have open *100 kb segments from chromosomes 1, 3 and 4 ORFs that could encode a protein with 345 revealed homologous pairing as 7^10 times less amino acids. Genebank searches revealed that frequent than association of homologues, not these proteins shared closed protein-sequence more often that expected at random and not identity with the transposase of the Mos1 from simultaneous along the chromosomes. D. mauritanica, the first mariner element descri- Therefore, predominantly random arrangement bed. The results of in situ hybridization techni- of Arabidopsis CTs di¡ers from that found in Dro- ques revealed the existence of several elements of sophila (characterized by frequent somatic pairing this type in all the chromosomes. of homologues), in spite of their similar genome size and chromosome number. Nevertheless, in PO:06:28 up to 31.5% of Arabidopsis nuclei homologous sequences share positions which might be close Arrangement of chromosome enough for homologous recombination. territories and homologous pairing in somatic nuclei of Arabidopsis thaliana PO:06:29 A. Pecinka1, V. Schubert1, A. Meister1, Tandem repeats inserted into two G. Kreth2, M. Klatte1, M. Lysak1, J. Fuchs1 distinct loci are frequently associated and I. Schubert1 with each other and with 1Institute of Plant Genetics and Crop Plant heterochromatic chromocenters in Research (IPK), Corrensstrasse 3, Gatersleben, 2 Arabidopsis thaliana interphase nuclei D-06466, Germany; Kirchhoff Institute for 1 2 1 Physics, University of Heidelberg, Im A. Pecinka , N. Kato , A. Meister , Neuenheimer Feld 227, Heidelberg, D-69120, A. Probst3, I. Schubert1 and E. Lam2 Germany 1Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, Gatersleben, Chromosome painting (CP) by fluorescence in 2 D-06466, Germany; Biotechnology Center for situ hybridization (FISH) allows to study the Agriculture and Environment, Rutgers, The State arrangement of chromosomes in interphase University of New Jersey, 59 Dudley Rd., nuclei. Interphase chromosomes form spatially New Brunswick, NJ 08901, USA; 3Laboratory separated chromosome territories (CTs) which of Plant Genetics, University of Geneva, may be arranged in a specific manner. Geneva 4, CH-1211, Switzerland We have analysed the side-by-side arrange- ment of CTs and of homologous *100 kb chro- The green-fluorescent-protein (GFP)/lac-repressor mosome segments, which might be of special (LacI) þ lac-operator array (LacO) chromatin interest for DNA repair via homologous recombi- tagging system has been used in various organ- nation, in Arabidopsis thaliana root and isms to study the dynamics and organization of leaf nuclei of various ploidy levels. The observa- the chromatin. tions were compared to the computer model We have investigated the e¡ects of the GFP/ simulations for random CT arrangement. Side- LacI þ LacO system on the chromosome organi- by-side arrangement of all heterologous CT com- zation of transgenic Arabidopsis thaliana 2C binations and of homologous arm territories of nuclei, in which the 10 kb LacOs were inserted 15th ICC: Abstracts 33 into the top arm of chromosome 3, 4.2 Mb and microduplications, particularly if they apart. We analysed GFP spots and FISH signals involve single clones, can be very challenging. for LacO and transgene-£anking sequences. All To achieve reliable array analysis in any popula- GFP signals colocalized with the FISH signals, tion of patients a catalogue of clones showing but on *17% of the tagged loci no GFP spots recurrent gain or loss (polymorphic clones) is were detectable. The FISH analysis further required. In the past year we have been screening revealed that *35% of the allelic tagged loci the genome of patients with mental retardation were paired in homozygous nuclei without, and for sub-microscopic chromosomal gains and los- *45% of loci in nuclei with GFP/LacI expres- ses using the commercial 1Mb array (Spectral sion. The £anking regions paired in *5% of Genomics). In this process we have detected wild-type nuclei. About 40% of the tagged loci clones that show recurrent change. Although were found associated with heterochromatin. some clones showed close proximity to the cen- The regions adjacent to the transgene were 4 tromere and terminal ends of the chromosome, times less frequently associated with heterochro- which are known to be rich in repeat sequences, matic chromocenters, similar as in wild type the majority were found along the chromosomal nuclei. The results demonstrate that transgenic arms. The chromosome distribution and genetic tandem repeats may preferentially associate with content of these clones will be discussed. Ideally, each other and with heterochromatin in Arabi- the creation of a database containing all the dopsis interphase nuclei. Expression of GFP/ clones with recurrent gains and losses will facil- LacI increases the LacO association, probably itate the interpretation of single clone losses and due to increased protein association. gains and enhance application of array analysis in clinical diagnostic laboratories.

PO:06:30 PO:06:31 Genomic variability detected using the Chromosome arrangement in 1 MB BAC array interphase nuclei can be caused by the E. Rajcan-Separovic1, C. Tyson2 and differences in scaffold-associated C. Harvard1 region (SAR) composition 1 2 2 1Children’s and Women’s Health Centre of BC A. Rodionov , S. Galkina and N. Lukina and University of British Columbia, Department 1 Botanical Institute of Russian Academy of of Pathology, Cytogenetics, 4480 Oak Street, 2 Sciences; Biological Institute of St. Petersburg Vancouver, V6H 3V4, Canada; 2Cytogenetics, State University, Russia Southmead Hospital, Bristol, BS10 5NB, UK Habermann and coauthors (2001) have shown The development of 1Mb whole genome BAC that territories of chicken microchromosomes arrays offers an opportunity to screen the entire (MI) in cell nuclei were clustered within the genome for subtle chromosomal changes at a center of the nucleus, while territories of macro- resolution 10–20 higher than conventional cyto- chromosomes (MA) were located toward the genetic analysis. The number of reports describ- nuclear periphery. In humans, small chromo- ing patients with cryptic chromosomal somes ## 17, 19, 20 (but not # 18 and Y) are rearrangements detected using the array but mis- located in the nuclear center, whereas the chro- sed by routine karyotyping is increasing and mosomes # 1–5 lie near nuclear envelope clearly shows the tremendous advantage of this (Cremer et al., 2001). method in comparison to conventional cytoge- Earlier, we have shown that: netics. However, due to the currently still limited knowledge of genetic variability at the level of a 1. Chicken MIs are similar to T-bands of human single DNA clone, usually 200 kb in size, the chromosomes, enriched in short GC-rich sequen- interpretation of array detected microdeletions ces and ‘heavy’ isochores (Andreozzi et al., 2002); 34 15th ICC: Abstracts

2. The chicken MA gene orthologs are located in required to analyse 3D digital images and to G-bands and also in T-, chromomycin-, H3-, determine appropriate parameters to quantify the and Alu-negative R-bands of human chromo- architecture and organisation. A software pack- somes, i.e., MA-gene orthologs lie on chromo- age was developed specifically for FISH labelled somes of nuclear periphery. In contrast, the cell nuclei and 3D-fluorescence microscopy. For chicken MI gene orthologs are in GC-rich processing a huge package of 3D-image data an Giemsa-light, T-, chromomycin-, H3-, Alu- user-friendly shell script has been developed, positive regions of human chromosomes which makes it possible to process a lot of 3D- (Rodionov et al., 2001); images in a very short time. To ensure that mea- 3. H3-positive R-regions of chicken MAs and MIs surements of the parameters are threshold inde- correspond to that of lampbrushes consisting of pendent, the whole intensity range is analysed. ‘AT/GC-neutral’ chromomeres. Moreover The segmentation of the FISH signals is based on the chicken lampbrush MIs do not contain a local 26 relationship. The criterion for a voxel DAPI-positive chromomeres, which are the to be inside a local 26 neighbourhood of another characteristic feature of MAs and correspond voxel is, that the voxels must be connected at to theirs G-bands (Galkina et al., 2001). their sides, edges or corners. Because of the inten- sity dependent parameters it is feasible to get As the bright chromomere £uorescence after information about the topology and the interior DAPI/AMD-staining is mainly due to presence organisation of chromatin, e.g. the radial position of AT-rich sequences in SARs, we suppose that 1) in the cell nucleus, relative distances to other there are di¡erent types of SARs in T- and G- marker, or the shape of the labelled site. The soft- bands; 2) di¡erentiation in SARs composition of ware was successful tested and applied on terri- avian MA/MI and in correspondent targets of tories of chromosome #18, #19 and #X in nuclear matrix can determine the chromosome ter- human lymphocytes and fibroblasts, on territories ritory position in interphase nuclei. of chromosome #8 in nuclei of pancreatic tumour cells in formalin-fixed, paraffin-embedded tissue PO:06:32 sections, and on H3K9 marked heterochromatin. The results indicate the possibility to characterise 3D-image analysis of the topological differences in the architecture and organisation of organisation of chromatin in human the FISH labelled objects in order to distinguish lymphocytes, ¢broblasts and in cancer normal and aberrant cell nuclei. cells of formalin-¢xed, paraf¢n-embedded tissue sections Reproduction Symposium S. Stein1, I. Solovei2, M. Cremer2, R. Zinner2, S. Timme3, A. Walch3, M. Werner3, M. Hausmann3 and C. Cremer1 L47 1Kirchhoff-Institute of Physics, University of Investigation of aneuploidy Heidelberg, Im Neuenheimer Feld 227, D-69120 mechanisms by FISH and CGH Heidelberg, Germany; 2Institute of Antropology and Human Genetics, University of Munich, analysis of oocytes and polar bodies Richard-Wagner-Str. 10, D-80333 Munich, J. Delhanty1, E. Fragouli1, D. Wells3, Germany; 3Institute of Pathology, University C. Conn1, K. Whalley2, J. Mills2 and Hospital, Albertstr. 19, D-79104 Freiburg, 2 Germany M. Faed 1Department of Obstetrics & Gynaecology, UCL, The higher order chromatin architecture is not 86–96 Chenies Mews, London, WC1E 6HX, UK; completely understood. FISH techniques and 2Ninewells Hospital Dundee, Dundee, DD1 9SY, high resolution microscopy contribute to its Scotland, UK; 3St Barnabas Medical Center, further elucidation. Therefore, novel tools are West Orange, NJ 07052, USA 15th ICC: Abstracts 35

Molecular cytogenetic investigations of cleavage deletions) diagnosed by standard karyotyping stage embryos have revealed anomalies affecting all and molecular investigations using somatic tis- sizes of chromosomes. The aim was to investigate the sue samples (usually blood samples). By this variety of anomalies arising during maternal meio- approach, however, the underlying cause sis I by analysis of unfertilised oocytes and polar remains unknown in an outstanding majority of bodies to gain insight into aneuploidy mechanisms. infertile men with impaired spermatogenesis. On Sequential FISH analysis was carried out with the other hand, direct meiotic investigations speci¢c probes derived from eight chromosomes, using testicular biopsy samples, revealed that representing all sizes. Only imbalance due to a gain around 1 in 5 such men with severe oligo- or of a whole chromosome or chromatid, represented azoospermia show a delay in meiosis1 matura- by extra signals, was counted, to avoid artefact. In a tion during the prophase stages leptotene and separate approach, DNA was extracted from zygotene and a partial arrest at the subsequent oocytes and polar bodies for use in CGH analysis. pachytene stage in addition drastic reduction in Data were obtained from FISH analysis on 236 chisma frequency at metaphase1 and increased eggs from 124 patients of average age 32.5 years aneuploidy at metaphase2. There are also indi- (range 22 to 44). Ten patients (average 32.6 years) cations that milder variants may affect men had abnormal eggs. The abnormality rate for with less severe disturbance of spermatogenesis. oocytes and for polar bodies was close to 4% for This condition, which we have termed OLIGO, each. Fourteen hyperploidies were found, seven is characterised by increased parental con- involving additional single chromatids. The abnor- sanguinity and familiar occurrence of reduced malities a¡ected chromosomes 13,16,18, 21 and X fertility, and is suggested to represent the first butnotchromosomes1,9,or12.CGHresultsare recognised (recessive) meiotic mutation in available for a small number of samples to date. humans. It seems likely OLIGO contributes sub- Anomalies have been found for a wider range of stantially to the increased rate of aneuploid off- chromosomes but patient speci¢c factors are spring by ICSI; hopefully recent scientific involved. advances (including identification of recombina- The data provide evidence for several aneu- tion foci by MLH1 immunofluorescence) and ploidy mechanisms including classical non- increased knowledge on meiotic mutants in mice disjunction of whole univalents; pre-division of will soon allow recognition of the genetic muta- chromatids prior to anaphase I, leading to imbal- tion per se, facilitating adequate diagnosis and ance detected at metaphase II; germinal mosai- genetic counselling in the first instance in male cism for a trisomic cell line and preferential factor infertility. involvement of the smaller chromosomes. L49 L48 Segregation study of Robertsonian and OLIGO meiotic maturation arrest and reciprocal translocations in sperm and recombination de¢ciency causative of pre-implantation embryos male factor infertility E. Van assche1, G. Ogur1, C. Staessen2, 1 M. Hulte´n M. Bonduelle1, A. Michiels1, G. Verheyen2, 1 2 1Department of Biological Sciences, University of K. Keymolen , P. Devroey , Warwick, Coventry CV47AL, UK A. Van Steirteghem2 and I. Liebaers1 1Center for Medical Genetics, University Infertility affects around 1 in 6 couples with Hospital, Dutch-speaking Free University of more than 50% attributed to factors leading to Brussels, Laarbeeklaan 101, 1090 Brussels, disturbance of spermatogenesis. Attention has Belgium; 2Center for Reproductive Medicine, focussed on chromosome abnormalities (such as University Hospital, Dutch-speaking Free XXY, XYY, translocations, inversions and University of Brussels, Laarbeeklaan 101, 1090 insertions) and DNA mutations (such as Yq Brussels, Belgium. 36 15th ICC: Abstracts

In the present study the meiotic segregation beha- The incidence of aneuploidy in sperm of men viour in spermatozoa, from 14 Robertsonian with male factor infertility is one of the most (Rob) and 20 reciprocal (Rcp) translocation car- contentious issues in reproductive medicine. In riers was determined by two- or three- colour particular, the risk of ICSI treatment of these FISH, with selected centromeric and/or region- men unwittingly giving rise to aneuploid off- specific and/or sub-telomeric probes according to spring has raised concerns in the scientific and the translocation type. Pre-implantation genetic popular press. To the best of our knowledge diagnosis (PGD) was performed in 6/14 Rob car- however there are no reports of treatment riers (14 cycles) and in 7/20 Rcp carriers (16 regimes purporting to successfully reduce the cycles). The different types of meiotic products incidence of sperm aneuploidy. Through the use (alternate, adjacent 1 and 2 and 3:1 segregation) of three colour FISH, we have evidence of a sig- were identified and compared in sperm and pre- nificant reduction in sperm aneuploidy fre- implantation embryos. In the different Rob car- quencies for chromosomes 21, X and Y in a riers, a high rate of balanced spermatozoa (range: cohort of six patients undergoing TCM treat- 76% –89.4%) and consequently a relatively low ment. In each patient initial aneuploidy fre- rate of unbalanced spermatozoa (range: 10.6% – quencies were significantly higher than controls 24%), was observed. In contrast, the incidence of for at least one of the investigated chromosomes, unbalanced sperm in the different Rcp carriers however coincident with TCM treatment, aneu- was highly variable from one translocation to ploidy levels fell significantly. In addition, experi- another (range: 10.4% –79.6%), but for 19/20 car- ments were undertaken to determine whether riers the rate was often higher than 50%. During individual herbs and/or the combinations used PGD, 53.4% of the analysed embryos seemed to for treatment exhibited biological activity. All be normal in the Rob carriers and only 21.5% in herbs were tested for anti-oxidant activity and the Rcp carriers. Considering the meiotic beha- endocrine properties (including oestrogenic, anti- viour of the different translocations, comparable oestrogenic, androgenic and anti-androgenic segregation patterns were observed in sperm and activity). Analysis of the biological activity of the embryos. In all cases, the frequency of imbalances herbs revealed that the majority of the individual in sperm and embryos was above the average rate herbs and all of herb prescriptions tested pro- of imbalance observed at term or in prenatal duced both anti-oxidant activity and anti- diagnosis demonstrating the presence of an effi- oestrogenic activity. These results provide cient selection against unbalanced embryos/foe- evidence of a reduction in aneuploidy frequencies tuses further in pregnancy. coincident with TCM treatment and suggestions of possible biological pathways through which L50 they may be acting. We propose that placebo controlled clinical trials should be considered Evidence for a signi¢cant reduction in using this treatment with a view to ultimately sperm aneuploidy levels coincident with providing an alternative or an adjunct to ICSI. treatment by traditional Chinese herbal medicine and for endocrine and anti-oxidant activity of the therapeutic L51 herbs Cytogenetic analysis of human H. Tempest1, D. Christopikou1, blastocysts M. Dalakiouridou1, X. Zhai2, S. Homa2 H. Clouston2, A. Murdoch3 and and D. Griffin1 J. Wolstenholme1 1Cell and Chromosome Biology Group, 1Department of Cytogenetics, Institute of Human Department of Biological Sciences, Brunel Genetics, International Centre for Life, Newcastle University, Uxbridge, UB8 3PH. UK; upon Tyne, UK; 2(current address of HJC) North 2112 Harley Street, London, UK Trent Cytogenetics Service, Sheffield Childrenaˆs 15th ICC: Abstracts 37

Hospital, Sheffield, UK; 3Newcastle Fertility 1Genetic and Molecular Research Laboratory, Centre, International Centre for Life, Newcastle Melbourne IVF, Australia upon Tyne, UK FISH studies have demonstrated a high fre- The blastocyst is a relatively understudied stage quency of aneuploidy in early embryos from in of human development, but one that appears key vitro fertilisation (IVF) patients. Most of these in the aetiology of many abnormal karyotypes. aneuploidies are lethal and would prevent the In the largest study so far, 671 spare IVF/ICSI embryos from implanting and/or developing to blastocysts, were subjected to thymidine-based term. IVF clinics perform preimplantation synchronisation of cell division and acetic acid genetic diagnosis for aneuploidy screening (PGD- disaggregation[1,2]. Metaphases were obtained AS) using FISH for chromosomes X, Y, 13, 16, from 477 blastocysts. Of these, 248 blastocysts 18, 21 and 22 to test single cells biopsied from were successfully karyotyped, and partial analysis embryos. This allows selection of embryos most of a further 199 blastocysts generated ploidy likely to be viable for transfer to the patient. An information. Almost 30% of blastocysts demon- obvious limitation is that FISH is restricted to strated diploid:tetraploid mosaicism. identification of a few chromosomes and the full Among fully karyotyped blastocysts, two extent of aneuploidy in early embryos remains (0.8%) were triploid and one (0.4%) was fully tet- largely unknown. We have developed single cell raploid. Nine blastocysts (3.6%) had aneuploidies CGH and successfully used it to fully karyotype con¢rmed in multiple cells, and four (1.6%) were cells from embryos for PGD-AS (Wilton et al., classi¢ed as trisomic based on a single cell analy- Fertil Steril 2003 80:860–8). Consequently, we sis. Two blastocysts (0.8%) were con¢rmed have data describing chromosome abnormalities mosaic trisomies and a further twelve (4.8%) were in more that 300 aneuploid cells from approxi- possible mosaic trisomies (a single trisomic cell mately 120 3-day old (*8-cell) human embryos. was found alongside normal cells). Sequential This includes multiple cells from more than 60 FISH techniques enabled further characterisation embryos. of abnormal ¢ndings, in particular the clari¢ca- Every chromosome was involved in aneuploidy. tion of ambiguous results and the investigation of Approximately 40% of cells had a single aneu- mosaicism in interphase nuclei. ploidy, 10% had double aneuploidy and about Comparison with abnormality rates at earlier 50% showed extensive aneuploidy of >3 chromo- and later stages of gestation allows two main con- somes. Analysis of multiple cells from the same clusions to be drawn. Firstly, it appears that sig- embryo showed that approximately 25% of ni¢cant selection against abnormal embryos embryos had consistent aneuploidy in each cell, occurs prior to blastocyst development. Secondly, which was presumed to be meiotic in origin. the general range and incidence of most main About 75% of embryos had di¡erent aneuploidies groups of chromosome abnormality observed in in di¡erent cells, which probably occurred post- ¢rst trimester pregnancies appear to be in place zygotically. Many embryos had aneuploidies that by the blastocyst stage. would not have been identi¢ed using FISH highlighting the bene¢t of full karyotyping for 1. Clouston et al, (1997) Hum Genet 101: 30–36 PGD-AS. 2. Clouston et al, (2002) Prenat Diagn 22: 1143–52

L52 Full karyotyping of human L53 preimplantation embryos using Deletions and duplications between comparative genomic hybridisation palindromes P5 and P1 in subfertile (CGH) men L. Wilton1 K. Writzl1, B. Zorn2 and B. Peterlin1 38 15th ICC: Abstracts

1Division of Medical Genetics, Department of The increase in aneuploidy with maternal age Obstetrics and Gynecology, UMC Ljubljana; leads to an increased risk of producing aneu- Ljubljana, Slovenia; 2Andrology Centre, ploid offspring and spontaneous abortions as Department of Obstetrics and Gynecology, UMC well as a decrease in implantation rates. To Ljubljana; Ljubljana, Slovenia determine the mechanisms that produce aneu- ploidy in oocytes and establish which chromo- Microdeletions of the long arm of human Y chro- somes are more prone to aneuploidy, 33 mosome represent an important cause of male unfertilized oocytes from women 35 years old infertility. Analysis of large deletions of the Y (mean, 38.2) were analysed. The whole chromo- chromosome [including azoospermia factor b some complement of the first polar body (1PB) (AZFb) and AZFc loci] has shown that large was analyzed by comparative genomic hybridisa- palindromes on Yq (named P1–P5) serve as sub- tion (CGH), while the corresponding MII oocyte strates for non-allelic homologous recombination. was analyzed by fluorescent in situ hybridisation This form of male infertility can thus be classified (FISH) to confirm the results. Matched CGH- as a genomic disorder. The reciprocal products of FISH results were obtained in twenty-nine doub- these deletion events, duplications, which are lets 1PB-MII, twenty-five (86.2%) showing reci- expected to occur at the same frequency as dele- procal results. The aneuploidy rate was 60.6%. tions, have not been reported to date. FISH analysis revealed that as much as two Therefore, our objective in this study was to deter- thirds of the aneuploid events were chromatid mine whether the reciprocal duplications between abnormalities, while only one third were due to palindromes P5 and P1 occur in subfertile men. non-disjunction of bivalents. Interestingly, the Ninety patients with male idiopathic sub- chromosomes more frequently involved in aneu- fertility (44 azoospermics and 46 oligozoos- ploidy were, in order, chromosomes 16, 1, 4 and permics) were analysed for deletion/duplication 15. Classifying the chromosomes as large (chro- of the sY125 locus using real time PCR. Two mosomes 1–12 and X) or small (chromosomes azoospermic men had deletion of the sY125 locus 13–22), small chromosomes were more prone to (also detected by PCR screening of microdele- aneuploidy (Chi2 test, P < 0.025). Four out of tions of the Y chromosome). No duplication of twenty aneuploid doublets (20%) would have the sY125 locus was detected. been missdiagnosed as normal using FISH with Duplications of the region between palin- 9 chromosomes probes (1, 13, 15, 16, 17, 18, 21, dromes P5 and P1 don’t seem to be a signi¢cant 22 and X). In this study, the combination of two risk factor for spermatogenic failure. different techniques, CGH and FISH, for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome L54 abnormality, as well as helping to determine the Age-related aneuploidy in unfertilised mechanisms involved in the genesis of maternal oocytes: Comparison of CGH and aneuploidy. FISH analysis C. Gutie´rrez-Mateo1, D. Wells2, J. Benet1, PO:06:33 P. Colls3,J.Sa´nchez-Garcia1, M. Bermu´dez2, Chromosomal anomalies in referred S. Munne´2 and J. Navarro1 patients with male infertility 1Departament de Biologia Cellular, Fisiologia i S. Bheemineni1, J. Solanki2, R. Uppala1 Immunologia, Unitat de Medicina, Universitat and R. Uppala1 Autonoma de Barcelona, E-08193 Bellaterra, Spain; 2The Institute for Reproductive Medicine 1Green Cross blood bank & Genetic Research and Science, St. Barnabas Medical Center, 101 Centre, Anil Kunj, Paldi, Ahmedabad-380014, Old Short Hills Road, Suite 501, West Orange, NJ India; 2Department of Animal Genetics and 07052, USA; 3Reprogenetics, Hoboken, New Breeding, Veterinary College, Gujarat Agriculture Jersey, USA University, Anand-388001, India 15th ICC: Abstracts 39

Infertility is a well-known clinical manifestation Orange, NJ 07052, USA; 3Servei de Medicina de of various ethical factors and is defined as an la Reproduccion, Institut Universitari Dexeus, inability to conceive after 12 months of unpro- Barcelona, Spain tected intercourse. The prevalence of infertility couples ranges between 2 and 16%, with 50% of Preimplantation Genetic Diagnosis (PGD) of cases due to male infertility. Many cases of male aneuploidies has achieved a reduction of sponta- infertility are due to identifiable severe defects in neous abortions and increased implantation and sperm production, which are classified as azoos- pregnancy rates in certain groups of patients. permia, oligospermia, abnormal motility or mor- The technique most commonly used has been phology and some are idiopathic. Several factors fluorescent in situ hybridisation (FISH), but the are associated with infertility few of them inclu- number of chromosomes that can be analysed is ded infection, immunological factors, anatomic a clear limitation of this method. We evaluated abnormalities, environmental etiologies, endocri- the limitations and reliability of using compara- nologic dysfunctions and some are influenced by tive genomic hybridisation (CGH) to detect the genetic factors. We have analyzed 80 male infer- whole set of chromosomes, as an alternative to tility patients for possible chromosomal anoma- PGD using FISH. Fifty single cells were ana- lies. After the detailed family history and lysed, corresponding to 25 metaphase II oocytes pedigree analysis, routine PHA stimulated per- (MII) and their complementary first polar bodies ipheral blood cultures were set up and karyotyp- (1PB). The aneuploidy rate found was 48%. ing was done after GTG banding and other Dividing the data into two maternal age groups banding techniques were performed as required. (<37 and 37 years old), more chromosome A high incidence of 20% (n ¼ 16) of chromoso- abnormalities were found in the older group mal abnormalities was observed among 80 infer- (23% and 75%, respectively, P < 0.02). Six out of tility patients referred (i.e. both structural and twenty-five doublets 1PB-MII (24%) did not dis- numerical). Those included 5% with balanced play reciprocal results, as one cell showed one or reciprocal translocations (N ¼ 4), 3.75% with sex two missing chromosomes while their sibling cell reversals (n ¼ 3) and 12.5% with gonosomal showed a normal profile for those chromosomes. aneuploidies/mosaicisms (n ¼ 9). These data sug- About 33% of the doublets 1PB-MII diagnosed gests that patients with history of infertility as aneuploid by CGH would have been incor- should be investigated cytogenetically. DNA rectly diagnosed as normal if FISH with 9 chro- microsatellite and FISH analysis are in progress mosome probes had been used. In this study we in few patients found with rare chromosomal demonstrate the reliability of 1PB analysis by translocations and also Y chromosome micro- CGH to detect almost any chromosome abnorm- deletions analysis. ality in oocytes in a time frame compatible with regular in vitro fertilization. The application of CGH in PGD of aneuploidy to select chromoso- PO:06:34 mally normal oocytes could help to increase implantation and pregnancy rates of patients Comparative genomic hybridisation undergoing in vitro fertilization treatment. analysis of ¢rst polar bodies and metaphase II oocytes PO:06:35 C. Gutie´rrez-Mateo1, D. Wells2, J. Benet1, The role of chromosomal aberrations J. Sa´nchez-Garcia1, M. Bermu´dez2, I. Belil3, to the no-fertilization in oocytes after J. Egozcue1, S. Munne´2 and J. Navarro1 FIVET or ICSI techniques 1Departament de Biologia Cellular, Fisiologia i G. Nannetti1, S. Bonifacio1, E. Lisi1, Immunologia, Unitat de Medicina, Universitat S. Carniani1, N. Nadalin1 and F. Torricelli1 Autonoma de Barcelona, E-08193 Bellaterra, Spain; 2The Institute for Reproductive Medicine 1UO Citogenetica e Genetica, AO Careggi, and Science, St. Barnabas Medical Center, West Firenze Italy 40 15th ICC: Abstracts

Techniques such as FIVET or ICSI have allowed two cytogenetic rearrangements which occurred a practical treatment of infertility. The purpose simultaneously: a reciprocal translocation of our study was to detect numerical chromoso- between the chromosomes 12 and 13 and an mal aberrations in oocytes subjected to ICSI or insertion of a part of the derivative translocation FIVET but not outcomed in embryos. chromosome 13 into chromosome 7. The com- The cytogenetic results obtained by FISH plexity of this ‘three-way’-rearrangement gives a allowed us to evaluate chromosomal anomalies high risk for unbalanced karyotypes in offspring that could explain the failure of fertilization. (ranging from spontaneous abortions to unba- The study has been structured on the study of lanced/affected life-born) and explains the 54 oocytes: 39 of them had not presented fertiliza- experienced repetitive abortions in this 36 year- tion after FIVET and 15 after ICSI. old woman. Oocytes were hybridized with locus speci¢c Here, we present the results of the (molecular) probe for chromosome 21 and alpha satellite cytogenetic analyses, propose a mechanism of probes for chromosomes X and Y after a pretreat- occurrence of the rearrangements and discuss the ment with HCl and Tween 20 solution. Y chromo- clinical consequences of the various unbalanced some probe was used to detect sperm DNA. karyotypes. About 95% of the analyzed oocytes exhibited discernible FISH signal. For metaphase II ICSI oocytes, with or without Comparative Genomics / Chromosome pronuclear formation, and for the FIVET Evolution Symposium oocytes, our objective was to determine their chro- mosomal status. Our results con¢rms the utility of FISH techni- L55 que for determining the relatively low fertilization rates. Contribution of cross-species chromosome painting to comparative genomics PO:06:36 M. Ferguson-Smith1 Complex balanced chromosomal 1Cambridge University Centre for Veterinary rearrangement explains reproductive Science, UK dif¢culties (recurrent abortions) in a healthy female: a case report Comparison between human and representative species of all extant mammalian orders using A. Simons1, H. Mieloo1, F. Van Erp1 and 1 chromosome painting reveals the remarkable D. Smeets conservation of genomes throughout evolution. 1Dept. of Human Genetics, Division of Large regions of chromosome, including whole Cytogenetics, UMCN St. Radboud Nijmegen, PO chromosomes and chromosome arms, are often box 9101, 6500 HB Nijmegen, The Netherlands shared between species. Differences in chromo- some number and structure result from rare Routine cytogenetic analysis was performed on events of illegitimate meiotic recombination lead- cultured peripheral lymphocytes of a woman and ing to chromosome fusion, fission and other her partner, because of the occurrence of two inter- and intra-chromosomal rearrangements. subsequent spontaneous abortions. While her Characteristically, these events occur at sites of partner showed a normal male (46,XY) karyo- DNA duplication. They have been used in con- type, a complex balanced chromosomal rear- structing phylogenetic trees and in the prediction rangement involving chromosomes 7 (q31.2), 12 of the likely ancestral mammalian karyotype. (q22) and 13 (q21.2 and q22.3) was found to be Recent studies have used chromosomal painting present in the healthy woman. Regular GTG- to help explain the occasional fertility of mules, banding and additional FISH analyses showed to interpret the complex meiotic multivalent 15th ICC: Abstracts 41 produced by the ten unpaired chromosomes of support of the Botechnology and Biological Sciences Research the male platypus and to identify the homo- Council (BBSRC) morphic sex chromosomes of the crocodile that is homologous to the avian Z chromosome. Sim- ple comparative maps have been made between L57 mapped and unmapped species, and DNA micro- The moving landscape of comparative array painting is being used to characterise evo- genomics in mammals lutionary breakpoints. The current challenge is to 1 overcome the limitations of chromosome paint- S. O’Brien ing between mammalian species and those of 1NCI-Frederick, MD, USA birds, reptiles, marsupials and monotremes. The full genome sequence of human, mouse, dog and rat genomes combined with dense physical L56 genetic maps have been complemented by precise gene homologue alignment of dense physical The chicken genome: A resource for genetic maps of livestock, companion animals chicken and evolutionary biology and additional mammal species. Comparative 1 genetic assessment expands the utility of these D. Burt maps in gene discovery, in functional genomics, 1Roslin Institute, Edinburgh, Scotland, UK and in tracking the evolutionary forces that sculptured the genome organization of modern In this paper we review progress in chicken geno- mammalian species. mics and introduce ChickNET: an international organisation devoted to the analysis and applica- tion of genomics to avian biology. The chicken L58 genome project has its roots in a decade of map Primate chromosomes: phylogenetic building by genetic and physical mapping meth- ods. Chicken genetic markers for map building landmarks, evolutionary breakpoints have generally depended on labour intensive and comparative nuclear architecture screening procedures. In recent years this has all S. Mu¨ller1 changed with the availability of *500K EST sequences, a draft sequence of the entire chicken 1Department Biology II, Human Genetics, genome and a map of over 1 million SNPs. Ludwig-Maximilians University, Munich, Clearly, the future for the chicken genome and Germany avian research is an exciting one. Through the integration of these resources, it will be possible To date, over 80 mammalian species were investi- to solve challenging scientific questions exploiting gated by multi-directional chromosome painting, the power of a chicken model the aim of among them approximately 60 primates from ChickNET is to drive this vision. In this talk I every major phylogenetic lineage. A more detailed wil review progress and some of the conclusions comparative analysis employing chromosome bar from the analysis of this genome sequence in codes allowed the assignment of subregional terms of its evolution and comparison with other homologies between various higher primate spe- genomes. cies. BAC and YAC contigs have been used for the establishment of high-resolution comparative maps and a detailed analysis of evolutionary breakpoints in several human homologous chro- Acknowledgements mosomes. The resulting chromosomal homology maps between numerous primate species allowed I am grateful to my colleagues and many collaborators who continue to contribute to the development of chicken genome the detailed reconstruction of evolutionary chro- (see ChickNET www site) and acknowledge the financial mosome rearrangements. It has also become 42 15th ICC: Abstracts possible to draw increasingly accurate conclusions within the South American groups is proposed, about the direction of these changes and to establish based primarily on number and position of 18S- chromosomal phylogenies. Moreover, this knowl- 25S rDNA translocations. Cytogenetic analysis of edge has been prerequisite to address open ques- the North African species, Hypochaeris angustifo- tions concerning evolutionary conserved motives of lia, reveals the same basic chromosome number as higher order chromatin arrangements and their pos- South American taxa (n ¼ 4), and thereby serves sible consequences on the nature and direction of to connect Old World (e.g., H. maculata) and chromosome evolution. New World taxa. Polyploids (all tetraploids) are The presentation provides an overview of tech- more common among South American Hypo- nological developments in primate comparative chaeris than previously reported and are often cytogenetics and gives a reconstruction of the present within diploid populations (cytotype mix- major landmarks in primate karyotype evolution. ture), suggesting autopolyploid origins. Tetra- Further, recent results obtained from the analysis ploids often differ karyotypically from their of evolutionary chromosomal breakpoints and diploid relatives, some representing unique kar- from comparative studies of primate nuclear yotypes, and appear to undergo more genome architecture are summarized rearrangements than diploids, as judged by rDNA sequence localization. Elimination and L59 translocation of 18S-25S-rDNA loci are pronounced trends. Pathways of rapid karyotype evolution in Hypochaeris (Asteraceae), with L60 special emphasis on polyploids 1 1 Precise characterization of pericentric H. Schneeweiss , T. Stuessy ,K. inversions that distinguish human and Tremetsberger1, S. Siljak-Yakovlev2 and J. Parker3 chimpanzee genomes J. Szamalek1, V. Goidts1, C. Sandig1, 1Department of Higher Plant Systematics and N. Chuzhanova2,S.Ta¨nzer3,S.Mu¨ller4, Evolution, Institute of Botany, University of 3 5 1 Vienna, Rennweg 14, A-1030 Vienna, Austria; M. Platzer , D. Cooper , H. Hameister 2De´partement Evolution & Syste´matique, and H. Kehrer-Sawatzki1 UPRESA CNRS 8079, Universite´ de Paris-Sud, 1 3 Department of Human Genetics, University of Orsay Cedex, France; Botanic Garden, 2 Cambridge University, Cambridge CB2 IJF, UK Ulm, Ulm, Germany; Department of Computer Science, Cardiff University, Cardiff, UK; 3Department of Genome Analysis, Institute of The genus Hypochaeris (Compositae) has a dis- Molecular Biotechnology, Jena, Germany; junct distribution with approximately 10 species 4Institute of Anthropology and Human Genetics, in Eurasia and NW Africa and 50 species in University of Munich South America. The genus arrived in South (Ludwig-Maximillians-University), Munich; America from the Old World within the past 3–5 5Institute of Medical Genetics, University my, and evolved there rapidly within 1–2 my. of Wales College of Medicine, Cardiff, UK The European species possess symmetrical kar- yotypes with x ¼ 3, 4, 5, and 6, whereas the Although human and chimpanzee karyotypes are South American species have asymmetric kar- similar, some important differences are apparent. yotypes with x ¼ 4. Chromosome number and These are the fusion that gave rise to human morphology are similar in all South American chromosome 2, variations in the amount of species, but molecular cytogenetic analyses of constitutive heterochromatin and nine pericentric more than 20 species reveal differentiation of inversions. These inversions have attracted karyotypes based on 18S-25S and 5S rDNA loca- considerable interest, since they may have influ- lization and allow discrimination of five kar- enced the parapatric speciation of early hominids yotypic groups. A model of karyotype evolution and chimpanzees. Breakpoint analysis of the 15th ICC: Abstracts 43 pericentric inversions is a prerequisite to deter- the short-beaked echidna by chromosome painting mine their biological consequences and to explore of mitotic cells. The use of this technique for iden- the mechanisms underlying primate chromosome tification was necessary as many of the monotreme evolution. We isolated breakpoint-spanning chromosomes are small metacentrics and the order BACs from the human and the chimpanzee gen- and relationship of the unpaired chromosomes omes and identified the breakpoints. Comparative require the identification of small homologous pair- sequence analysis revealed that the inversion ing regions. The existence of a large set of unpaired breakpoints of chromosomes homologous to chromosomes, which have to segregate in meiosis, human chromosomes 4, 5, 9, 16, and 17 occurred raises questions about the evolution of the sex- in regions rich in high-copy repeats. The human- chromosome system in monotremes. specific inversion of chromosome 18 appears to The platypus and echidna are the only extant have been mediated by segmental duplications of monotreme species. The Australian platypus 19-kb. By contrast, the inversion of the chimpan- (Ornithorhynchus anatinus) belongs to the Orni- zee chromosome homologous to HSA12 is asso- thorhynchidae family, whilst the short-beaked ciated with two duplications of 86-kb and 23-kb echidna (Tachyglossus aculeatus) and the long both of which occurred in the chimpanzee line- beaked echidna (Zaglossus bruijnii) belongs to the age. None of the pericentric inversions disrupts Tachyglossidae family. the protein-coding region of a gene. In the platypus unpaired chromosomes are pre- Interestingly, seven of the nine pericentric inver- sent in the male only (data on female echidna is as sions are chimpanzee-speci¢c, whereas humans yet not available). The females have two copies of have retained the ancestral, unrearranged state of the odd-numbers in the male multivalent chain, the respective chromosomes. Thus, the human i.e. element numbers 1,3,5,7, and 9; the even num- karyotype is closer to an ancestral primate kar- bers are absent. This requires an alternating pat- yotype than that of the chimpanzee. FISH with tern of chromosome segregation in male meiosis. breakpoint-spanning BACs indicated that Large sections of the odd numbered chromo- bonobo (Pan paniscus)andPan troglodytes share somes do not have homologous regions with their the same inversions. Therefore, their ¢xation pre- even numbered neighbours. Thus these regions dated the separation of these two lineages and are present as one copy in male and two copies in would have occurred in the time interval from female and behave like ‘X-chromosome regions’. 5^6 to 1.8-Mya. We have used cross species painting to show the degree of chromosome conservation between L61 these two extant monotreme species. Unusual features of the platypus and L62 echidna karyotypes revealed by Evolution of genes located on the cross-species chromosome painting 1 2 1 Y-chromosome in the cat family W. Rens , F. Grutzner , P. O’Brien , Felidae H. Fairclough1, J. Graves2 and 1 2 1 M. Ferguson-Smith1 J. Pecon-Slattery , V. King , W. Murphy , A. Pearks Wilkerson1 and S. O’Brien1 1Molecular Cytogenetics Laboratory, Centre for Veterinary Science, Department of Clinical 1Laboratory of Genomic Diversity, National Veterinary Medicine, University of Cambridge, Cancer Institute-Frederick, Frederick MD 21702, Madingley Road, Cambridge, UK; 2Research USA; 2The Center for the Advancement of School of Biological Sciences, Australian National Genomics, Rockville MD, 20850 USA University, Canberra, Australia The recent radiation of the cat family Felidae We have determined the identity of the unpaired offers a unique opportunity to examine patterns of chromosomes that form a multivalent chain in evolution within Y-chromosome genes. Genomic male meiotic cells of the duck-billed platypus and segments from single copy X-Y homologues 44 15th ICC: Abstracts

SMCY, UBE1Y and ZFY (3604 bp) were ampli- spread synaptonemal complexes (SC) and stan- fied in 36 species of cat. These genes are located dard cytogenetics, in seven males. Synapsis with within the X-degenerate region of the NRY and regular bivalent formation and no evidence of are thought to be ‘fossils’ that ceased conventional multivalents, was found. Nevertheless, gross recombination with the X-chromosome early inequalities in length of the lateral elements of within the placental mammal evolution 65–104 SCs were observed, producing ca. 19–22 hetero- MYA. The pattern and tempo of evolution at these morphic bivalents, also present at diakinesis. In three genes is significant in light of the recent, G-banded mitotic chromosomes, the number of rapid evolution of the family over approximately heteromorphic pairs was consistent with the 12 MY and provides exceptional support for each meiotic results. Regular male meiotic behaviour of the eight recognized felid lineages as well as (bivalent formation) of T. barrerae does not sup- clear diagnostic substitutions identifying nearly all port a polyploid status of the species at the chro- species. Bootstrap support and Bayesian posterior mosomal level, although the high degree of probabilities are uniformly high for each lineage. A chromosomal heteromorphism is unusual for similar analysis of the male-determining gene SRY mammals and suggestive of hybridsation (and and its adjacent genomic flanks (2957 bp) suggests perhaps, allopolyploidisation). Indeed, allote- this reproductively important gene to be tightly traploids may undergo evolutionary diploidisa- linked with speciation. Adaptive evolution of SRY tion but, how is the high heteromorphic status is assessed by codon analyses. No specific sites are mantained in the case of T. barrerae? One under selection, but an abundance of w > 1 answer is that the observed chromosomal poly- (w ¼ Dn:Ds) among all branches within the SRY morphisms are not different from those observed tree suggests most speciation events are marked by in many animal species, and are neutral or man- nonsynonymous changes. The precise phylogenetic tained by balancing selection. The other, pro- results obtained using the well-characterized Feli- poses T. barrerae as a polyploid of hybrid origin: dae as a species tree suggest genes within the NRY heteromorphic autosomal pairs would be home- are powerful markers of evolution within species. ologous chromosomes from the parent species. But, how is this multiple heteromorphism man- tained through meiotic segregation? A further L63 hypothesis predicts multiple reproductive interac- Multiple chromosomal tions of the putative parent species, along evolu- tionary time. Polyplody notwithstanding, heteromorphisms in Tympanoctomys T. barrerae presents several challenges to animal barrerae,2n¼ 102 (Caviomorpha, evolutionary cytogenetics. Octodontidae): polyploidy or polymorphism? L64 C. Bidau1, M. Gallardo2, C. Lanzone3, Reconstruction of karyotype evolution R. Stanyon4 and J. Santos5 in Arabidopsis thaliana and its close 1Universidade do Estado do Rio de Janeiro, relatives by multicolour chromosome Brazil; 2Universidad Austral de Chile, Chile; painting 3 Universidad Nacional de Misiones, Argentina; 1 2 1 4 5 M. Lysak , R. Schmidt , A. Pecinka , National Cancer Institute, USA; Universidad 1 1 Complutense de Madrid, Espana J. Fuchs and I. Schubert 1Institute of Plant Genetics and Crop Plant Tympanoctomys barrerae, is a desert specialist Research (IPK), Gatersleben, Germany; 2Max restricted to salt flats of west central Argentina. Planck Institute of Molecular Plant Physiology, Its 102 chromosome karyotype, DNA content Golm, Germany and exceedingly large sperms have suggested a tetraploid origin. Male meiosis of T. barrerae The model plant Arabidopsis thaliana is one of was studied by electron-microscopy of surface- the few species of the Brassicaceae family with 15th ICC: Abstracts 45 chromosome number 2n ¼ 10. Little is known The karyotypes of birds, turtles and snakes are about evolutionary history of the Arabidopsis characterized by two distinct chromosomal com- karyotype and processes that contributed to the ponents, macrochromosomes and microchromo- variation of basic chromosome numbers (n ¼ 5–8) somes. This close karyological relationship in the genus Arabidopsis and related taxa. The between birds and reptiles has long been the topic unique organization of the A. thaliana genome and of speculation among cytogenetists and evolu- the availability of chromosome-specific BAC librar- tionary biologists, however there is no evidence ies allowed multicolour painting of all Arabidopsis for orthology at the molecular level. To compare chromosomes and comparative painting studies in chromosome organization between birds and rep- related species with a similar genome structure. tiles, we constructed comparative cytogenetic For the ¢rst time, comparative chromosome maps of the Chinese soft-shelled turtle (Pelodiscus painting was successfully used to identify all chro- sinensis) and the Japanese four-striped rat snake mosomes of an entire complement in genomes of (Elaphe quadrivirgata) with EST clones, which related plant taxa. Multicolour FISH using Arabi- were isolated from cDNA libraries of the two dopsis BAC contigs arranged according to the species. Chromosome homology between the tur- comparative genetic map between A. thaliana and tle and chicken chromosomes was highly con- Capsella rubella (n ¼ 8) revealed homeologous served, the six largest chromosomes being almost chromosomes/chromosome regions in pachytene equivalent each other. In contrast, the conserved complements of ¢ve cruciferous species with 6 to 8 linkage homology to chicken chromosomes was chromosome pairs (Hornungia alpina n ¼ 6, lower in the snake than that in the turtle. The Turritis glabra n ¼ 6, Neslia paniculata n ¼ 7, Pseu- turtle chromosome 6 and snake chromosome 2 doarabidopsis toxophylla n ¼ 7andA. lyrata revealed the linkage homology with the chicken n ¼ 8). The chromosome homeology pattern sex Z chromosome. These results suggest that the found for both, C. rubella and A. lyrata,wasiden- avian and turtle genomes have been well con- ti¢ed as the ancestral karyotype which became served for over 250 million years in the phylogeny rearranged in other taxa in connection with of Arcosauria, and that the avian and snake sex chromosome number reduction from n ¼ 8to5. Z chromosomes were derived from the different Recurrent chromosome number reduction has chromosomes of the common ancestor. The pre- been accompanied with gross genome reshu¥ing sent approach for comparative mapping using an andgenomesizedecrease.Wehavereconstructed extensive number of ESTs is a new strategy to diverse evolutionary scenarios including chromo- investigate chromosome homologies between rep- some translocations, fusions and inversions that tiles and birds. The disposition of conserved chro- led to diversi¢cation of chromosome numbers mosome segments among reptiles, birds and from n ¼ 8ton¼ 7, 6, and n ¼ 5. mammals provides a clue for clarifying the phylo- genetic hierarchy of vertebrate genome evolution. L65 Karyological relationships between PO:06:37 birds and reptiles inferred from On the distribution of the interstitial comparative gene mapping telomeric sites and chromosome Y. Matsuda1, C. Nishida-Umehara1, evolution in birds H. Tarui2, A. Kuroiwa1, K. Yamada1, S. Derjusheva1, A. Kurganova1, T. Isobe1, J. Ando1, T. Saito3 and F. Habermann2 and E. Gaginskaya1 2 K. Agata 1Biological Institute of St. Petersburg University, 2 1Center for Advanced Science and Technology, St. Petersburg, Russia; Technical University of Hokkaido University, Sapporo, Japan; 2RIKEN Munich, Germany Center for Developmental Biology, Kobe, Japan; 3National Institute of Radiological Sciences, Recently, chicken macrochromosome painting Chiba, Japan probes were successfully applied to chromosomes 46 15th ICC: Abstracts of sixteen avian species. The species belonged The genus Cypripedium (Cypripedioideae; Orchi- mainly to the order Galliformes, a few other daceae) consists of approximately 45 species, and avian orders were represented by a single species we have carried out phylogenetic analyses using per order. In our study, we used chicken macro- matK and the trnL-F region of plastid DNA and and microchromosomal paints to analyze homo- the ITS region of nuclear ribosomal DNA. Most logies between chromosomes of the chicken (Gal- species of Cypripedium (including the native lus gallus domesticus: Galliformes), pigeon lady’s slipper orchid, C. calceolus) have large (Columba livia: Columbiformes), chaffinch (Frin- genomes (in C. calceolus,1C¼ 32.4 pg). As a gilla coelebs: Passeriformes) and redwing (Turdus result of these large genomes, AFLP fingerprints iliacus: Passeriformes). It was found that in are suboptimal, and are characterised by a few investigated birds, individual chromosomes, with strongly amplifying bands and many unscorable, a few exceptions, remain chromosomal integrity weakly amplifying bands. The strong bands are of ancestral synteny. We revealed that chicken present in all individuals within species, and in chromosome 4 paint hybridized with chromo- some cases are shared between species. We have some 4 and one pair of microchromosomes in investigated the use of cloned copies of these pigeon, redwing and chaffinch. These data pre- bands as a means of identifying high-copy segments sent an evidence of assumption that chicken 4q of DNA, some of which have now been shown to represents an ancestral type of avian chromo- be fragments of retrotransposons. Combining these some 4. We showed also that fission of chicken data with the phylogenetic hypotheses from chromosome 1 seems to be typical of passerine sequence data and FISH, we will be able to gain a karyotypes. Using FISH-mapping, we analyzed greater understanding of genome size evolution in the distribution of TTAGGG-repeat in pigeon, Cypripedium. chaffinch and redwing karyotypes. Interstitial telomeric sites (ITSs) were found to localize in PO:06:39 centromeric and/or interstitial CMA-positive bands on certain chromosomes of all investigated Origin of B and sex chromosomes in species. We did not find any correlation between plants the distribution of ITSs and chromosome rear- 1 rangements revealed by comparative chromo- H. Goswami some painting. In chaffinch, ITSs co-localize with 1Department of Genetics, Barkatullah University, a tandem repeat GS (AF427996, AF427997), Bhopal, India monomers of which contain internal TTAGGG motif. Our data clearly demonstrate that, at least Despite discovery of a sex chromosome in a in the chaffinch karyotype, interstitial telomeric plant in 1917 by Allen (Sphaerocarpus donelli) sequences are generated by the expansion of not many plants have been known to possess sex TTAGGG-microsatellite into monomers of satel- chromosomes. Among genera, sex chromosomes lite DNA. This work was supported by RFBR are known in Fucus ; Sphaerocarpus and March- (project 02-04-49980). antia ; Isoetes pantii; Cycas and Ginkgo and sev- eral genera among Cucurbits and Melandrium. This paper presents a ‘modus operandi’ for the PO:06:38 origin of a sex chromosome in natural popula- Towards an understanding of genome tions of aquatic pteridophytic weed Isoetes pantii Goswami (1975) suggesting molecular mechan- size evolution in Cypripedium isms operative within the genome wherein a ‘sex- (Orchidaceae) chromosome has evolved denovo’. Following events appear to be directly correlated with the M. Fay1, P. Cook1, J. Greensmith1, 1 1 1 ‘appearance’ of sex chromosome(s) recurrent het- R. Cowan , L. Hanson and I. Leitch erochromatinisation, chromosome breaks, trans- 1Jodrell Laboratory, Royal Botanic Gardens, locations and chromosome polymorphisms. Sex Kew, Richmond, Surrey, TW9 3AB, UK chromosomes are very rare but B chromosomes 15th ICC: Abstracts 47 are common in almost all groups of plants (Jones chromosomal syntenies. In order to obtain a & Rees, 1982). Two primary source for B- deeper insight into the evolution of the chromosomes have been considered. (A) Either karyotypes of neotropical members of the family an intragenomic fragement acquires the char- Muridae we investigated Akodon cursor,2n¼ 14, acteristics of a B or (B) Interspecific hybridiza- 15 and 16 and A. montensis,2n¼ 24 and 25 tion provides foreign DNA that evolves into a B (genus Akodon) by cross-species chromosome chromosome. Isoetes pantii (natural hybrid) painting employing mouse and Chinese hamster infact supports combination of both, hybridiza- chromosome-specific painting probes. tion theory as well as acquisition of B- chromsomes as foreign DNA. As per this hypothesis the B-chromosomes from both the PO:06:41 parents viz: I. coromandelina and I. sampathku- marini have contributed to the genomic reshuffle Human chromosomal thereby triggering appearance (origin) of X chro- Q-heterochromatin regions in mosome(s). Comparative studies on DNA methy- individuals of various age lation also indicate inherent role of genomic 1 imprinting. A. Ibraimov 1National Center of Cardiology and Internal Goswami H.K. 1975. Cytologia 40: 543–552. Medicine, Togolok Moldo str., 3, Bishkek, Goswami H.K. 2001 Annals de Genetique 44: 392 Jones R.N. & Rees H.C. 1982 B-chromosomes Acad Press, 720040, Kyrgyzstan, C.I.S. London. The quantitative content of chromosomal Q-heterochromatin regions (Q-HRs) was studied in individuals of different age groups taking into PO:06:40 account their racial and ethnic affiliation. It was Reconstruction of karyotype changes shown that chromosomal Q-HRs are most during the evolution of neotropical numerous in the genome of neonates, while they are the least numerous in the genome of elderly rodents (Akodontini, Muridae, subjects (aged 60 years and older) regardless of Rodentia) by cross-species the ethnic features of the individuals studied. Our chromosome painting data indicate that 1) in a population there is a 1 2 1 clear-cut tendency towards a decrease in the I. Hass , I. Sbalquiero and S. Mu¨ller number of chromosomal Q-HRs with age, regard- 1Department Biology II, Human Genetics, less of racial and ethnic features on the indivi- Ludwig-Maximilians University, Munich, duals; 2) of all the age groups the genome of Germany; 2Departamento de Gene´tica, neonates contains the greatest number of Q-HRs; Universidade Federal do Parana´, Curitiba, Brasil 3) decreases in the number of Q-HRs with age are not due to the ‘loss’ of Q-heterochromatin on Rodent genome and chromosome organization is individual loci or chromosomes, but occur simul- a major focus of basic and applied research since taneously in all the seven Q-polymorphic auto- certain members of this mammalian order are somes, in keeping with all our previous among the most important biomedical model observations. It should be noted that decreases in species. Despite this fact, very little is known the number of chromosomal Q-HRs with age do about the evolution of rodent chromosomes. not occur smoothly apparently. Thus, according Evidence from comparative sequence analysis, to our data, in the Kyrghyz and Russian popula- however, revealed that mouse and rat have tions 39.6% and 47.6% of chromosomal Q-HRs acquired numerous chromosome rearrangements ‘disappeared’ by the age of 60 years, respectively. compared to other mammalian orders, whereas Of them the portion of new-born Kyrghyz and Zoo-FISH experiments indicated that the squirrel Russian infants amounted to 70.6% and 58.4%, conserved most of the ancestral mammalian respectively. It is supposed that the lesser amount 48 15th ICC: Abstracts of Q-HRs in the genome of elderly subjects is PO:06:43 due to the selective advantage in their survival to old age. The possible selective value of chromo- Condensed chromatin and cell somal Q-HRs is discussed. thermoregulation A. Ibraimov1 PO:06:42 1Laboratory of Human Genetics, National Center Chromosomal Q-heterochromatin of Cardiology and Internal Medicine, Togolok Moldo str., 3, Bishkek, 720040, Kyrgyzstan, variability in neonates deceased C.I.S. during ¢rst year of age A. Ibraimov1 Based on own original investigations of chromo- somal Q-heterochromatin regions variability in 1National Center of Cardiology and Internal human populations, as well as on the analy- Medicine, Togolok Moldo str., 3, Bishkek, sis of existing literary data on the condensed 720040, Kyrgyzstan, C.I.S. chromatin (CC), structure of interphase nucleus and redundant DNA in the genome of higher There is analyzed the amount of chromosomal eukaryotes, an attempt is made to justify a Q-heterochromatin regions (Q-HRs) in a gen- view about possible participation of CC in cell ome of neonates deceased during first years of thermoregulation. CC (tissue-specific condensed life. Chromosome preparations were made from chromatin, facultative and constitutive hetero- umbilical-cord blood with the subsequent Q- chromatin regions), being the densest domains staining in 145 Kyrghyz and 37 Russian neo- in a cell, apparently conducts heat between the nates. During the first year of life 17 Kyrghyz cytoplasm and nucleus when there is a differ- and 5 Russian neonates have deceased from ence in temperature between them. The various diseases in stationary conditions, the assumed heat conductivity effect of CC is sti- diagnosis of which is confirmed by pathological pulated by its principal features: condensed anatomical dissection. Mean numbers of Q-HRs state during the interphase, association with the per individual in newborn population were lamina and the inner nuclear membrane, repli- 3.16 0.13 in Kyrghyz and 3.59 0.23 in Rus- cation at the end of the S period of a cell ¼ sian, whereas in neonates died 4.58 0.23 and cycle, formation of the chromocenter, genetic ¼ 4.80 0.37 respectively. Neonates are char- inertness, wide variability in the quantitative acterized by a high range of variability in the contents both within and between species. Our distribution of Q-HRs (from 0 up to 7). But idea proposed in this paper is reduced to the died neonates, besides high value, differ by evolution of genome structure and physiology extremely narrow range of variability of Q-HRs of the whole organism in higher eukaryotes in population: number of Q-HRs in a karyotype going in parallel to counteract changes of tem- changes from 4 up to 6. Q-HRs in all samples perature in the ambient environment for more studied are encountered with an expected fre- effective preservation of constancy of tempera- quency on all the potentially Q-polymorphic ture of the internal environment. The outcomes autosomes, i.e. in no group there was pre- of such parallel evolution were: a) appearance of ferential Q-HR localization, and this is again different kinds of CC (C- and Q-hetero- fsuggesting that Q-heterochromatin is not locus- chromatin, G þ and Q þ bands, sex chro- specific material in the genome. It is supposed matin body), at a genome level the effect of that individuals with the greatest amount of which is generally subject to laws of physics; b) Q-HRs in the newborn population have greater formation at an organism level of a complex probability to die in the first years of life other organ-based physiological system of thermo- conditions being equal. regulation. 15th ICC: Abstracts 49

PO:06:44 torius (2n ¼ 22), Phodopus sungorus (2n ¼ 28), P. campbelli (2n ¼ 28), and P. roborovskii (2n ¼ 34), Molecular cloning and and one Calomyscinae species, Calomyscus mys- characterization of novel highly tax (2n ¼ 44). These results suggest that both the repetitive DNA sequences of the repetitive DNA sequences were species-specific greater long-tailed hamster and rapidly diverged. We compare them with the site-specific repetitive sequences cloned from (Tscherskia triton, Cricetridae) M. auratus, and discuss the molecular evolution E. Kamimura1, K. Yamada2, K. Tsuchiya3, of repetitive sequences in the Cricetinae. C. Nishida-Umehara2 and Y. Matsuda2 1Laboratory Animal Center, Yamagata PO:06:45 University School of Medicine, Yamagata, Japan; 2Laboratory of Animal Cytogenetics, Center for Genome downsizing in polyploid plants Advanced Science and Technology, Hokkaido I. Leitch1 and M. Bennett1 University, Sapporo, Japan; 3Laboratory of Wild Animals, Department of Zootechnical Sciences, 1Jodrell Laboratory, Royal Botanic Gardens, Faculty of Agriculture, Tokyo University of Kew, Richmond, Surrey TW9 3DS, UK Agriculture, Atsugi, Japan Polyploidy is a key factor in angiosperm evolu- The diploid chromosome number of the greater tion leading to the formation of new species. All long-tailed hamster (Tscherskia triton, Crice- else being equal, polyploids are expected to have tridae) is 28, and its karyotype consists of eleven larger C-values (DNA amount in the unrepli- pairs of acrocentric chromosomes, two pairs of cated gametic nucleus) than their diploid pro- small metacentric chromosomes, and middle- genitors, increasing in direct proportion with sized subtelocentric X and metacentric Y chro- ploidy. This expectation is observed in some mosomes. We isolated a new family of site- polyploid series, especially those newly formed, specific highly repetitive DNA sequences from but there are examples suggesting that C-values TaqI-digested genomic DNA of the greater long- in particular polyploids are lower than expected. tailed hamster, and characterized them by chro- The availability of the angiosperm DNA mosome in situ hybridization, sequence analysis C-values database (http://www.rbgkew.org.uk/ and filter hybridization. The TaqI-family of repe- cval/homepage.html) has allowed this question titive sequences was classified into two types by to be addressed across a broad sample of angios- their genome organization and chromosomal dis- perms and has revealed striking results deviating tribution; 1) centromere-specific repetitive from expectation: (i) Mean 1C DNA amount did sequence, and 2) sex chromosome-specific repeti- not increase in direct proportion with ploidy, and tive sequence. The size of the centromere-specific (ii) Mean DNA amount per ‘basic genome’ ( ¼ repetitive sequence was 112 bp, and the element 2C divided by ploidy) tended to decrease with was arranged into large tandem arrays and loca- increasing ploidy. The same striking results were lized to centromeric C-positive regions of all also observed for six monophyletic subgroups acrocentric autosomes and subtelocentric X chro- (monocots, all eudicots, Caryophyllales, core mosome. The sex chromosome-specific repetitive eudicots, asterids and rosids) and six families sequence, which was composed of 30–32bp inter- (Poaceae, Alliaceae, Ranunculaceae, Fabaceae, nal repeats, was distributed on the short arm of Solanaceae, Asteraceae). These results suggest X chromosome and the long arm of Y chromo- that DNA loss following polyploid formation, or some. Both the centromeric satellite DNA and genome downsizing, maybe a widespread phe- the sex chromosome-specific repetitive sequence nomenon of considerable biological significance. did not cross-hybridize with genomic DNAs of Recent advances in understanding the molecular other seven Cricetinae species, Mesocricetus aur- events which take place following polyploid for- atus (diploid number: 2n ¼ 44), M. brandti mation, together with new data on how DNA (2n ¼ 44), Cricetulus griseus (2n ¼ 22), C. migra- amounts can change, provide some insights into 50 15th ICC: Abstracts how genome downsizing may take place. In monilophytes were characterised by intermediate- searching for the nature of the evolutionary for- sized ancestral genomes. More in-depth analyses ces which may drive DNA loss, the basic genome within each land plant group provided evidence size itself appears to play some role in determin- for several independent increases and decreases ing the extent of genome downsizing following in genome size. Thus in agreement with several polyploidization focused studies within angiosperm families and genera showing that C-values may both increase and decrease, it is apparent that this dynamic PO:06:46 pattern of genome size evolution is repeated on a Genome size evolution across land broad scale across land plants. plants (Embryophyta) I. Leitch1, D. Soltis2, P. Soltis3 and PO:06:47 1 M. Bennett Dynamic mobility of the rDNA spacer 1Jodrell Laboratory, Royal Botanic Gardens, around the genome in the evolution of Kew, Richmond, Surrey TW9 3DS, UK; 2Department of Botany and the Genetics Nicotiana Institute, University of Florida, Gainesville, 1 2 2 3 K. Lim , K. Skalicka , B. Koukalova , Florida 32611-5826 USA; Florida Museum of R. Volkov3, V. Hemleben3, A. Leitch1 and Natural History and the Genetics Institute, 2 University of Florida, Gainesville, Florida A. Kovarik 32611-7800 USA 1Queen Mary University of London, UK; 2Institute of Biophysics, Brno, Czech Republic; Genome sizes (DNA C-values) of land plants 3Center of Plant Molecular Biology, Tu¨bingen, (Embryophyta; comprising bryophytes, lyco- Germany phytes, monilophytes, gymnosperms and angios- perms) range over 1000-fold from c. 0.1 pg to The 26-18S rDNA intergenic spacer (IGS) of 127.4 pg. To understand the evolutionary sig- Nicotiana contains A1/A2 subrepeats. To our nificance and to investigate the evolutionary for- surprise we observed large amounts of subrepeats ces responsible it is essential to evaluate the (termed A1/A2 satellite) outside of rDNA loci in phylogenetic component of this huge variation. species of Nicotiana section Tomentosae but not Such an analysis is timely given the recent from species in other sections. The chromosomal improved consensus of relationships between and location, patterns of genomic dispersion and within different land plant groups, and increased copy numbers of A1/A2 satellite varied con- availability of C-values (data are currently avail- siderably between species in section Tomentosae. able for over 4500 species, the majority of which At the species level we found differences in the are accessible in the Plant DNA C-values data- chromosomal distribution of the A1/A2 satellite base (release 2.0, Jan. 2003; http://www.rbgke- even between accessions of N. tomentosiformis w.org.uk/cval/homepage.html). Consequently, suggesting a rapid rate and large-scale sequence C-values were superimposed onto well-supported redistribution within timeframes shorter than phylogenies and analysed by character state speciation events. The A1/A2 satellite and the reconstruction using MacClade. Using the defini- A1/A2 sequences at the rDNA locus can be tions of very small (1.4 pg), small (3.5 pg), inter- separated by gel electrophoresis and units from mediate (3.5 to 14.0 pg); large (14 pg) and very both genomic fractions sequenced. We were large (35 pg) to characterise C-values (see Soltis astonished to find that the sequences occurred in et al. 2003 Am. J. Bot 90: 1596-1603), analysis the same populations. The best model to fit the revealed the ancestral C-value of all land plants data is that there is recurrent integration of was probably very small, a state that also char- repeats into the genome from the rDNA loci and acterised ancestral angiosperms and bryophytes. that this is associated with copy number expan- In contrast gymnosperms and the majority of sions, contractions and deletions, all occurring 15th ICC: Abstracts 51 over relatively short time frames (probably less and the plastidic 5-trnL(UAA)-trnF(GAA) than 60,000 years). There was also a trend region to infer a robust phylogenetic hypothesis towards the elimination of the A1/A2 satellite in based on two independent markers. The pre- N. tabacum (tobacco, 10,000 years old), an allo- liminary results confirm the monophyletic origin tetraploid with parents closely related to the of the Brassiceae, however, it is also suggested diploids N. sylvestris and N. tomentosiformis. that Conringia and Calepina might be placed out- This process may have already commenced in an side this clade. This is congruent with the S3 generation of synthetic tobacco. absence of chromosome triplication.

PO:06:48 PO:06:49 Chromosome triplications indicate that Karyotypic evolution of hamster the tribe Brassiceae is descended from species (Cricetinae, Muridae, a hexaploid ancestor: evidence from Rodentia) inferred from comparative comparative chromosome painting FISH analyses M. Lysak1, M. Koch2, A. Pecinka1 and K. Matsubara1, K. Tsuchiya2 and Y. Matsuda1 1 I. Schubert 1Lab. Anim. Cytogenet., Center for Advanced Sci. 2 1Institute of Plant Genetics and Crop Plant and Tech., Hokkaido Univ.; Lab. Wild Anim., Research (IPK), Gatersleben, Germany; 2Institute Fac. Agri., Tokyo Univ. Agri., Japan for Plant Sciences, University of Heidelberg, Heidelberg, Germany Hamster species belonging to the Cricetinae are distributed from South Europe to East Asia and Based on comparative genetic mapping between 18 species of these taxa are now recognized in Arabidopsis thaliana and Brassica species, it was this subfamily. The diploid chromosome number suggested that diploid Brassica genomes are des- of Cricetinae species is 2n ¼ 22–44, and their fun- cended from a hexaploid ancestor and this might damental number ranges from 44 to 80. The be valid also for other members of the Brassiceae diversity of karyotypes has been achieved by the tribe. To prove this assumption we employed accumulation of chromosome rearrangements comparative chromosome painting using a BAC that occurred during evolution, and thus it has contig spanning 8.7 Mb of the bottom arm of been possible to define the history of karyotypic Arabidopsis chromosome 4 in 21 species tradi- changes by comparing the karyotypes among the tionally placed in the tribe Brassiceae. With the extant species. In this study, to identify the chro- exception of the genera Calepina and Conringia, mosomal homologous regions among the six the analyzed BAC contig labels segments of three Cricetinae species, Chinese hamster (Cricetulus (to four) chromosome pairs in species with griseus), Armenian hamster (C. migratorius), 2n ¼ 14, 16, 18, 20, 22, 28, 30 and six chromo- Syrian hamster (Mesocricetus auratus), Djungarian some pairs in amphidiploid Brassica species with hamster (Phodopus campbelli), Desert hamster 2n ¼ 34, 36 and 38. The multiple copies corre- (P.roborovskii) and Greater long-tailed hamster spond structurally to the BAC contig of A. thali- (Tscherskia triton), we conducted comparative ana or more often they are rearranged in a chromosome painting with the chromosome- species-specific manner. Our data bring new evi- specific probes of Chinese hamster. Comparative dence that most of the diploid Brassiceae species analysis revealed that many chromosome rear- (including diploid brassicas) are descended from rangements had occurred among the six species. an Arabidopsis-like ancestor which underwent The chromosomal locations of the 5S and 18S- polyploidization (hexaploidy) followed by sub- 28S ribosomal RNA (rRNA) genes were also sequent chromosome fusions/fissions and inver- compared. The chromosomal location of the 5S sion/translocation events. We generated sequence rRNA genes on the region homologous to data for ITS 1 and 2 regions of nuclear rDNA Chinese hamster chromosome 3 was highly 52 15th ICC: Abstracts conserved in all the species. In contrast, there L. Mora1, M. Garcia1 and M. Ponsa1 was variation in the location of the 18S-28S 1 rRNA genes. The karyological data were com- Institut de Biotecnologia i Biomedicina. Departament de Biologia Celular, Fisiologia i pared with the molecular phylogenetic data, and Immunologia. Universitat Autonoma de karyotypic evolution in Cricetinae is discussed. Barcelona, Spain

PO:06:50 In this work, chromosomes conserved from the Evolution of the heterochromatin ancestor of Primates are studied to know its organization in the Drosophila position in interphase nucleus in order to eluci- date if the position is conserved in different spe- melanogaster species group cies. For this purpose we have analysed human L. Monti-Dedieu1, S. Aulard2, N. Chaminade2, (HSA) chromosome 17 and 9 and its homo- C. Montchamp-Moreau2 and F. Lemeunier2 loguos chromosomes in Lagothrix lagothricha (LLA) and Saimiri sciureus (SSC) cell lines. 1 2 IUFM, Paris, France; CNRS, Populations, Although ancestral HSA 17 is conserved among Ge´ne´tique, Evolution, Gif, France the three species, ancestral HSA 9 is conserved in LLA and HSA but fusionated with another In Drosophila, the heterochromatic sequences are chromosome fragment in SSC. located to restricted regions of the chromosomes Fibroblast cell lines from the three species were and subjected to a very rapid evolution. Previous grown on slides, ¢xed and permeabilized main- data on karyotype descriptions were mainly taining the 3D structure. Nuclei were hybridised obtained by classical cytogenetic techniques. using human whole-chromosome probes and Using molecular approach, we are now able to observed by laser confocal microscopy. Distances describe more precisely the chromosomal mor- from the centre of each signal to the centre of the phology and the distribution of heterochromatin. nucleus were measured and divided by its relative Because the ribosomal DNA genes (rDNA) are radius. Around 100 nuclei were analysed for each embedded in the heterochromatic regions, they probe and species. can be used as convenient markers of the evolu- Results show that the average position of ances- tion of genes in heterochromatin. tral HSA 17 in human, LLA and SSC cell lines is In order to understand the occurring of hetero- 62.72%, 55.84% and 45.35% of the nuclear radius chromatic rearrangments within the melanogaster respectively, being the position of SSC sig- group, we used £uorescent in situ hybridisation ni¢cantly di¡erent. Average positions of ancestral (FISH) and DAPI staining. We studied species HSA 9 in HAS, LLA and SSC are 49.08%, belonging to 8 of the 11 subgroups from the 63.51% and 68.58% respectively, being HSA melanogaster group. position signi¢cantly di¡erent. The results show that the heterochromatin dis- Our data show that although both chromo- tribution is species speci¢c and that the Y chromo- somes are phylogeneticaly conserved its position some is mainly concerned by rearrangements. in the nucleus is not always conserved. Results are They suggest also that the ancestral con¢guration discussedinrelationtothesize,morphologyand consists of one nucleolus organizer (NOR) on phylogenetic parameters of each chromosome. each sex chromosome. This scheme is found in most subgroups but some of them present excep- tions that will be discussed in the light of species PO:06:52 evolution. Comparative cytogenetic studies of PO:06:51 regeneration organisms, newt and Position studies in interphase nuclei of two planarian species conserved chromosomes from different T. Murakami1, Y. Umesono1, A. Sanchez2, primate species C. Umehara3, Y. Matsuda3 and K. Agata1 15th ICC: Abstracts 53

1Center for Developmental Biology, Riken; 1Center for Advanced Science and Technology, 2Department of Neurobiology and Anatomy, Hokkaido University, Sapporo, Japan; University of Utah School of Medicine; 2Immunology Section, National Research 3Chromosome Research Unit, Hokkaido Institute of Aquaculture, Fisheries Research University Agency, Tamaki, Japan; 3RIKEN Center for Developmental Biology, 4 Freshwater planarian flatworms have high ability Kobe, Japan; Department of Biological Sciences, of regenerating complete organisms from tiny Brunel University, Uxbridge, UK fragments of the bodies. This regenerative compe- tence is based on stem cell population that is pre- Bird karyotypes are mainly classified into two sent in the adult planarian. Recently, to promote types, the conservative karyotype and the var- the molecular analysis of regeneration and stem iant karyotype. The conservative karyotype that cell regulation in planarians, over 9,000 ESTs has is composed of two major components, macro- been generated from a clonal line of Dugesia japo- chromosomes and microchromosomes, is com- nica that is originated from Japan and also 3,000 moninmostofavianordersandreptiles.The unique ones from Schmidtea mediterranea that is variant karyotypes are observed in the Accipi- originated from Spain and has been kept at tridae and Falconidae of Falconiformes, which U.S.A., respectively. Although distinctive results have few microchromosomes and a large num- using this ESTs database have been developed ber of middle-sized chromosomes. To investigate independently, there is no data whether one clone the chromosome conservation and process of generated from one species is able to fit others or chromosome rearrangements in birds with the not. Therefore, we need to confirm that these dis- two different karyotypes on a molecular level, tinct family-level different flatworms can share the we compared chromosome homologies in birds functional genes each other. Furthermore, chro- of five orders (Struthioniformes, Tinamiformes, mosome research in planarian has been very few Galliformes, Strigiformes, Falconiformes) by in this decade and there is no report FISH analy- comparative chromosome painting with chicken sis for the organisms. Consequently, we estab- DNA probes of chromosomes 1-9 and Z. Each lished basic chromosome staining techniques chicken probe painted one chromosome pair followed by applied FISH analysis for both spe- except for chromosome 4 paint that hybridized cies. Then, we could make ideograms and it will to one macrochromosome pair and a pair of be standard for identifying each gene localization. microchromosomes in four ratite species of Telomere sequences were conserved and could Struthioniformes (ostrich, common rhea, Dar- observe larger signals on the end of all chromo- win’s rhea, double-wattled cassowary) and Tina- some than other organisms. In S. mediterranea miformes (elegant crested tinamou). Also in there was translocation from the first chromosome Galliformes and Strigiformes, chromosome rear- to third ones in asexual strain. This is outstanding rangements have occurred in low frequency. In for revealing the mechanism of evolution of sexual contrast to the chromosome conservation in the reproduction. Finally we were able to hybridize 10 four orders, high frequencies of chromosome functional genes on each chromosome, and will rearrangements were observed in the Accipi- establish the complete genome maps. tridae and Falconidae species. Chicken chromo- some 1 probe painted six and two pairs of PO:06:53 chromosomes in mountain hawk eagle of the Accipitridae and peregrine falcon of the Falco- Comparative cytogenetic studies on nidae respectively, and chromosomes 2 and 3 karyotypic evolution in birds probes painted three and four chromosome pairs 1 1 in the eagle, and two and two pairs in the fal- C. Nishida-Umehara , M. Shibusawa , con, respectively. These results indicate that 1 1 1 Y. Shimada , J. Ando , J. Ishijima , macrochromosomes have been divided by 2 3 4 A. Fujiwara , T. Murakami , J. Masabanda , sequential chromosome rearrangements in these D. Griffin4 and Y. Matsuda1 species. 54 15th ICC: Abstracts

PO:06:54 1/3, 1/16, 2/12, 2/20, 4/15, 5/15, 7/11, 9/15, 10/11, 10/22; whereas 3/14 and 9/17 were found New results in Platyrrhini chromosome only in Aotus nancymai. evolution based on comparative chromosome painting: The case of Aotus PO:06:55 A. Ruiz-Herrera1, F. Garca2, M. Aguilera3, Effects of the genetic background, M. Garcia1 and M. Ponsa`1 karyotype and sex of Rb heterozygous 1Departament de Biologia Cellular, Fisiologia i mice (Mus musculus domesticus) on the Immunologia, Universitat Auto`noma de nondisjunction rate in meiosis I Barcelona. 08193, Cerdanyola del Valle`s 1 2 3 2 M. Scascitelli , F. Pacchierotti , F. Spirito , (Barcelona). Spain; Institut de Biotecnologia i de 1 1 Biomedicina (IBB). Universitat Autonoma de B. Gustavino and M. Rizzoni Barcelona. 08193, Cerdanyola del Valle`s 1Department of Biology, University of Rome, Tor (Barcelona). Spain; 3Grupo BIOEVO,  Vergata, via della Ricerca Scientifica snc, 00133 Universidad Simo´n Bol var (Caracas). Venezuela Rome, Italy; 2Section of Toxicology and Biomedical Sciences, ENEA CR Casaccia, via The owl monkey, Aotus, is the unique genus Anguillarese 301, 00060 Rome, Italy; from Subfamily Aotinae (F. Cebidae, Platyr- 3Department of Genetics and Molecular Biology, rhini). Taxonomy of the genus Aotus is especially C. Darwin, University of Rome, la Sapienza, intricate since cytogenetic studies based on Piazzale Aldo Moro 5, 00185, Rome, Italy G- and C-banding comparisons have character- ized 18 different karyotypes, which chromosome The anaphase I nondisjunction rate of specific diploid numbers range from 46 to 58 chromo- chromosome arms of Robertsonian metacentrics somes. In an attempt to clarify the chromosomal was estimated by dual-colour FISH (fluorescence relationships between Aotus species in relation to in situ hybridisation). Spermatocyte II meta- the rest of New World monkey species, we have phases were obtained from laboratory mice (Mus used human chromosome specific-paints to deli- musculus domesticus) heterozygous for two and neate, for the first time, the homologous chromo- three Robertsonian centric fusions. somal segments present in Aotus. Data from double heterozygotes were com- Heparinized peripheral blood samples were pared with previous ones from heterozygotes for taken from one male Aotus nancymai (2n ¼ 54) the same number and type of Rb chromosomes, and one male Aotus sp. (2n ¼ 50) from Venezuela but obtained with a di¡erent mating scheme. It in order to obtain chromosome preparations. All allowed to check the possible in£uence of the two specimens were chromosomally characterized genetic background on the nondisjunction rate for after sequential G- and C-banding. Whole chro- speci¢c chromosome arms and/or on the overall mosome paints for all human chromosome were amount of aneuploid spermatocytes. used for FISH on Aotus metaphases. On the other hand the comparison of triple With the exception of human chromosome Y, heterozygotes for Rb chromosomes involving dif- all human chromosome-speci¢c painting probes ferent acrocentrics allowed to understand if alter- are hybridized on all Aotus chromosomes. Based native combinations of Rb centric fusions a¡ect on G-banding comparisons and the £uorescent the NDJ rate of speci¢c chromosomes and/or the ‘in situ’ hybridizations results, the chromosomal incidence of aneuploidy during meiosis I. homologies between two Aotus species have been We studied the overall frequency of aneuploid established. From the putative ancestral Platyr- oocytes from individuals with multiple Rb hetero- rhini associations (2/16, 3/21, 5/7, 8/18, 10/16, zygous chromosomes: we found higher values 14/15), two of them, 2/16 and 10/16 were not compared to the male germ system with the same found in the two Aotus species. In addition, some karyotype. Finally we analysed their role in an derived chromosomal associations were detected: evolutionary context with mathematical models. 15th ICC: Abstracts 55

Telomeres Symposium L68 Regulation of telomerase and the L66 mechanism of telomere length Protection and maintenance of human homeostasis telomeres J. Lingner1, M. Arneric1,K.Fa¨rstemann1, 1 1 1 T. De-Lange C. Kelleher , I. Kurth , P. Sperisen and T. Teixeira1 The Rockefeller University, 1230 York Avenue, 1 New York, NY 10021, USA Swiss Institute for Experimental Cancer Research (ISREC); CH-1066 Epalinges s/Lausanne, No abstract was submitted for this talk. Switzerland

L67 Telomerase activity is regulated at individual chromosome ends in cis, to achieve telomere Dangerous entanglements: roles of length homeostasis. This regulation involves telo- telomeres in promoting S-phase meric proteins and perhaps higher order DNA progression structures. Based on cellular studies, the human telomeric single-strand DNA binding protein K.M. Miller, M. Ferreira and J. Cooper protection of telomeres 1 (POT1) has been impli- Telomere Biology Laboratory, Cancer Research cated in positive and negative regulation of telo- UK, London WC2A 3PX, UK merase. To determine its effects at the molecular level, we used recombinant POT1 and analyzed Fission yeast Taz1 is the only known ortholog of its effects on telomerase activity in vitro. We show the mammalian telomere proteins TRF1 and that POT1 negatively effects telomerase activity TRF2. Taz1 regulates telomeric DNA synthesis, by reducing the processivity of the enzyme, while position effects on nearby transcription, and it does not prevent telomerase from accessing the meiotic chromosome movements. Furthermore, telomeric DNA primer. The negative regulation Taz1 controls two types of detrimental associa- of telomerase by POT1 requires the DNA bind- tions between chromosome ends. First, Taz1 acts ing protein to be in molar excess over the primer with Rap1 to prevent DNA repair reactions from suggesting that POT1 modulates telomerase pro- acting inappropriately on telomeres. Deletion of cessivity while bound to the DNA substrate. To taz1þ or rap1þ leads to end-fusions during G1 analyze the mechanism of telomerase regulation arrest, when nonhomologous end-joining activ- in vivo we developed a method to measure telo- ities are elevated. Second, Taz1 itself regulates mere elongation at nucleotide resolution in replication fork progression through telomeres. Saccharomyces cerevisiae. We found that the Deletion of taz1þ leads to replication fork stal- number of nucleotides added to a telomere in a ling at telomeres, while rap1 cells do not suffer single cell cycle is independent of telomere this impediment. This replication defect is asso- length. Telomerase does not act on every telo- ciated with telomeric entanglement at cold tempera- mere in each cell cycle, however. Instead it exhi- tures. These entanglements, which can be suppressed bits an increasing preference for telomeres as by a specific mutation in topoisomerase II, are their lengths decline. Deletion of the telomeric distinct from covalent telomere fusions but none- proteins Rif1 or Rif2 gives rise to longer telo- theless lead to chromosome breakage and meres by increasing the frequency of elongation missegregation. We will discuss the origin and reso- events. We conclude that telomere length home- lution of this newly recognized threat to ostasis is achieved via a switch between telomer- unprotected chromosome ends. ase extendible and non-extendible states. 56 15th ICC: Abstracts

L69 Telomeres are physical ends of chromosomes responsible for chromosome stability main- Telomere cytogenetics in senescence tenance. The evidence is accumulating that telo- and ALT mere maintenance may be altered in cells with Y. Zou1, J. Shay1 and W. Wright1 DNA damage response defects including cells from patients with ataxia telangiectasia (AT), 1 Univ. Texas Southwestern Medical Center, USA Nijmegen breakage syndrome (NBS) and Fan- coni anemia (FA). To investigate the effect of Progressive telomere shortening is the counting DNA damage response deficiencies on telomere mechanism that controls replicative aging. Using maintenance further we used several primary Q-FISH, we have identified the shortest telo- fibroblast cell lines from radiosensitive patients meres in BJ normal human fibroblasts. We show with novel DNA damage response defects includ- that the shortest telomeres co-localize with DNA ing defects in Artemis, ligase I, ligase IV, and damage signals in senescent cells, and that the 10 two unknown factors that cause radiosensitivity. shortest telomeres account for >90% of all telo- We monitored the rates of telomere shortening in meric end-associations observed when senescent the above cell lines using Southern blot and flow- checkpoints are abrogated by expressing human FISH techniques. Our results revealed acceler- papilloma virus 16 E6 þ E7 proteins. We have ated telomere shortening in cells from the above also examined the timing of telomere replication patients in comparison with control cells. In using a newly developed CO-FISH technique. addition we found evidence of telomere dysfunc- Yeast telomeres are late replicating and telomeres tion in cells with accelerated telomere shortening. at each end of yeast chromosomes exhibit coordi- We also analyzed telomere maintenance in mouse nated replication timing. In contrast, individual embryonic stem cells deficient in BRCA1. Simi- human and Indian Muntjac telomeres replicate at larly we observed accelerated telomere shortening specific times throughout S-phase. Although and end-to-end chromosome fusions in these replication timing of p or q arm telomeres of cells. We are currently attempting to suppress both chromosomal homologues is similar (e.g., BRCA1 in human mammary epithelial cells using maternal and paternal 2p telomeres replicate at siRNA and analyze effect of BRCA1 suppression approximately the same time), the timing of p on human telomeres. and q arms of individual telomeres are not coor- dinated (e.g., there is no coordination between 2p and 2q replication). Finally, we will describe the PO:06:56 presence of ultra-high frequency sister chromatid Physical mapping of the terminal exchange within telomeres (T-SCE) in ALT cells. ALT cells use a recombination-based mechanism regions of individual rye chromosomes of telomere elongation in order to become O. Alkhimova1, J. Dolezel2 and A. Vershinin3 immortal. In contrast to normal mortal or telo- 1Institute of Molecular Biology and Genetics, merase-expressing immortal cells, ALT cells show 2 SCE at typically >50% of all telomeric ends per 03143 Kiev, Ukraine; Institute of Experimental Botany, 77200 Olomouc, Czech Republic; metaphase. 3 Institute of Cytology and Genetics, 630090 Novosibirsk, Russia L70 Telomeres are specialized chromosome structure, Telomere maintenance in DNA which play important role in chromosome stability damage response de¢cient cells and behaviour. Together with subtelomeres, they 1 1 form terminal regions of chromosomes. These are E. Cabuy and P. Slijepcevic dynamic regions and might help organisms to 1Department of Biological Sciences, Brunel adapt to new environmental conditions and pro- University, Uxbridge, Middlesex, UB8 3PH, mote genomic rearrangements. Large heterochro- UK matic blocks, which are present near the telomeres 15th ICC: Abstracts 57 of rye chromosomes, are notoriously difficult for Telomeric-like sequences have been found in physical mapping because of enrichment by differ- non-terminal regions of chromosomes (so-called ent types of repetitive DNA sequences. We used ITS) in a large amount of different vertebrate wheat-rye addition lines and performed fluores- species. ITS have been considered the result of cence in situ hybridization (FISH) on metaphase ancient reorganisations, alternative sites for telo- flow-sorted chromosomes and extended DNA mere formation or markers of unstable regions. fibers for comparison of the terminal regions of the Because of the great importance of rat and other individual rye chromosomes. Each wheat-rye addi- rodent species in biomedical research, the study tion line produced a chromosome-specific large- of the distribution of ITS will contribute to the scale organization of the tandem repeat arrays. The better understanding of genomic architecture and FISH signals on DNA fibers showed the multiple rodent evolutionary process. patterns of co-linear monomers arrangement of Metaphasic chromosomes from ¢broblasts of repetitive families as well as of authentic telomeric three foetuses from three Rattus norvegicus probe from Arabidopsis. The primary structure of Sprague^Dawley females (2n ¼ 42) were obtained some spacer sequences revealed the scrambled by standard procedures. Fluorescent in-situ hybri- regions of similarity to various known repetitive disation with a long synthetic (TTAGGG)n probe elements. Using PCR analysis, we determined the was performed. Only double spot signals were DNA sequences adjacent to the tandem repeats scored and the analysis was performed at a resolu- array (spacers). They are enriched of short direct, tion of 238 bands/haploid genome. complementary, inverted and symmetrical repeats A total of 305 metaphases was analysed and, as whichappeartobeassociatedwithrecombination expected, all telomeres hybridised with the probe. events. These spacers may be a powerful source of In addition, 121 intrachromosomal loci with dif- tandem array rearrangements. We mapped some of ferent hybridisation frequencies were detected them on metaphase stretched chromosomes, and along all chromosomes with the exception of chro- the distribution of the signals was in good agree- mosome RNOY. A non-random ITS distribution ment with the localization of the breakpoints for through the karyotype of RNO was observed. A deletions and for translocations of 1R long arm preliminary comparative study has shown that 18 with different wheat chromosomes. ITS were located in those chromosomal bands a¡ected by X-radiation in R. norvegicus; so the This work was supported by the INTAS grant (03-51-5908). 75% of irradiation breakpoints coincide with ITS. Considering ITS as origin or result of chromosomal reorganisations, the large amount PO:06:57 of ITS found in the Rattus norvegicus karyotype Distribution of intrachromosomal would be explained by the high rate of chromo- some reorganisations which characterises the telomeric sequences (ITS) on Rattus evolutionary history of rodents. norvegicus chromosomes N. Camats1, A. Ruiz-Herrera3, F. Garcı`a1, PO:06:58 3 2 J. Egozcue and M. Garcia Telomere length control in Arabidopsis 1Institut de Biotecnologia i Biomedicina (IBB). plants Dept de Biologia Celular, Fisiologia i Immunologia. Universitat Auto´noma de G. Maillet, C.I. White and M.E. Gallego Barcelona (UAB). Spain; 2Unitat de Biologia, CNRS UMR 6547, Universite Blaise Pascal, Facultat de Medicina, Departament de Biologia Aubie`re, France Cellular, Fisiologia i Immunologia, Universitat Auto´noma de Barcelona (UAB), Spain.; 3Unitat de Biologia, Facultat de Cie`ncies, Telomeres are nucleoprotein complexes at the end Departament de Biologia Cellular, Fisiologia i of eukaryotic chromosomes. They play essential Immunologia, Universitat Auto´noma de roles in maintaining genomic stability by permit- Barcelona (UAB), Spain ting full replication of chromosomes, preventing 58 15th ICC: Abstracts unwanted fusion events and protecting chromo- between chromosome rosettes, indicating associa- somes ends from degradation. Telomeres are tion of Tel of different chromosomes into one maintained at a species-specific equilibrium cluster. Decondensation of the centromeric het- length. We have found that the telomere length erochromatin consists of major satellite (maSat) equilibrium differs between different Arabidopsis and minor satellite (miSat) as well as telomeric thaliana ecotypes. To investigate the genetic reg- heterochromatin went on in the middle and late ulation of telomere length, plants with differing S phase. During S/G2 transition the correspon- telomere sizes were crossed and the telomere dence of Tel with the chromocenters is most length in the hybrids analysed. For all of the apparent. In G0/G1 Tel aggregates colocalized hybrids, a new equilibrium in telomere length is with maSat and with miSat to lesser extend. Tel established, intermediate between that of the two aggregates are embedded into the maSat granules parental plants. A regulation mechanism thus at G0/G1. MTBP shift according to Tel is visible shortens the longer telomeres and lengthens the in prophase. In prometaphase, part of MTBP shorter. To analyze the possible involvement of doesn’t coincide with telomere as well. Most of the Rad50, Ku80 and Lig4 proteins in the estab- Tel are colocalized with MTBP/TRF2 through- lishment of the new telomere length, Ws ecotype out all stages of the cell cycle though it is possi- (long telomeres) heterozygotes for the RAD50, ble to find 1-2 telomere that are not covered with KU80 and LIG4 genes were crossed with wild the protein. Prominent MTBP shift in respect to type Col ecotype plants (short telomeres). the Tel is visible in prophase. In prometaphase RAD50+/ inter-ecotype plants are unable to part of MTBP doesn’t coincide with Tel due to elongate the shorter telomeres while retaining the the preparation of part of MTBP move to the ability to shorter longer ones. No haplo-insuffi- nuclear envelope remnant in mitosis. Biochemical ciency was detected for the KU80 þ/ inter-eco- features of MTBP/TRF2 suggest it participation type plants. Unexpectedly, LIG4 þ/ inter- in Tel clusterization. ecotype plants maintain the original size of the parental telomeres. This result is the first evidence PO:06:60 of a role for Lig4 in telomere homeostasis. Subtelomeric duplication 7q36-qter and monosomy 13q32-qter detected by PO:06:59 FISH in two brothers with severe Telomere and TRF2/MTBP mental retardation localization in respect to satellite DNA A. Nucaro2, G. Crisponi3, L. Minafra1, during cell cycle of the mouse cell line R. Rossino4, A. Cao2 and C. Cianchetti1 L929 1Dipartimento di Neuroscienze, 1 1 I. Kuznetsova , A. Voronin and sez. Neuropsichiatria Infantile, Universita’ di O. Podgornaya1 Cagliari, Italy; 2Istituto di Neurogenetica e 1 Neurofarmacologia, CNR, Selargius, Cagliari, Institute of Cytology RAS Italy; 3Servizio di Puericultura, Universita’ di Cagliari, Italy; 4Dipartimento di Scienze The mouse chromosomes are acrocentric, mean- Biomediche e Biotecnologie, Universita di ing that each centromere is adjacent to telomere Cagliari, Italy (Tel). The location of satellite DNA, telomeres and TRF2/MTBP protein was studied during We describe two patients with submicroscopic cell cycle in cell line L929. During the cell cycle, duplication of 7q36.3qter and monosomy Tel underwent movement and rearrangement: 13q32qter, detected by subtelomeric FISH probes. most of them tend to aggregate into conglomer- The patients, a 28 old man and his 21 year old ates in G0/G1. In prometaphase during meta- sister, were referred to the Neuropsychiatric phase plate formation half of Tel together with Department for severe mental retardation and CENs arranged in circle. Clusters located dysmorphic features. 15th ICC: Abstracts 59

Both had markedly delayed psychomotor devel- avidin-CY3, all q-arm probes with digoxigenin- opment, severe mental retardation, bilateral pyr- 11-dUTP and detected with antidigoxigenin- amidal signs, slight ataxia. Subcortical brain FITC. Furthermore the q-arm of acrocentric and atrophy and partial agenesia of corpus callosum Y chromosomes were labelled simultaneasly with was also present at CT scan. The dysmorphic fea- biotin-16-dUTP and digoxigenin-11-dUTP so tures were: abnormal posture, microcephaly, nar- that we could identify these regions as an yellow row forehead, low hairline, facies signi¢cantly signal due to the fusion of the red signal (CY3) abnormal, long and £at face, prognathism, nar- and the green signal (FITC). row nasal bridge, tented upper lip, dental caries, We have used three slides per person: every brachydactyly. slide was divided into six sections, every section Standard cytogenetics analysis revealed an appar- was cohybridized with two or three probes di¡er- ently normal karyotype, but a familiar history of ently labelled. This method was validated on nega- mental retardation persuaded us of the opportunity tive and positive controls. to investigate all members of the family. We report the results of the study of the sub- FISH with speci¢c 7 and 13 painting probes and telomeric regions, using the method described, in telomeric probes revealed a balanced translocation 60 children with idiopathic mental retardation. in the mother and in a brother of the two patients. A son of this brother showed a severe mental retardation and dysmorphic features. Molecular PO:06:62 and cytogenetics investigation revealed the pre- How do Alliaceae maintain their sence of unbalance karyotype 46, XY, der(7)t (7;13)(q36;q32).(partial trisomy 13q32qter and telomeres? ^ revisited partial monosomy 7q36qter). E. Sykorova1, K. Neplechova2, M. Sklenickova1, Y. Lim3, M. Chase4, PO:06:61 A. Leitch3 and J. Fajkus1 Mental retardation: detection of 1Institute of Biophysics, Czech Academy of Sciences, Kralovopolska 135, 61265 Brno, Czech subtelomeric rearrangements 2 Republic; Department of Functional Genomics D. Parrini1, M. Betti1, G. Marseglia1, and Proteomics, Masaryk University, Kotlarska 3 A. Nutini1, R. Ciampi1 and F. Torricelli1 2, 61137 Brno Czech Republic; School of Biological Sciences, Queen Mary University of 1UO Citogenetica e Genetica, AO Careggi, London, Mile End Road, E1 4NS London, UK; Firenze, Italy 4Jodrell Laboratory, Royal Botanic Gardens, Kew, TW9 3AB Richmond, UK Chromosomal rearrangements involving the sub- telomeric regions have been found in 7,4% of The search for missing typical plant telomeres in children with moderate to severe mental retarda- Asparagales revealed that this monocotyledonous tion. The detection of these rearrangements plant order can be subdivided into three groups requires a FISH-based assay with a set of chro- of plants differing substantially in the type of mosome-specific probes, localized as close as their telomeres-plant-type (e.g. orchids), human- possible to the telomere. type (e.g. asparagus), unknown type (e.g. onion). We have used a set of 42 BAC/PAC probes spe- The presence of different types of telomere corre- ci¢c for the subtelomeric regions: these probes sponds with phylogeny and it defines two evolu- were alternative to those published by Knight tionary points where the change had happened et al. Every probe has been tested for sensibility (Sykorova et al., Proc. R. Soc. Lond. B, 2003, and speci¢city. The following step was the devel- 270, 1893–1904). The first evolutionary point opment of a method that allows the simultaneous divides the families with the plant and the human analysis of all the subtelomeric regions. Using type of telomeres, the second point divides the data from literature, all p-arm probes were label- families with the human and the unknown type led with biotin-16-dUTP and detected with of telomeres (Alliaceae). To further define this 60 15th ICC: Abstracts switch point we started screening the Alliaceae to be phosphorylated at its Ser-219 by ATM family to find out where the loss of human type kinase and we have shown that inhibition of this of telomeres had happened. Our current results phosphorylation by wortmannin leads to sup- of slot-blot and in situ hybridisations suggest the pression of mobility of GFP-TRF1 bound to ‘big change’ happened within Allium. This find- ITRs in Chinese hamster V79 cells and to an ing move us closer to a solution for the mystery increased frequency of spontaneous chromosome of onion telomeres and their maintenance. aberrations (SCA) which are clustered at ITRs. Although the previously suggested models of func- Similar increase of SCA was also observed after tion of alternative onion telomeres based on satel- transfection of V79 cells with the expression plas- lite repeats or transposable elements could explain mid encoding tankyrase 1 which suppresses bind- the mechanism of replenishment of chromosome ing of GFP-TRF1 to ITRs. We suggest that end sequences, the other telomeric functions dynamic binding of TRF1 induces formation of (e.g., the end capping) would require substantial compact chromatin at ITRs limiting spontaneous adaptation of the protein machinery involved. DNA breakage by nucleases. This breakage may depend on endonuclease of L1 retrotransposons This work was supported by Leverhulme Trust, GACR (204/ which is expressed at high level in somatic mam- 04/P105, 204/02/0027) and the institutional support malian cells and stimulated by ectopic parallel (MSM143100008, Z5004920) pairing of (TTAGGG)n repeats promoted by immobile TRF1. PO:06:63 Sequence-speci¢c control of stability PO:06:64 of internal (TTAGGG)n repeats in Localization of telomere repeats in mammalian cells chromosomes of two species, Sorex 1 1 1 N. Tomilin , R. Krutilina , A. Smirnova , araneus and Sorex granarius S. Oei2 and M. Bradbury3 N. Zhdanova1, T. Karamisheva1, 1Institute of Cytology, Russian Academy of 1 1 2 2 N. Astakhova , N. Rubtsov , J. Minina , Sciences, 194064 St. Petersburg, Russia; Institute P. Lansdorp3 and M. Kammori of Biochemistry, Free University of Berlin, 14195 Berlin, Germany; 3Department of Biological 1Institute of Cytology and Genetics of SB RAS; Chemistry, UC Davis School of Medicine, Davis, 2Novosibirsk State University, Russia; 3Terry Fox CA 95616, USA Laboratories, BC Cancer Research Centre, Vancouver, BC, V5Z 4E6, Canada A major fraction of the genome in mammals is represented by tandem and dispersed repeats According to molecular clock, S.araneus and which provide segments for unequal homologous S.granarius diverged few tens of thousand years recombination (UHR) leading to chromosome ago. Their karyotypes consist of practically iden- aberrations. UHR is known to be initiated by tical chromosomal arms. In S.araneus (Novosi- double-strand DNA breaks (DSBs) which forma- birsk race) these elements combines together as tion within repeats can be suppressed by chroma- metacentrics, whereas karyotype of S.granarius tin. Alternatively, DNA ends of already formed contains a few metacentrics, de and tu, the DSBs can be fixed in chromatin to prevent ecto- remaining chromosomes represented by individual pic homologous interactions and misjoining. In arms. All S.araneus telomeres exhibited hybridiza- some mammalian species tandem (TTAGGG)n tion signals with labeled telomere repeat probe gen- repeats are located not only in telomeres but also erated by PCR. In addition, ITSs were found in the within internal chromosome bands and here we centomere of metacentics of middle size, go, hn, ik, present evidence that internal (TTAGGG)n gl, mp, and gr. In contrast, ITSs have not revealed repeats (ITRs) can be protected by telomeric pro- in large, af, bc de, and small, tu, metacentrics, which tein Pin2/TRF1. This essential protein is known are the results of more ‘‘ancient’’ Robertsonian 15th ICC: Abstracts 61 fusion. It is appears, that in S.araneus Rb transloca- sequence is used to programme complex nuclear, tions were formed with the preservation of telomere cellular and tissue functions throughout differ- repeats. Probably, they could be deleted in entiation and development. There are many ‘‘ancient’’ metacentrics. Contrary, strong signals approaches to these issues but we have con- have been detected only in the pericentromeric centrated on understanding how a single mam- regions of S.granarius acrocentrics. The use of PNA malian gene cluster is activated or silenced as telomeric probe showed, that these regions contain stem cells undergo lineage commitment, differ- up to 300 kb of telomeric DNA, while the telomeres entiation and maturation. In particular we have of long arms 3.8 kb only. To study the structure of analysed the alpha globin cluster which is expres- S.granarius long telomeres, we generated micro- sed in a cell type- and developmental stage- library from 6 copis of pericentromeric regions of a specific manner in the haemopoietic system. Our chromosomes. The microlibrary painted pericen- studies include analysis of the transcriptional tromeric regions of the all S.granarius acrocentrics. programme which accompanies globin gene acti- Using two color F-FISH with telomeric PCR probe vation focussing on the expression of relevant and microlibrary, we observed both cohybridization transcription factors and co-factors. Binding of and sequential localization of two probes. We these factors to the chromatin domain containing assume, that multiplication and settling of telomeric the alpha cluster has been characterised by chro- repeats simultaneously with the other repeated matin immunoprecipitation. In addition, we have sequences from pericentromeric regions of a arm monitored the epigenetic modifications (e.g. occurred during the formation of S.granarius nuclear position, timing of replication, chromatin karyotype. modification, DNA methylation) that occur as the genes are activated (in erythroid cells) or silenced (e.g. in granulocytes) as haemopoiesis Epigenetics Symposium proceeds. Together these observations provide a uniquely well characterised model illustrating the L71 mechanisms which regulate and memorise pat- terns of mammalian gene expression as stem cells The biology of proteins that bind to undergo lineage specification, differentiation and methylated DNA terminal maturation. A. Bird L73 University of Edinburgh, Scotland, UK Role of histone modi¢cations in No abstract was submitted for this talk. chromatin function and cancer T. Kouzarides1, S. Saunders1 and L72 L. Hughes-Davies1 How transcriptional and epigenetic 1Wellcome/Cancer Research UK Gurdon programmes are played out on an Institute. Tennis Court Road, Cambridge, UK individual mammalian gene cluster Chromatin is covalently modified at many sites during lineage commitment and within histones. Our efforts are concentrated on differentiation understanding the mechanism by which histone 1 modifications control chromatin. In addition, D. Higgs since chromatin modifying enzymes are impli- 1Weatherall Institute of Molecular Medicine, cated in cancer we would like to know how dis- University of Oxford, UK ruption of the pathways that leads to histone modifications contribute to cancer. In the post-genome era a great deal of work has Our analysis of lysine methylation has led us to been focussed on understanding how DNA examine SET domain containing proteins in the 62 15th ICC: Abstracts yeast S.pombe. We are analysing a protein SET9, opment: In zygote, the reprogramming processes which we ¢nd can methylate histone H4 at K20. are targetted to male pronucleus thus creating an We ¢nd that a SET9 deletion or a point mutation epigenetic asymmetry between parental genomes. in the catalytic domain of SET9, shows loss of via- In blastocyst, with the ¢rst wave of di¡erentiation bility when exposed to DNA damaging agents epigenetic reprogramming occurs in the plur- such as UV and gamma irradiation. Our results ipotent cells of inner cell mass (ICM) as opposed indicate that SET9 (which has uncharacterised to the di¡erentiating cells of trophectoderm human homologues) represents a member of a (future extraembryonic tissues). The genome wide new family of lysine methylases, which play a role reprogramming commences later once again dur- in DNA damage response. ing the development of germline, when the devel- We are also extending our studies on the oping germ cells undergo a complete erasure EMSY breast cancer gene. EMSY associates with of all epigenetic information prior to the onset of the BRCA2 tumour suppressor and has a role to sexual di¡erentiation. play in transcriptional repression and DNA These naturally occuring reprogramming repair. The EMSY protein forms a binding plat- events are, at least to some extent, recapitulated form with chromatin regulators such as HP1. The in vitro in somatic nuclear transfer and cell fusion domain of EMSY necessary for binding BRCA2 experiments. Search for common factors should and HP1 is under structural analysis and the thus shed light on molecular mechanisms under- relative contribution of EMSY to transcription lying these complex processes. and DNA repair is being investigated.

L74 L75 Epigenetic reprogramming ^ taking The role of SDE4 in chromosomal a lesson from an embryo silencing in plants 1 1 P. Hajkova1, P. Western2, S. Erhardt3, A. Herr and D. Baulcombe S. Sullivan1, F. Cesari-Weimar1, J. Brenton4 1Sainsbury Laboratory and A. Surani1 1 Certain types of chromosomal silencing in Wellcome Trust/Cancer Research UK Gurdon plants are associated with features of RNA Institute, Cambridge, UK; 2ARC Center for silencing. Small interfering RNAs (siRNAs) Development and Biotechnology, Murdoch Childrens Research Institute, Melbourne, can be found from the silenced locus and com- Australia; 3UCSF, LBNL Berkeley, US; 4Cancer ponents of the silencing machinery are Genomics Program, Department of Oncology, required both for the production of siRNA University of Cambridge, Hutchison/MRC and the silencing of the gene. Loci undergoing Research Centre, Cambridge, UK chromosomal silencing, such as AtSN1 (Arabi- dopsis Sine element 1), produce only the longer Nuclear cloning experiments brought new class of siRNAs (24-25nts), whereas both size insights into genome wide reprogramming con- classes (21/22 and 24/25) can be found in nected with changing plasticity of genome and cytoplasmic-silencing systems. These size differ- re-gaining of cellular pluripotency. Such repro- ences reflect distinct silencing pathways. Trans- gramming is characterised by change in global gene silencing in Arabidopsis requires the gene expression accompanied by genome wide RNA-dependent RNA-polymerase RDR6 changes of epigenetic modifications and chroma- (SDE1/SGS2) and argonaute 1 (ago1). tin structure. However, little is known about Chromosomal silencing requires SDE4, the molecular basis of this process. argonaute-like protein AGO4, and the RNA- Genome reprogramming events occur naturally dependent RNA-polymerase RDR2. Intrigu- several times during mammalian embryonic devel- ingly, mutants of the chromosomal silencing 15th ICC: Abstracts 63 pathway delay the onset of GFP transgene L77 silencing and suggest that there is cross-talk between these two pathways that enhances Association of glutathione silencing efficiency. Map-based cloning of S-transferase and chromosomal SDE4 suggests a model in which SDE4 helps aberration as a means to determine to produce an aberrant RNA substrate that is occupational exposure recognized by RDR2. A. Movafagh1, F. Maleki, S. Mohamadzadehghobadloo and S. Fadaei L76 1Shahid Beheshti Medical University Department Changes in the arrangement of Of Genetics, Evin, Tehran, Iran chromosome territories in human cell clones It is well known that exposure of the body to ionizing radiation such as X-rays produces an D. Ka¨hler1, J. Gao2, J. Mattes2, R. Eils2, 1 1 extremly wide range of mutational events.The T. Cremer and I. Solovei influence and status of glutathione S-transferase 1Department of Biology II, Ludwig Maximilians (GST) on the rate and frequency of chromosomal University (LMU), Munich; 2iBioS, German aberrations in peripheral blood lymphocytes of 50 Cancer Research Center (DKFZ), Heidelberg medical radiotherapy workers over 5 years periods handling X-ray machines and 43 control indivi- The inheritance of chromosome territory duals was subjected to our studied. GST is divi- arrangements (CT-A) from one cell generation to ded into four classes; the mu and theta showed the next one has remained a controversial issue genetic polymorphism, that involves a gene dele- (Gerlich et al [2003] Cell 112: 751–764 ; Walter tion. This deletion has been shown to be asso- et al. [2003] J Cell Biol, 160: 685–697). We per- ciated with the susceptibility to mutagen induced formed 3D-FISH experiments with sets of chro- cytogenetic damages. GST activity in serum was mosome specific probes on human cell clones, estimated by improved Habig method, also the including normal diploid fibroblasts (2-cell frequency of chromosomal aberrations was eval- clones), binucleated fibroblasts, normal epithelial uated in blood lymphocytes by conventional Tryp- cells (2- and 4-cell clones) and the cancer cell sin G-banding technic. Interpretation of this study lines DLD and HeLa (2-, 4-, 8-, 16-, and 32-cell yields of dicentrics acentrics followed by ring clones). Nuclear image stacks were recorded with chromosomes and of total chromosomal aberra- a confocal microscope and used for the 3D- tions and GST activity were significantly higher in reconstructions of CTs and the determination of the radiotherapy workers P ¼ 0.04 and P ¼ 0.001 the gravity centres of each CT. To estimate the respective compare to controls. Increase GST con- similarity of CT-As between cells from the same centration and chromosomal damages are corre- clone we determined (i) the 3D distance differ- lated in the present study. The mean frequencies ences between homologues CT gravity centres of different chromosomal aberrations were higher and (ii) the ‘bending energy’, a measure of topo- in radiotherapy workers with over 10 years occu- logical alterations required to transform the CT- pation exposure (P ¼ 0.05) compare to less than A of one nucleus to the CT-A of another. Both 10 years with similar condition. GST activity in approaches consistently confirmed very similar male were more frequent (P ¼ 0.04) than female in CT-As in sister nuclei of 2-cell clones or binu- radiotherapy workers. Age and sex were not cleated cells. For clones with larger number of found to be significant predictor for GST activity cells, higher values of both parameters were and chromosomal abnormalities in controls. found, steadily increasing from 2- to 16-cell Despite the limited number of blood sample the clones. At this stage dissimilarities between CT- results seem to indicates an association between As reached the level observed for randomly cho- chromosomal aberration and GST activity, sen cells from the original cell population. although for firm conclusion, it needs more data. 64 15th ICC: Abstracts

L78 PO:06:65 Parental origin-dependent phenotype Preferential inactivation of the of mice transgenic on bovine satellite parental hypermethylated X DNA chromosome in hybrid females of the I. Suchkova1, N. Slominska1 and E. Patkin1 genus Phyllotis (Rodentia: Muridae) 1 1 1The Institute of Experimental Medicine M. Acevedo and L. Walker of RAMS, St. Petersburg, Russia 1Instituto de Ciencias Biome´dicas, Facultad de Medicina, Universidad de Chile. Santiago, Chile We produced two lines of transgenic mice har- bored repeated unit (3.8 kb) of bovine satellite IV Though is well documented that dosage compensa- (Sat) in hemizygous state. Whereas Sat transmis- tion of X-linked genes in mammals, is achieved by sion from Sat hemizygous females did not cause random inactivation of one X chromosome, scarce the statistically significant deviation from theore- information is available concerning this process in tically expected 50%, the transmission of Sat hybrid females. DNA methylation is one epigenetic from male resulted in the decrease of the fre- mechanism that maintain the inactive X state; quency of newborn offsprings up to 4,5%–13,3% however, it is not clear whether inactive (iX) or in comparison with theoretically expected 50%. active X (aX), is the hypermethylated one. To find This could be a result of our observation of out if the X chromosomes are randomly inacti- abnormal and delayed embryos following vated in Phyllotis hybrid females, we evaluated the implantation. The second striking effect was an methylation state of these chromosomes in males appearance of mammary gland tumors in trans- and females of the parental species, P. darwini and genic females following first lactation. In theory P. xanthopygus and in their hybrid females, assum- such effects could be ascribed to deleterious ing that X male methylation state corresponds to effect of Sat on one of the imprinted genes. G- an aX chromosome. banding and FISH showed that Sat was mapped Metaphases were treated with SPRINS techni- to chromosome 12 in nearest vicinity to gene que: after HpaII digestion of non methylated R- Pax9, which upregulation was shown to be asso- bands, digoxygenin-labelled nucleotides were nick ciated with both abnormal development and can- translated and detected with £uorescence- cer. Thus, if this gene is imprinted, Sat conjugated antibodies (Boehringer Mannheim). integration resulted in loss of imprinting, and HpaII-SPRINS bands intensity of parental spe- subsequent increasing of this gene product simi- cies and hybrid X chromosomes, were compared larly to described earlier imprinted genes. Alter- using an epi£uorescence microscope. natively Sat could operate as transactivator of The X chromosomes of both parental females remote imprinted genes for example via attract- showed evident di¡erences in the intensity of their ing methylation groups from imprinted genes HpaII-SPRINS bands, being the strongest £uores- and/or a formation of unusual chromatin struc- cence of the hypomethylated one, similar to that ture. Our results of heterochromatin and methy- showed by the respective male X chromosome. lation analysis of Sat mice at least partly Since X male is genetically active, the iX in these evidence in favor of last proposal. Sat was com- females must be the hypermethylated one. The pletely methylated in abnormal embryos and hybrid female X-chromosomes also had clear mice with tumors, and nuclei had another hetero- di¡erences in their HpaII-SPRINS bands, being chromatin morphology in comparison with the darwini X chromosome more £uorescent than normal mice. the xanthopygus X one. Thus, in these hybrid females the xanthopygus hypermethylated X chro- The work was supported by RFBR grant N 04-04-48169 mosome is preferentially inactivated. 15th ICC: Abstracts 65

PO:06:66 C. Bacher1, E. Heard2 and R. Eils1 Interchromatin compartment 1Divisions of Intelligent Bioinformatics Systems1, visualized by induced-chromatin- German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany; 2Mammalian condensation (ICC) in living Developmental Epigenetics Group2, UMR HeLa cells 218-Nuclear Dynamics and Genome Plasticity, Curie Institute-Research Section, Paris, France H. Albiez1, L. Schermelleh1, I. Solovei1, 1 1 H. Leonhardt and T. Cremer X-chromosome inactivation ensures dosage com- 1Department of Biology II, Ludwig Maximilians pensation between XX females and XY males. University, 80333 Munich, Germany The inactivation of an entire X-chromosome takes place while its homologue remains geneti- It was earlier demonstrated that incubation of cally active in the same nucleus. We are examin- cells in a hyperosmotic medium induces strong ing undifferentiated female and male mouse condensation of chromatin (Robbins et al. JCB, embryonic stem cells to investigate the spatial 40:400–416 (1970)). The effect was completely localisation and distribution of the X-inactivation reversible after restoring physiological osmotic center (Xic) during the initiation of X inactiva- conditions. We have employed this approach, tion using FISH. We have acquired 3D multi- called Induced-Chromatin-Condensation (ICC), colour image sets of the fixed cell nucleus in living HeLa cells. ICC was obtained in less labelled with DAPI and primary transcripts at than 1 min following the incubation of cells in the Xic using confocal laser scanning microscopy hyperosmotic medium (525 mOsm/l) and likely during the first 4 days of ES cell differentiation. resulted from an increase of divalent ions in the We used in-house developed software tools for cell interior. Transcription as well as replication image processing and for the quantitative analy- was discontinued during ICC but restored as sis of the Xic signal distances to the nuclear soon as the normal condensation state was re- periphery of female and male cells and the inter established. ICC was accompanied by an enlarge- Xic locus distances in female cells, respectively. ment of the interchromatin compartment in the Positional changes of the Xic loci in female ver- nucleus. In accordance with the chromosome ter- sus male cells will be presented, with a view to ritory–interchromatin compartment (CT-IC) understanding the potential implication of model and experimental data from other groups nuclear localisation in the X-inactivation process. (Cmarko et al. Histochem Cell Biol. 113:181–187 (2000)) nascent transcripts formed prior to ICC were observed at the surface of condensed chro- PO:06:68 matin lining the enlarged interchromatin com- partment, while splicing speckles and nuclear Spatio-temporal dynamics of X bodies were retained in its interior. Cells kept in inactivation in differentiating mouse the ICC state for up to 30 min and then released ES cells. into isotonic medium retained their viability. 1 1 1 These features indicate that ICC provides a J. Chaumeil , P. Le Baccon and E. Heard useful approach to study the structure-function 1Curie Institute, UMR 218, Mammalian relationships of chromatin domain surfaces and Developmental Epigenetics Group, Pavillon the interchromatin compartment. Pasteur, 26 rue d’Ulm, 75248 Paris cedex 05, France

PO:06:67 X-chromosome inactivation, the mechanism by Spatial aspects of chromosomal which dosage compensation for the sex chromo- somes between males and females is achieved in inactivation during stem cell mammals, involves the stable silencing and differentiation the heterochromatinization of one of the two 66 15th ICC: Abstracts homologous X chromosomes during early female residues in the histone H3 and H4 amino termini development. It represents a remarkable example such as H3-K9, H3-K27 and H4-K20 has been of epigenetic gene regulation. Coating of the X linked with the formation of constitutive and chromosome by Xist RNA is essential for the facultative heterochromatin as well as with speci- initiation and propagation of X-inactivation. fically repressed single gene loci, whereas methy- However, little is known about the mechanisms lation of H3-K4, H3-K36 and H3-K79 is that transform this signal into transcriptional associated with transcriptionally competent silencing. Using female embryonic stem cells as a euchromatin. Using antibodies directed against system model, where in vitro differentiation is different lysine methylation sites, we visualized accompanied by X-inactivation, we have carried the nuclear distribution pattern of chromatin out an integrated analysis of the order of appear- flagged by these methylated lysines in 3D pre- ance of early events underlying X-inactivation, served nuclei of normal and malignant cell types. showing that multiple histone H3 and H4 mod- Optical confocal serial sections were used for a ifications appear immediately after Xist accumu- quantitative evaluation. We demonstrate distinct lation. This data support an early role for differences of these histone methylation patterns histones in the X-inactivation process and sug- among nuclei of different cell types. Changes in gest that they form at least part of the cellular the pattern formation were also observed during memory of the inactive state on the X chromo- the cell cycle and after exit of the cell cycle. Our some. We present new data showing that the data suggest an important role of methylated his- transcription machinery is excluded from the Xist tones in the reestablishment of higher order chro- RNA coated X chromosome, prior to the onset matin arrangements during telophase/early G1. of histone modifications and gene silencing. We Cell type specific histone methylation patterns have analysed the position and dynamics during are possibly causally involved in the formation of differentiation of several X-linked genes on the X cell type specific heterochromatin compartments, chromosome. We find that there may be a tight composed of (peri)centromeric regions and chro- correlation between the location of genes on the mosomal subregions from neighboring chromo- X chromosome and their activity status during X some territories, which contain silent genes. inactivation.

PO:06:69 PO:06:70 Three dimensional analysis of histone Cytological study of DNA methylation methylation patterns in normal and and of histone methylation state in tumor cell nuclei human retinoblastoma and in blood cells M. Cremer1, R. Zinner1, H. Albiez1,  1 2 2 A. Peters5, T. Jenuwein2, R. Brack-Werner3, M. Skalnkova´ ,E.Ba´rtova´ , S. Kozubek 1 M. Speicher4, K. Pfleghaar4 and T. Cremer1 and M. Kozubek 1 1Faculty of Informatics, Masaryk University, LMU Dept. Biol. II, Munich, Germany; 2 2Institute of Molecular Pathology, Vienna, Botanicka´ 68a, Brno, Czech Republic; Institute Austria; 3GSF, Institute of Molecular Virology, of Biophysics, Academy of Sciences, GSF, Munich, Germany; 4Institute of Human Kra´lovopolska´ 135, Brno, Czech Republic Genetics, Technical University and GSF, Munich, Germany; 5Friedrich Miescher Institute for Epigenetic silencing in eukaryotes is associated Biomedical Research, Basel, Switzerland with the formation of a transcriptional repressive higher-order chromatin structure that is also Histone modifications represent an important characterized by methylation of DNA at cytosine epigenetic mechanism for the organization of nucleotides. Differential methylation of CpG higher order chromatin structure and gene reg- islands has been inversely correlated with gene ulation. Methylation of position-specific lysine activity: transcriptionally active genes were found 15th ICC: Abstracts 67 to be hypomethylated, whereas transcriptionally Prader-Willi/Angelman Syndrome (PWS/AS) in silent genes were hypermethylated (Singal R., human cells (LaSalle and Lalande [1996] Science 1999). 272: 725–728). We re-investigated this ‘kissing In our experiments, the detection of sub-nuclear phenomenon’ using 3D-FISH with locus-specific localization of DNA methylated regions was per- probes and replication labeling for a precise S- formed using indirect immuno£uorescence techni- phase staging. Average relative distances (ARDs) que combined with FISH. The selected metaphase between the two PWS/AS loci were determined chromosomes HSA 6, 10, 11, 18, 19, 20 and 22 were in 3D confocal image stacks. In PHA-stimulated determined using FISH. Sequential 5-methylcytosin lymphocytes ARD was significantly smaller in rich regions were visualised using immuno- late S-phase compared to early S-phase and £uorescent labelling in di¡erent cell types. We have quiescent lymphocytes. A corresponding decrease found, for example, that the HSA 19, 20 and 22 was found for chromosome 15 centromeres, involved heavy methylated regions. On the other which is located in close vicinity to both riboso- hand, HSA 18, 10, 11 and 6 were found to be unme- mal genes on 15p and to the PWS/AS region on thylated in CpG islands in our experiments, which is 15q11-13. In lymphoblastoid cells of a PWS in accordance with Barbin et al., (1994). N-myc patient with a complete deletion of the PWS/AS ampli¢cation detected by PCR/FISH in the retino- region in paternal #15, we noted the same blastoma cell line Y79 was found to be heavy methy- decrease of ARD between #15 centromeres in latedinCpGislands. late S-phase. In gorilla lymphoblastoid cells, Another modi¢cation linked to silencing is however, where the human #15 homolog bears methylation of histone H3 on lysine 9 (H3-K9). the PWS/AS locus but no ribosomal genes, the Domains of euchromatin is characterised by ARD between the two loci remained unchanged methylation of H3-K4 (Mattei M.G., 2003). during S-phase. Significant ARD changes during Therefore, in situ immuno£uorescence, was per- S-phase of lymphoblasts were also not detected oformed using antibody against H3-K4. We for another imprinted locus, the Beckwith-Wiede- observed orientation of methyl groups of H3-K4 mann region on 11q15.5. In summary, our data into the interchromatin space in the interphase provide strong evidence against the postulated nuclei of human K-562 cells. a˜kissing phenomenona¨.

This work was supported by the GA CR (Grant No. 202/03/ D033) and partly also by the Min.Edu CR (Grant No. MSM- Plant Chromosomes Symposium 143300002)

L79 PO:06:71 Dissecting large genomes of cereals by Topology of the imprinted chromosome sorting Prader-Willi/Angelman and J. Dolezel1, M. Kubalakova1, J. Bartos1, Beckwith-Wiedemann Syndrome J. Safar1, J. Janda1, J. Cihalikova1, 1 1 loci in cycling cells P. Kovarova and P. Suchankova 1 K. Teller1, I. Solovei1, K. Buiting2, Laboratory of Molecular Cytogenetics and 2 1 Cytometry, Institute of Experimental Botany, B. Horsthemke and T. Cremer Sokolovska 6, CZ-77200 Olomouc, Czech 1Department of Biology II, Ludwig Maximilians Republic University, Munich; 2Institute of Human Genetics, University of Essen, Essen, Germany Cereals provide a major portion of our diet. While some of them like rice and sorghum Spatial association of oppositely imprinted possess small genomes, the genomes of barley, regions in late S-phase, but not at other stages rye and wheat are complex, consisting mainly of of the cell cycle, was reported for the various classes of repetitive DNA sequences. In 68 15th ICC: Abstracts addition, the recent evolution of common wheat cross-pollinated Ae. speltoides and annual self- involved two episodes of polyploidization. These pollinated Ae. sharonensis are located 30 meters features hamper physical mapping and gene iso- apart on different soil types. Despite the close lation. Purification of individual chromosomes by proximity of the two species and their close relat- flow cytometry can greatly simplify these tasks edness, no mixed colonies are known due to sea- by providing small and defined genome fractions. sonal, ecological, and biological isolation. This lecture will review a development of the Comparative molecular cytogenetic analysis methodology and its potential for genome map- based on intra-population variability of rDNA ping in barley, rye and wheat. Chromosome ana- chromosomal patterns of individual Ae. spel- lysis by flow cytometry permits quantitative toides genotypes revealed directional and rapid detection of structural and numerical chromo- process of chromosomal repatterning. The some changes. Due to small differences in DNA mechanisms for repatterning are heterologous content, only one chromosome can be dis- recombination and/or transposable elements criminated and sorted in each of the species. mediated rDNA transfer. FISH experiments However, cytogenetic stocks facilitate separation revealed the inter- and intra-chromosomal trans- of other parts of the genomes as individual chro- fer of 5S rDNA in complex with En/Spm mosomes and chromosome arms. Sorted chromo- elements. In its turn, arising of a new rDNA site somes have been used for discovery of rare may induce a number of intra-genomic events structural changes and high-resolution cytoge- consequent from heterologous synapses and netic mapping using FISH. The use of sorted recombination between chromosomes that carry chromosomes for HAPPY mapping, targeted iso- extended rDNA arrays. The process is in an lation of low-copy sequences, and high-through- equilibrium state o¨ chromosomal mutations can put physical mapping of ESTs are attractive arise de novo and can be eliminated. Analysis of options. As millions of chromosomes with intact the progeny of the investigated genotypes testifies DNA may be sorted, construction of BAC librar- that inheritance of de novo rDNA sites happens ies is possible. Subgenomic, chromosome-specific frequently. Stabilization of new rDNA clusters is and chromosome arm-specific BAC libraries have achieved by homozygotization. Consequently, already been constructed and represent unique several modified genomic forms intermediate resources for wheat genomics. between Ae. speltoides and Ae. sharonensis per- manently arise in studied wild populations, that This work has been supported by the Grant Agency of the make it possible to recognize Ae. sharonensis as a Czech Republic (grants 522/03/0354 and 521/04/0607), and derivative species of Ae. speltoides, as well as to the Ministry of Agriculture of the Czech Republic (grant QC1336). propose rapidness and canalization of the specia- tion process in Sitopsis species. L80 Speciation-related chromosomal L81 repatterning in a small peripheral plant Variable organization of the terminal population: assumption from rDNA regions in rye chromosomes clusters variability. O. Alkhimova1 and A. Vershinin2 O. Raskina1, E. Nevo1 and A. Belyayev1 1Institute of Molecular Biology and Genetics, 1Institute of Evolution, University of Haifa, Israel Kiev 03143, Ukraine; 2Institute of Cytology and Genetics, Novosibirsk 630090, Russia We present a particular case of the chromosomal repatterning in connection with sympatric specia- Terminal regions, telomeres and subtelomeres, of tion in the Sitopsis section of genus Aegilops rye chromosomes consist mostly of highly repeti- (Poaceae, Monocotyledones). Two small, peri- tive tandemly organized DNA sequences. Little is pheral, isolated, wild populations of annual known about internal structure of tandem arrays, 15th ICC: Abstracts 69 including total array size and the pattern of histone H4, at four lysine residues, K5, K8, K12 monomer distribution within arrays. We addres- and K16. Dynamic changes of histone acetyla- sed these issues for telomere associated hetero- tion throughout mitosis and its relationships to chromatin of individual rye chromosomes, using mitotic chromosome were analyzed using a three- wheat-rye addition lines and rye specific DNA dimensional immunofluorescent method to facil- probes. FISH on meiotic rye chromosomes itate a comprehensive understanding of histone revealed a specific mosaic arrangement of H4 acetylation. Basically two patterns of dynam- domains for each chromosome arm where differ- ics were revealed according to the region specific ent tandem repetitive families localized pre- acetylations and their stages in mitotic barley dominately without obvious tendency in domains cells. One of the pairwise acetylation at K5/K12 order, size and spacer distribution. Each wheat- during mitosis is shown in the nucleolar organiz- rye additional line studied by pulse field gel elec- ing regions (NORs). The NORs escape from full trophoresis produced a chromosome-specific condensation when the whole chromosomes are overall blot hybridization display indicating a fully condensed, pairwise acetylation of K5/K12 unique large-scale organization of the tandem in H4 at the NORs should serve to avoid tight repeat arrays on each rye chromosome. The condensation as found in the other chromosomal FISH signals on DNA fibers showed the multiple regions. The second pairwise acetylation involves patterns of co-linear monomers arrangement of K8/K16, which were observed in the distal repetitive families as well as of authentic telo- regions of chromosomes, although their dynam- meric probe from Arabidopsis. The majority of ics was not identical. The distal regions are cor- the arrays consisted of the monomers of different responding to gene-rich regions, thus this families in various alterations separated by pairwise acetylation should involve in transcrip- spacers. The primary structure of some spacer tional regulation. In addition, the pairwise acet- sequences revealed the scrambled regions of simi- ylation of K5/K12 is involved in acetylation of larity to various known repetitive elements indi- centromeric regions together with K8 acetylation. cating the concerted action of several DNA These findings indicate that combinations of break-repair mechanisms. This level of complex- acetylation correlating chromosome structure ity in the long-range organization of tandem seem to have functional roles in centromere func- arrays has not been previously reported for any tion, and transcription in the NORs and the plant species. The various patterns of the tandem gene-rich regions. Therefore, it is concluded that arrays internal structure is likely to have resulted the combinations of histone H4 acetylation from the evolutionary interplay, array homo- could serve as novel histone codes for chromo- genization and the generation of heterogeneity some structure and related functions during mediated by double-strand breaks (DSBs) and mitosis. associated repair mechanisms. L83 L82 Dynamic analyses of Aurora kinases Dynamic changes of histone H4 in plants acetylation in mitotic cells of barley S. Matsunaga1, A. Kawabe1, K. Nakagawa1, 1 2 T. Wako and K. Fukui A. Yoneda2, D. Kurihara1, S. Uchiyama1, 2 1 1National Institute of Agrobiological Sciences S. Hasezawa and K. Fukui 2 and Graduate School of Engineering, 1 Department of Biotechnology, Graduate School Osaka University, Japan of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Covalent modifications in nucleosome histones 2Department of Integrated Biosciences, play various roles in cellular functions. One of Graduate School of Frontier Sciences, the most important modifications is histone acet- University of Tokyo, 5-1-5 Kashiwanoha, ylation at lysine residues which occurs, in case of Kashiwa, Chiba 277-8562, Japan 70 15th ICC: Abstracts

Aurora kinases are serine/threonine protein kina- 8C, 16C and 32C leaf nuclei up to 100% of ses with essential roles in cell division through nuclei revealed sister chromatid separation. eukaryotes. They regulate the interaction between Simultaneous FISH with BACs from different cytoskelton and chromosomes at the kinetochore, positions along chromosome 1 has shown that the spindle assembly and the cytokinesis. We sister chromatid separation can be punctual and identified three Aurora kinases in Arabidopsis is more frequent in mid arm compared to term- thaliana, AtARK1, AtARK2 and AtARK3. The inal or pericentromeric positions. The high fre- kinase domain of AtARK1 shares 95% and 63% quency of spatially separated sister chromatid amino acid identity with AtARK2 and AtARK3, arms in nuclei with 4C or higher DNA content respectively. There are two transcriptional forms suggests that sister chromatid cohesion along the of AtARK2. A deletion type of transcript entire chromosome arms is not obligatory at (AtARK2S) lacks the fourth exon that encodes a least in differentiated cells. part of kinase domain compared to complete type Di¡erential labelling of chromosome 1 top arm (AtARK2L). These two transcripts were detected territory and BAC T2P11 therein revealed in in almost all tissues but the AtARK2S expressed 5.3% of 4C leaf nuclei separated sister chromatid very low amount. The AtARK2S expressed rela- arm territories and in 38.1% punctual non-c tively higher in roots, leaves and stems. To reveal ohesion at the segment corresponding to the the localization of AtARKs during plant mitosis, T2P11 sequence. In 12.8% of nuclei (n ¼ 359) dynamic analyses was performed using GFP- BAC signals were found to be looped out from fused proteins in tobacco BY-2 cells. The locali- the arm territory. zation of AtARK proteins was classified into two classes. One class showed subcellular localization of mitotic spindles during cell division. The other L85 class showed the localization on chromosomes. Quantum speciation in the wild The localization of AtARK proteins was different from that of animal Aurora kinases, reflecting the Aegilops species on the base of difference in mitotic mechanisms between plant chromosomal rearrangements and animal. directional selectivity A. Belyayev1, O. Raskina1 and E. Nevo1 L84 1Institute of Evolution, University of Haifa, Israel

Sister chromatid non-cohesion is The evolution of diploid and polyploid wheats is common in Arabidopsis thaliana connected with significant remodeling of rDNA interphase nuclei sites. In light of this, we explored deviations in the distribution of 5S and 45S rDNA sites in V. Schubert, M. Klatte1, A. Pecinka1 and 1 natural populations of diploid Sitopsis species of I. Schubert the genus Aegilops (Ae. speltoides, Ae. longissima, 1Institute of Plant Genetics and Crop Plant Ae. searsii, Ae. sharonensis, and Ae. bicornis). Research (IPK), Corrensstrasse 3, D-06466 Chromosomal rearrangements involving 5S and Gatersleben, Germany 45S rDNA sites as well as long arms and the satellites of chromosomes 1, 5, and 6 were deter- It is widely assumed that sister chromatids are mined in homo- and heterozygotes in Ae. spel- cohesed from replication in S phase until the toides populations. These rearrangements are beginning of anaphase. However, FISH with permanent for different populations, and geno- individual BACs (*100 kb) from different posi- types with rearranged rDNA patterns are similar tions along chromosomes 1 and 4 to 4C root and to other sitopsis species. The progeny of plants leaf nuclei yielded frequently more than two carrying modified rDNA patterns mainly fol- hybridization signals indicating non-cohesion of lowed parental genotypes after selfing. However, sister chromatids in 32.5% to 59.6% of nuclei. In we regularly observed both the emergence of new 15th ICC: Abstracts 71 mobile rDNA sites as well as genome ‘‘normal- 1Institute of Plant Genetics and Crop Plant ization’’, i.e., elimination of the additional rDNA Research (IPK), 06466 Gatersleben, Germany sites. Modified rDNA clusters have been stabi- lized in populations in two alternative ways: (i) Post-translational modifications of histone H3 by selfing or crossing with similar genotypes, and have been linked to chromatin dynamics and the (ii) by non-Mendelian epigenetic mechanisms of chromosome segregation for a number of eukar- En/Spm mediated intra-genomic transfer of yotes. Using specific antibodies we have analysed rDNA. Directional selectivity of chromosomal the spatio-temporal distributions of phosphory- rearrangements in wild populations and finding lated H3 at positions serine 10, 28 and threonine intermediates between Ae. speltoides and other 11 during mitosis and meiosis. Interestingly, Sitopsis species genotypes testify existence of cer- although histone H3 and most of its post- tain evolutionary channels leading from the plas- translational modifications are highly conserved, tic Ae. speltoides genome to other Sitopsis a number of significant differences exist between species. animals and plants. The temporal and spatial pattern of histone H3 phosphorylation at serine 28 and serine 10 is similar in plants and the cell L86 cycle-dependent phosphorylation at both serine Molecular cytogenetics and plant positions are likely to be involved in the same process, such as sister chromatid cohesion but genome organization differs between mono- and polycentric chromo- P. Heslop-Harrison1 somes. In contrast, phosphorylation at threonine 11 temporally correlates with condensation of 1 Department of Biology, University of Leicester, mitotic and meiotic chromosomes in plant cells. LE1 7RH, UK In order to gain more insights into the function of histone H3 phosphorylation we have identi¢ed Plant genomes consist of genes (which have Aurora-like kinases in Arabidopsis thaliana.Aur- many similarities over a wide range of species), ora/Ipl1 kinases are characterized for yeast, Dro- low copy and regulatory sequences, and repeti- sophila, Caenorhabditis elegans and a number of tive DNA sequences. The repetitive sequences vertebrates. In this species the function of these include highly conserved units – such as the very kinases is closely linked to microtubule dynamics, abundant retroelements which are found in vir- chromosome segregation and histone H3 phos- tually all organisms. I will discuss the evidence phorylation. To analyse whether the modulation for the major classes of retroelements including of kinase activity in£uences the phosphorylation pararetroviruses in plants, and their evolutionary status of histone H3 and segregation behaviour of mechanisms in the context of chromosomal chromosomes, overexpression/inactivation stu- distribution. I will also discuss the nature and dies of AtAurora-kinases were performed. Expres- evolution of tandemly repetitive DNA, which is sion patterns, in vitro kinase assay and protein rapidly evolution and often specific to one localization analyses suggest that AtAurora kina- genome or group of genomes. ses are involved in the cell-cycle regulated phos- phorylation of histone H3. References to our work are available on www.molecularcyto- genetics.com, and I thank CREST, British Council, IAEA- Tropical Fruits and EU FP VI for support of aspects of the L88 work. Genetic and epigenetic consequences of a paracentric inversion in Arabidopsis L87 thaliana Histone H3 phosphorylation and P. Fransz1, G. Linc1, H. Ali2, I. Schubert2, chromosome dynamics in plants J. Wennekes3, H. De Jong3, M. Koornneef3, A. Houben1 and D. Demidov1 J. Peters4 and T. Gerats4 72 15th ICC: Abstracts

1University of Amsterdam, The Netherlands; 1Research Institute for Bioresources, Okayama 2IPK Gatersleben, Germany; 3Wageningen University, Kurashiki 710-0046, Japan.; 2Core University, The Netherlands; 4University of Research for Evolutionary Science and Nijmegen, The Netherlands Technology, Japan Science and Technology, Kawaguchi 332-0012, Japan Heterochromatic knobs are microscopically visi- ble as islands of compact chromatin on chromo- The 180-bp family of tandem repetitive sequences, some arms. The model plant Arabidopsis has a which comprises the major centromeric satellite in unique 700 Mb heterochromatic knob, hk4S, Arabidopsis thaliana, is thought to play important which is completely sequenced. So far, only two roles in kinetochore assembly. However, no direct accessions, Columbia (Col) and Wassilevskija evidence supporting this assertion has been (Ws), contain this heterochromatic island. We obtained, to date. In the cultured cells of A. thali- demonstrated by FISH that hk4S originated ana, drastic changes in chromosome number and from the pericentric heterochromatin after a structure have occurred; nevertheless no chromo- paracentric inversion event. Here we investigated somes without 180-bp repeats were found. To the extent of the inversion and the genetic and assess the centromere activities of the 180-bp epigenetic consequences. We applied FISH with repeats, we performed indirect fluorescence immu- BACs that span the region from the knob to the nolabeling with antibodies against AtCENP-A or pericentric heterochromatin and compared the HTR12 (Arabidopsis centromeric histone H3 var- knob accessions, Col and Ws, with knobless iant) and AtCENP-C (Arabidopsis CENP-C homo- accessions, Ler and C24. The FISH data confirm logue). The immunosignals from those antibodies a paracentric inversion event in chromosome arm appeared on all sites of the 180-bp repeats detected 4S. The inversion is flanked by the BAC regions by fluorescence in situ hybridization, except a num- F9H3 and C6L9 at the distal border of the knob ber of minor sites between the two major sites on and the pericentric heterochromatin, respectively. putative dicentric chromosomes. In addition to The inversion covers more than 1200 kb and these colocalizations of the antibodies with the 180- includes euchromatic and heterochromatic seg- bp repeats, anaphase-bridge formation by the puta- ments. By fiber-FISH we could finemap the distal tive dicentric chromosomes carrying two 180-bp breakpoint of the inversion at the proximal side sites indicated the close relationship between the of F9H3. The results indicate that the para- 180-bp repeats and centromere activity. However, centric inversion is followed by a 20 kb deletion some of the 180-bp repeat clusters, particularly in the F9H3 region of the knob accessions, and those that were long or stretched at interphase, led to a decrease in genetic recombination events. were not fully covered with the signals from anti- Moreover, AFLP analysis of the inversed region HTR12 or AtCENP-C. Chromatin fiber immuno- showed reduced polymorphism between Col and labeling clearly revealed that the centromeric pro- Ws compared to flanking regions, suggesting that teins localize only at the knobs on the extended the inversed region has been genetically stable chromatin fibers, which form a limited part of the since the inversion event. Finally, the inversion continuous 180-bp clusters. The chromatin immu- has altered the heterochromatin status of some noprecipitation assay using anti-AtCENP-A sug- segments and created new borders between gested that this sort of differential localization of heterochromatin and euchromatin. the centromeric proteins is caused by variation in DNA sequences of the 180-bp repeats.

L89 L90 Differential localization of the The centromere of rice chromosome 8 centromere-speci¢c proteins in spans *750kb of DNA and contains Arabidopsis thaliana active genes M. Murata1, F. Shibata2 and H. Sato2 J. Jiang1 15th ICC: Abstracts 73

1Department of Horticulture, University of Enhancer/Suppressor-mutator (En/Spm) trans- Wisconsin-Madison, Madison, WI 53706, USA posons belong to class II of transposable ele- ments (TE). They can be activated several times The centromeres in most multicellular eukar- in one generation and there is evidence that yotes are embedded within cytologically dis- duplication and insertion of transposons can tinctive heterochromatin and are associated occur at low levels in somatic and pre-germinal with long tracts of repetitive DNA. Thus cen- cells, but increases during meiosis. En/Spm tromeric regions typically do not contain active transposons are known to be linked with genes genes and are associated with suppression of and their expression, and transposition is con- genetic recombination. Centromeres often con- trolled by interacting auto regulatory and/or epi- tain megabase-sized array of satellite genetic mechanisms and is, therefore, relatively DNA, which resist mapping, cloning and independent of their chromosomal localization. sequencing. Rice centromeres contain a 155-bp Indeed FISH analysis of En/Spm transposons satellite repeat CentO that varies from *60 kb distribution on mitotic and meiotic chromosomes to 2 Mb among different centromeres. We of four wild Triticeae species namely Triticum sequenced a *1.5 Mb DNA contig that encom- monococcum, Triticum urartu, Aegilops speltoides passes the cytologically defined centromere of and Hordeum spontaneum reveal mobile cluster- rice chromosome 8 (CEN8). The CEN8 ing of En/Spm through both somatic and meio- sequence was successfully determined largely tic chromosomes. Nevertheless, our data indicate because of its unusually low abundance of the that there are certain preferences in En/Spm CentO satellite. A *740kbregionwithin transposons insertions. The majority of the huge CEN8 binds rice CenH3, the centromere-spe- En/Spm clusters are associated with 5S rDNA cific H3 histone. Surprisingly, thirteen predicted sites while no association with 45S rDNA sites and at least three active genes are interspersed has been detected. Second peculiarity is that this in the CenH3-binding domain. The retro- type of TE predominantly associated in mitosis transposons located within and outside of the and meiosis with mobile 5S rDNA sites on A CenH3-binding domain show similar ages and and B chromosomes, and part of de novo arisen structural dynamics. Thus CEN8 shows a dif- sites can be inherited. These data testify distinct ferent DNA structure from the centromeres of role of En/Spm transposons in the ongoing pro- most other model complex eukaryotes. We pro- cess of intraspecific chromosomal repatterning, pose that CEN8 may represent an intermediate which, in its turn, could lead to new character stage as centromeres evolve from genic regions, combinations and therefore to novel genetic as in human neocentromeres, to fully mature lineages-the main source for the speciation centromeres that accumulate megabases of process. homogeneous satellite arrays.

PO:06:73 PO:06:72 Chromosomal mapping of N-genome En/Spm-like transposons in wild speci¢c DNA sequences in the Triticeae species: preference to a Triticeae certain chromosomal regions K. Anamthawat-Jonsson1 1 2 2 A. Altinkut , O. Raskina , E. Nevo and 1 2 University of Iceland, Department of Biology, A. Belyayev Askja, Sturlugata 7, Reykjavik 101, Iceland 1Tubitak, Research Institute for Genetic Engineering and Biotechnology, 41470, Gebze, N-genome specific sequences (pLmIs1,pLmIs44 Kocaeli, Turkey; 2Institute of Evolution, and pLmIs54) were isolated from two species of University of Haifa, Mount Carmel, Haifa, 31905, Leymus Hochst. (Poaceae: Triticeae): tetraploid Israel northern American/Pacific L. mollis (Trin.) 74 15th ICC: Abstracts

Pilger, and octoploid northern European L. are- phylogenetic and systematic studies. The results narius (L.) Hochst. They are retroelement-like in the present study are based on twelve hepatics sequences and are found essentially in Leymus species collected in Poland. Chromosome num- and Psathyrostachys Nevski. Fluorescence in situ bers for five species had not been previously hybridization (FISH) experiment show dispersed counted. A comparison between chromosomes of pattern of sequence distribution on all Leymus three sibling species was made. Different techni- chromosomes, except at centromeres, telomeres ques were used to prepare gametophytic mitosis and NORs as expected. Southern genomic in situ for examination in all these species. For the hybridization experiments show that both genera small chromosomes (Conocephalum, Nardia, contain only the N-genome of Psathyrostachys, Cephalozia), the best results were obtained with no other Triticeae genomes are involved, and the DAPI staining method. Chromosomes with therefore the polyploid genus Leymus can only big heterochromatin fragments (e.g. Pellia, be autopolyploid or segmental allopolyploid con- Aneura) were analyzed and compared using the sisting of some variation of the basic N-genome. Giemsa C banding method. This method allows This molecular and cytogenetic evidence clearly us to distinguish chromosomes between sibling support the taxonomic integration of the diploid species from the Pellia epiphylla complex and to genus Psathyrostachys into Leymus. The poly- confirm that they are parental species of Pellia morphism within these sequences is found to be borealis-tetraploid liverwort originated through informative and reflecting phylogenetic relation- allopolyploidysation. In conclusion, different ships among the N-genome species investigated. cytological techniques are capable of providing We are now investigating genomic and genetic answers to many important questions, such as relationships among species of Leymus from both the origin of polyploid species or taxonomic Eurasia and N-America, in comparison to species status of cryptic species. of Psathyrostachys and a number of related taxa, using mainly the N-genome specific sequences isolated here. The N-genome species examined so PO:06:75 far form distinct cluster-group separated from other Triticeae genomes. The tetraploid wood The large-scale organization of Beta barley Hordelymus europaeus (Jessen) Harz is centromeres now shown to be one of the N-genome species, 1 1 1 Leymus D. Dechyeva , B. Weber , C. Dombrowski , presumably . The paper will also describe 1 1 1 molecular distribution, and mobility, of the T. Wenke , G. Menzel and T. Schmidt gypsy-like retroelement pLmIs44 in synthesized 1Institute of Botany, Technical University of hybrids between wheat (Triticum L.) and Ley- Dresden, D-01062 Dresden, Germany mus. The DNA composition of plant centromeres dis- PO:06:74 plays a high degree of variation, however, their long-range sequence organization apparently fol- Chromosome numbers of some Polish lows similar structural rules. Most plant cen- liverworts from genus Aneura, Pellia tromeres consist predominantly of highly and Conocephalum (Hepaticeae) repetitive DNA sequences. Characteristic sequence landmarks in the centromeric hetero- E. Chudzinska1 chromatin are rare, making the molecular analy- 1Adam Mickiewicz University, Poznan, Poland sis of these regions very difficult. To analyze a single centromere, we used mono- Material on studies in bryophyte cytology had somic chromosome fragments (minichromo- been relatively limited in Central Europe. Fur- somes) found in interspeci¢c Beta hybrids such as thermore only about 70 percent of species have a PRO1 and PAT2. These minichromosomes origi- designated chromosome number despite the fact nating from the wild beet Beta procumbens and that karyological analysis are very important in Beta patellaris, respectively, are stably maintained 15th ICC: Abstracts 75 in sugar beet (Beta vulgaris) and provide an experi- pairs of chromosomes, 11 of which were pre- mental system towards the molecular isolation of viously indistinguishable based solely on size. individual plant centromeres. Combinations of different probes and DAPI Beta centromeres have a complex structure and stain have made it possible to uniquely dis- consist of variable repetitive sequences, in parti- criminate among all 12 pairs of loblolly pine cular satellite DNA and retrotransposons. By chromosomes. A similar karyotype was devel- BAC sequencing and high-resolution FISH on oped for slash pine for direct comparison with pachytene chromosomes and extended DNA loblolly pine as well as for comparison with a ¢bres we analyzed the centromeric DNA of the previously published slash pine karyotype. This PRO1 and PAT2 minichromosomes. has allowed us to identify homologous chromo- Centromeric BACs contain highly ampli¢ed somes between loblolly and slash pines. Seven Ty3-gypsy retrotransposons as demonstrated by intercalary and ten proximal 18S rDNA sites the existence of conserved functional domains were identified. All intercalary 18S rDNA sites and the presence of LTRs (Long Terminal appeared to be major sites. Chromosome 1, con- Repeats). Most copies of the Ty3-gypsy retro- tained an intercalary major 5S rDNA site. Two transposons Beetle1 (6738 bp) are nested, rear- other independent metacentric pairs of chromo- ranged or inserted into Beetle2 (6684 bp). somes showed distal minor 5S rDNA sites. All The arrays pTS5 and pTS4.1 satellite of PRO1 but one chromosome showed DAPI positive have a length of approximately 350 kb and bands. Telomere repeat signals were observed, as 300 kb, respectively. Assuming that pTS5 is the expected, towards the end of each chromosomal functional centromeric satellite, the PAT2 cen- arm. With the exception of one or possibly two tromere is contained in 47^50 kb as shown by pairs, minor to major telomere signals were restriction analysis and might be represented by a observed near or around the centromeres. Statis- single BAC clone. tical analysis on chromosome arm lengths (short and long), and intercalary telomeric and rDNA This work is funded by grants from the BMBF (BioFuture positions has been carried out. Efforts are cur- grant 0311860). rently underway to combine our linkage maps with the karyotypes to provide a fully integrated cyto-molecular map. PO:06:76

Developing a standard karyotype for PO:06:77 loblolly pine using FISH and AT-rich banding Molecular cytotaxonomic study of 1 2 2 a new model grass, Brachypodium N. Faridi , C. Nelson and T. Kubisiak distachyon 1 USDA Forest Service, Southern Institute of 1 2 2 Forest Genetics, Forest Tree Molecular R. Hasterok , J. Draper and G. Jenkins Cytogenetics Lab, 1042 Agronomy Rd., College 1Department of Plant Anatomy and Cytology, 2 Station, TX77843, USA; USDA Forest Service, University of Silesia, Jagiellonska 28, 40-032 Southern Institute of Forest Genetics, Harrison Katowice, Poland; 2Institute of Biological Experimental Forest, Saucier, MS39574, USA Sciences, Edward Llwyd Building, University of Wales, Aberystwyth, Penglais, Aberystwyth, A standard karyotype has been developed for Ceredigion, SY23 3DA, UK loblolly pine based on fluorescent in situ hybridi- zation (FISH) using various labeled probes such Brachypodium distachyon has been proposed as as 18S-28S rDNA, 5S rDNA, Arabidopsis-like an alternative model organism to rice, with telomere repeat, and DAPI positive bands. potential for functional genomic analysis of tem- Somatic chromosomes were prepared from col- perate forage grasses and cereals. Its major chicine treated root meristems using an enzy- advantages are favourable phylogenetic matic digestion technique. Loblolly pine has 12 position as a ‘bridge species’ between tropical 76 15th ICC: Abstracts and temperate cereals, a genome as small and as SY23 3DA, UK; 4Institute of Grassland & economical as that of A. thaliana, short life cycle, Environmental Research, Plas Gogerddan, undemanding growth requirements, inbreeding Aberystwyth, Ceredigion SY23 3EB, UK habit and many other useful features. On taxo- nomic grounds Brachypodium is a genus of Brachypodium distachyon is a ubiquitous grass widely distributed temperate grasses with rela- species of ancient origin that occupies a dis- tively few species, two features indicating a very tinctive phylogenetic position before the radia- ancient origin. B. distachyon has a somatic chro- tion of the temperate cereals and grasses. Its mosome number of 10, but ecotypes have been highly desirable biological attributes such as an identified with multiples of this number. These exceptionally compact genome, inbreeding habit prompted taxonomists to suggest that this species and short generation time, are being exploited in had evolved a polyploid series based upon a new functional genomic initiative to use B. dis- 2n ¼ 2x ¼ 10, and that ecotypes which deviate tachyon as a ‘bridge’ species to gain access to from multiples of 10 are aneuploid. In order to important syntenic regions of the genomes of less confirm the genomic constitution of five putative tractable relatives such as wheat. As part of the polyploid ecotypes, we undertook molecular development of a functional genomics toolkit for cytogenetic analyses using fluorescence in situ this species, a BAC library has been constructed. hybridization (FISH) with total genomic (GISH) The library contains about 8,700 clones, with an and ribosomal DNA probes. The results average size of about 80 kbp, giving about 4.5 obtained challenge the assumption that the series times genome coverage. Regions in the rice gen- evolved simply by chromosome doubling and ome database have been identified that are synte- suggest that the ecotypes with 30 chromosomes nic to loci controlling agronomically important are in fact allotetraploids arising from inter- traits in wheat, such as disease resistance and specific hybridization between B. distachyon heading date. Primers of conserved regions have (2n ¼ 2x ¼ 10) and another Brachypodium species been designed and used to identify by PCR (2n ¼ 2x ¼ 20) with a genome resembling that of BACs containing orthologous regions in B. dis- a related diploid species, B. sylvaticum. tachyon, with a view to demonstrating the utility of this species for gene isolation. The level of The authors acknowledge financial support from the Royal microsynteny will be assayed by sequencing. Society (Joint Project 2001–2002 to R.H. and G.J.) and the BACs containing low copy or unique sequences Biotechnology and Biological Sciences Research Council (ISIS are also being anchored by FISH to the 10 chro- award to R.H. and J.D. 2003–2004). mosome arms of this species, and ‘landed’ on somatic chromosomes of wheat, rye and rice, in order to reconstruct the archetypal grass genome. PO:06:78 A bacterial arti¢cial chromosome The authors acknowledge financial support from the Royal Society (Joint Project 2001–2002 to R.H. and G.J.) and the (BAC) library for a new model grass, BBSRC (ISIS award to R.H. and J.D. 2003–2004). Brachypodium distachyon R. Hasterok1, A. Marasek2, G. Jenkins3, I. Donnison4, A. Thomas4, I. King4 and PO:06:79 3 J. Draper Genotoxic potential of soil of Amritsar 1 Department of Plant Anatomy and Cytology, using chromosomal aberration assay in University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland; 2Department of Biotechnology Allium cepa of Ornamental Plants, Research Institute of J. Katnoria1, A. Nagpal1 and R. Bhardwaj1 Pomology and Floriculture, 96-100 Skierniewice, Poland; 3Institute of Biological Sciences, Edward 1Dept. of Botanical and Environmental Sciences, Llwyd Building, University of Wales, Guru Nanak Dev University, Amritsar, Punjab, Aberystwyth, Penglais, Aberystwyth, Ceredigion, India 15th ICC: Abstracts 77

The environmental contamination with heavy Haemodoraceae was carried out using Feulgen metals has increased drastically in recent years. staining and fluorescence in situ hybridization Soils polluted with heavy metals can cause phy- (FISH). The somatic metaphase chromosome totoxicity and exhibit impaired microbial activ- number was 2n ¼ 2x ¼ 22 and the size of chromo- ities. Apart from being highly toxic to biological somes ranged from 1.27 to 3.80. Three pairs of systems, the subtle danger of presence of heavy chromosomes were relatively long in total length metals in soil lies in their being genotoxic. As and the others were short. Chromosome comple- plant bioassays are most sensitive in detecting ments comprise eight pairs of metacentic (chro- genotoxicity of environmental agents and serve mosome 2, 3, 6, 7, 8, 9, 10, and 11), two pairs of as the first alert for the presence of environ- submetacentric (chromosome 4 and 5), and one mental hazards, the present study was planned to pair of subtelocentric (chromosome 1). Using evaluate the genotoxic potential of extracts of FISH, the 17S and 5S genes were physically soil samples collected from different agricultural mapped on the metaphase chromosomes. Two fields of Amritsar (31370Nand74520E), pairs of 17S signal were detected on the terminal employing Chromosome Aberration Assay in regions of the short arms of homology 1 and 3. Allium cepa. This assay was validated in 1991, by One pair of 5S signal was detected on the short the International Programme on Chemical Safety arm of chromosome 3. This 5S site seemed to be (IPCS) under auspices of World Health Organi- the same locus as one of the 17S rDNA. sation and the United Nations Environment Pro- gramme. Extracts of soil samples were prepared Acknowledgement: This research was supported by a grant by suspending soil in distilled water in ratio of (Code #: PF001201-03: Pl J. W. Bang) from Plant Diversity 1:2 (w/v) for about 12 hours at room tempera- Research Center of 21st Century Frontier Research Program ture. Different concentrations of extract (10%, funded by Ministry of Science and Technology of Korean 25%, 50%, 75% and 100%) were used for treat- government. ment of roots. The extracts were found to be sig- nificantly genotoxic at higher concentrations. The genotoxic potential of the soil samples was corre- PO:06:81 lated with content of heavy metals like: chro- The distribiution of repetitive DNA mium, cobalt, copper, manganese, mercury, sequences in Chenopodium species nickel and zinc. A significant increase in ana- 1 2 2 phase aberration frequency was observed in roots B. Kolano , B. Gardunia , E. Jellen ,M. of Allium cepa with an increase in concentration Stevens2 and J. Maluszynska1 of heavy metals of soil extracts. Other physico- 1Department of Plant Anatomy and Cytology, chemical parameters like: pH, alkalinity, water University of Silesia, Katowice, Poland; holding capacity, bulk density and moisture con- 2Department of Plant and Animal Sciences, tent were also studied. Brigham Young University, Provo, Utah, USA

Domesticated Chenopodium species have recently PO:06:80 attracted interest as seed and fodder crops due to their nutrition value and wide adaptability to dif- Karyotype analysis and physical ferent ecological niches. Despite this increased mapping of 17S and 5S rDNA sites in interest the knowledge of their nuclear genome Anemarrhena asphodeloides Bunge organisation is still limited and need more 1 1 1 detailed studies. S. Kim , W. Lee and J. Bang Genomic organization of three kinds of repeti- 1Department of Biology, Chungnam National tive sequences: satellite, retroelement-like and University, Daejeon 305-764, Korea transposone-like were characterised in a C. qui- noa. Fluorescent in situ hybridization revealed Cytogenetic analysis of a medicinal plant Ane- that most of the mobile element-like sequences marrhena asphodeloides Bunge belonging to were localized mainly in pericentromeric region. 78 15th ICC: Abstracts

Interstitial or terminal localization occurred more technique were carried out. In In FISH experi- rarely. Satellite sequences were observed as a ment, one pair of 5S rDNA signals was detected week but discrete signals dispersed throughout on the pericentromeric region of chromosome 4 whole chromosome arms. and one pair of 45S rDNA signals was detected To compare C. quinoa genome organization on the telomeric region of chromosome 2 in B. with related species, hybridization experiments longeradiatum. In Angelica species, the numbers were conducted with the same probes on nuclear and size of 45S rDNA signals were varied genome of C. berlandieri (domesticated and wild between two species, however each signal of the subspiecies) and C. album. The distribution of the 5S rDNA was observed in two species, A. gigas sequences analysed by Southern hybridization and A. acutiloba. revealed that most of the DNA clones were pre- sent in C. berlandieri genome but not in C. album Acknowledgments: This work was supported by a grant from what may suggest that C. quinoa is closer related BioGreen 21 Program, Rural Development Administration, Republic of Korea. with C. berlandieri than with C. album.Somedif- ferences in genome organization between culti- vated and wild C. berlandieri subspecies were also shown. FISH to C. berlandieri chromosomes PO:06:83 exhibited very similar hybridization pattern to Chromosome sorting in durum wheat this observed in C. quinua but number and strength of signals were reduced. as a tool for wheat genome analysis M. Kubalakova1, P. Kovarova1, The research was supported by the Polish State Committee P. Suchankova1, J. Bartos1, J. Cihalikova1, for Scientific Research, grant No. 3 P04C 006 24, the Ezra T. 2 3 4 Benson Institute, and the McKnight Foundation. S. Lucretti , N. Watanabe , S. Kianian and J. Dolezel1 1Laboratory of Molecular Cytogenetics and PO:06:82 Cytometry, Institute of Experimental Botany, Cytogenetic analysis of eight species in CZ-77200 Olomouc, Czech Republic; 2ENEA, U.T.S. Biotecnologie per la Protezione Umbelliferae della Salute e degli Ecosistemi, 00060 S.M. di 3 D. Koo1, Y. Lee1 and J. Bang1 Galeria (Roma), Italy; Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan; 1Department of Biology, Chungnam National 4Department of Plant Sciences, University, Daejeon 305-764, Korea North Dakota State University, Fargo, ND 58105, USA Chromosome morphology can be used to clarify the relationships between species. Karyotypes Molecular analysis of the wheat genome has been and physical mapping of rDNAs as chromosome hampered by its large size and polyploid nature. marker were investigated from eight medicinal Previously, we demonstrated that flow cytometry plant species in Umbelliferae. Karyotype for- facilitated this task in hexaploid wheat by purify- mulas are as follows: Angelica acutiloba: ing chromosomes and chromosome arms. This K(2n) ¼ 22 ¼ 7m þ 1sm þ 3st; A. sinensis: K(2n) ¼ study evaluates the potential of flow cytometry 22 ¼ 6m þ 3sm þ 2st; A. gigas: K(2n) ¼ 22 ¼ for chromosome sorting in durum wheat (Triti- 5m þ 6sm; Bupleurum falcatum: K(2n) ¼ 26 ¼ cum durum,2n¼ 4x ¼ 28). Histograms of fluores- 12m þ 1sm; B. longeradiatum: K(2n) ¼ 12 ¼ 3m þ cence intensity (flow karyotypes) obtained after 3sm; Cnidium officinale: K(2n) ¼ 3x ¼ 33 ¼ the analysis of DAPI-stained chromosomes con- 5m þ 4sm þ 2st; Ostericum koranum: K(2n) ¼ 22 ¼ sisted of three peaks. Of these, one represented 4m þ 4sm þ 3st: O. karanum cv. Suwon: K(2n) ¼ chromosome 3B, a small peak corresponded to 3x ¼ 33 ¼ 6m þ 3sm þ 2st. Chromosomal localiza- chromosomes 1A and 6A, and a large peak tion of 5S and 45S rDNAs using multi-color represented the remaining eleven chromosomes. fluorescence in situ hybridization (McFISH) Chromosomes sorted onto microscope slides were 15th ICC: Abstracts 79 identified after fluorescence in situ hybridisation difference in chromosomal structure among (FISH) with probes for GAA microsatellite, the species. On the other hand, repetitive pSc119.2 and Afa repeats. Genomic distribution sequences of 45S rDNA were also mapped to of these sequences was determined for the first distal region(s) of chromosome(s) in O. sativa. time in durum wheat and it was found similar to Two-colored FISH of Os48 and rDNA was that of A- and B-genome chromosomes of hex- also carried out to determine the correlation aploid wheat. FISH with a probe for GAA between their loci and the difference in chro- microsatellite permitted unambiguous identifica- mosomal structure among japonica and indica tion of all chromosomes within the karyotype. varieties. These results indicated first, that Flow karyotyping in double ditelosomic lines of intra-species cytological diversity has accumu- durum wheat revealed that the lines could be lated in japonica and in indica rice and sec- used to sort any arm of the hexaploid wheat A- ond, that the molecular cytological diversity and B-genome chromosomes. Compared to hex- was narrow among japonica varieties, but aploid wheat, flow karyotype of durum wheat is broad among indica varieties. less complex. This results in better discrimination of telosomes and high purities in sorted fractions, approaching 100%. This work has been supported PO:06:85 by the Ministry of Education, Youth and Sports of the Czech Republic (grants ME527 and Meiotic studies of Brassica napus ME528) and the Ministry of Agriculture of the cultivars Czech Republic (grant QC1336). Z. Nourmohammadi1 and M. Sheidai1 1Biology Department, Shahid Beheshti University, PO:06:84 Tehran, Iran Molecular cytological diversity was Meiotic study was performed in 22 Brassica narrow among japonica varieties in napus cultivars considering chiasma frequency cultivated rice Oryza sativa and distribution as well as chromosome pairing. All cultivars possessed n ¼ 19 chromosome num- 1 S. Nakayama ber (4x) and showed a deviant course of pro- 1National Institute of Agrobiological Sciences, phase-I meiosis with synezetic knot and post Tsukuba, Japan pachytene diffuse stage. Chromosome stickiness occurred in most of the cultivars from early pro- In cultivated rice Oryza sativa, broad intra- phase to late telophase-II leading to the forma- species diversity has been demonstrated by tion of laggard chromosomes and micronuclei. physiological and molecular biological studies, The cultivars studied differed significantly in but a few papers have reported on molecular chiasma frequency and distribution as well as cytological diversity. To reveal the molecular bivalent formation indicating their genomic dif- cytological diversity in O. sativa, a tandem ferences. Cluster analysis and ordination based repetitive sequence Os48 (Wu and Wu 1987) on principal components analysis grouped those was visualized by fluorescence in situ hybridi- cultivars showing meiotic similarities. Some of zation (FISH) with tyramide signal amplifica- the cultivars showed the occurrence of unreduced tion in various rice varieties, including meiocytes and pollen grains. temperate and tropical varieties of japonica and boro and aman varieties of indica. Diver- sity was reflected by differences in the number PO:06:86 of FISH signals. The number of loci detected Cytological analyses in a fertile hybrid was almost the same among japonica varieties, but differed significantly among indica vari- between Vitis vinifera cv. Italia and eties. The difference in Os48 loci reflected a V. rotundifolia cv. Regale 80 15th ICC: Abstracts

N. Pierozzi1, C. Pinto Maglio1 and 1Chromatin, Inc, USA; 2The University of C. Pommer2 Chicago, USA 1 CPD Recursos Gene´ticos Vegetais. Instituto Integrative plant transformation has accelerated Agronimico (IAC). Campinas SP Brasil. the introduction of novel traits into plants and CEP:13001-970. PO Box 28.; 2CAPTA Frutas enabled the elucidation of gene function. But, (IAC) traditional transformation methods, including Agrobacterium mediated transformation and Vitis vinifera and V. rotundifolia were crossed in mechanical bombardment, result in imprecise an attempt to obtain hybrids with disease resis- gene integration into the plant host genome with tance present in the latter species. Several F1 uncertainty concerning transgene integrity, loca- seedlings were obtained, but only one survived tion, or copy number. The result is often unpre- (IAC 3-10) which was cytologically analyzed. dictable transgene expression, disruption of the Chromosome counts were performed using acetic host plant genome with uncertainty concerning orcein; chromosome characterizations were done transgene integrity, location, or copy number by C-band, -NOR and CMA3 techniques. Pollen that may only be eliminated after multiple gen- counts and measure were done by Alexander erations of event selection and breeding. To over- method. Leaf stomata number and measure were come these obstacles we have developed an obtained by cellulose acetate technique. It was autonomously replicating plant mini-chromo- confirmed 2n ¼ 38 for V. vinifera and 2n ¼ 40 for some that allows for more predictable, reliable, V. rotundifolia. The hybrid, however, showed and efficient introduction of genes into crop spe- 2n ¼ 38 chromosomes, an unexpected number. cies without gene integration into the host gen- The ideograms obtained showed little difference ome. Species-specific genomic DNA conferring among them. The C-band was concentrated centromere function were combined with a multi- around centromere. There are two pairs of chro- gene cassette to form autonomous plant mini- mosome with NOR in each species, after Ag- chromosomes which were delivered into cultured NOR and CMA3. Pollen grain viability mean plant cells. Whole plants were regenerated after value was 75.72% and grain size mean value was expansion of cell cultures and subsequently 19.87 1.51 mm, resembling those of V. vinifera. demonstrated to contain autonomous mini- Leaf stomata number mean values per squared chromosomes with consistent gene expression in area were 122.93 1.14 for V. vinifera, differentiated tissues. These results demonstrate 386.77 2.16 for V. rotundifolia and that autonomous plant mini-chromosomes can be 255.73 2.56 for the hybrid. Leaf stomata constructed from isolated plant genomic DNA to length mean values were 31.63 2.03 mm for V. produce well defined, heritable and stable linkage vinifera and 23.12 1.80 mm for V. rotundifolia groups supporting predictable gene expression in and a mix of both stomata length in the hybrid. plants without integration into the host genome. IAC 3-10 presents a breakthrough in table grape culture in Brazil due to its potential use directly in super humid regions, if proved resistant, or as an elite parent for the breeding program con- PO:06:88 tinuity, or as rootstock for elite grape cultivars. Chromosome and genome size Financial support: FAPESP. evolution in holoparasitic Orobanche (Orobanchaceae) and related genera PO:06:87 H. Schneeweiss1, J. Greilhuber2 and 3 Plant autonomous chromosome G. Schneeweiss transfer 1Department of Higher Plant Systematics and 1 1 1 1 Evolution, Institute of Botany, University of G. Rudgers , J. Mach , J. Jin , H. Zieler Vienna, Rennweg 14, A-1030 Vienna, Austria; and D. Preuss2 2Department of Systematic Karyology 15th ICC: Abstracts 81 and Embryology of Higher Plants, Institute of 1Biology Department, Shahid Beheshti University, Botany, University of Vienna, Rennweg 14, Evin, Tehran, Iran A-1030 Vienna, Austria; 3Department of Plant Biogeography, Institute of Botany, University of Cytogenetic studies were performed on about 40 Vienna, Rennweg 14, A-1030 Vienna, Austria Iranian pomegranate (Punica granatum L.) culti- vars considering the polyploidy level, chiasma Distribution of basic chromosome numbers and frequency and distribution, chromosome associa- karyotypes in Orobanche and related genera sup- tion and segregation, occurrence and effects of ports three lineages inferred from molecular phy- B-chromosomes and cytogenetical mechanisms of logenetic analyses. Orobanche sect. Orobanche unreduced (2n) gametes. All cultivars possessed ¼ plus Diphelypaea (Orobanche-group) have x 19 n ¼ 8 ¼ 2x chromosome number mainly formed and their chromosomes are small to medium- bivalents in metaphase but in a few of them sized mostly meta- to submetacentrics. Oro- quadrivalents were formed due to the occurrence banche sects. Gymnocaulis, Myzorrhiza and Trio- of translocation mainly between the largest chro- ¼ nychon (Phelipanche-group) have x 12 and mosome pair of the genome and one of the small small or medium-sized submeta- to acrocentric chromosome pairs. The cultivars studied differed ¼ chromosomes. Cistanche has x 20 with large significantly in their cytogenetical characters indi- meta- to submetacentric chromosomes. Genome cating their genomic difference. B-chromosome size estimates, newly obtained for 44 taxa (more (0-6) occurred in some of the cultivars which than 70 accessions) using Feulgen densitometry, were much smaller than the A-chromosome and agree with molecular phylogenetic hypotheses, did not pair with them. The effects of Bs on e.g., the phylogenetically distinct O. anatolica has chiasma frequency and distribution varied in dif- much larger chromosomes and thus higher DNA ferent cultivars and was related to the genotypic content than other species of sect. Orobanche. background of the cultivar. In some of the culti- Polyploidy is found in several lineages. In most vars unreduced (potential 2n) meiocytes and pol- cases, polyploids have signi¢cantly lower DNA len grains were formed due to cytogenetical content than related diploid species. For instance, abnormalities such as multipolar cell formation genome size of tetraploid species of sect. Myzor- and cytomixis. Details of such cytogenetic phe- rhiza is 2.16^2.53 pg (2C; corrected for ploidy nomena will be discussed. level) compared to 6.76^10.84 pg in the diploid species of sect. Trionychon. The mostly tetraploid Orobanche gracilis and relatives have the smallest PO:06:90 genomes of all species investigated (1.66^2.45 pg). Effect and exploitation of rye genome Genome size in the tetraploid cytotype of O. trans- caucasica is nearly twice as high as in the diploid in wheat improvement cytotype, suggesting rather recent polyploidisa- M. Sidhu1 and C. Satija2 tion. Polyploid Orobanche exhibit a clear trend 1 towards sequence elimination. Another evolutio- Department of Botany, Panjab University, Chandigarh, India; 2Department of Genetics and narily important trend is that species attacking Biotechnology, PAU, Ludhiana, India short-lived hosts, among them economically important weeds, tend to have lower DNA con- Rye genome is known to carry genes for resis- tents. tance to diseases and adaptability to abiotic stresses. Wheat-rye disomic/ditelosomic addition PO:06:89 lines have been used to incorporate specific rye chromosomes/part of chromosome to wheat. Cytogenetic features of Iranian The hybrid progenies of elite wheat genotypes pomegranate (Punica granatum L.) with wheat-rye disomic/ditelosomic addition lines in Chinese Spring background revealed dif- cultivars ferential effect of rye chromosome on morphol- M. Sheidai1 ogy, disease resistance and productive potential. 82 15th ICC: Abstracts

Crossability was fairly high among addition lines some acentric fragments. Multicolour FISH with with all wheat genotypes but the variation was BAC probes specific for individual chromosome genotypic dependent. Truthfulness of hybrid pro- arms showed that the anaphase bridges are often genies was established through the presence of formed by fusion of homologous chromosome awns as well as chromosome number (2n ¼ 43). arms. The frequency of chromosome fusions was Hybrid and their self-progenies with rye chromo- not reduced in mre11 ku70 double mutants, sug some 4 and 5 particularly short arm impart resis- gesting that plants possess robust DNA end- tance to Karnal bunt caused by Tilletia indica. joining activities independent of the Ku70/80 The rye chromosome 4 and 7 as well as long arm and Mre11 complexes. We also detected erro- of chromosome 2 and 4 has genes for increased neous synapsis in the pachytene stage and severe spike length. Chromosome 5 and short arm of chromosome fragmentation in the initial stages chromosome 3 have alleles for increasing number of male meiosis. This fragmentation was sub- of spikelets. Grain weight was influenced by stantially suppressed in mre11 spo11-1 double chromosome 6, 7 and short arm of 4 and addi- mutants, indicating that Mre11 is required for tion of these chromosomes results in high grain repair, but not for the induction of Spo11- weight. To exploit desirable parts of rye genome, dependent meiotic DNA breaks in Arabidopsis. the progenies having rye chromosomes need to The presented data play up differences in the be back crossed with wheat genotypes and select- function of Mre11 in the initial stages of meiotic ing for 42-chromosome constitution. The recombination between yeast and Arabidopsis. progenies with better performance for quality and resistance can be selected which will increase productivity with diversity. PO:06:92 Progress in £ow cytogenetics of barley 1 1 PO:06:91 P. Suchankova , M. Kubalakova , P. Kovarova1, J. Bartos1, J. Cihalikova1, Mre11-de¢cient Arabidopsis exhibit T. Endo2 and J. Dolezel1 aberrant meiosis and chromosomal 1Laboratory of Molecular Cytogenetics and instability in somatic cells Cytometry, Institute of Experimental Botany, 1 2 1 Sokolovska 6, CZ-77200 Olomouc, Czech J. Siroky , J. Puizina , P. Mokros , 2 2 2 Republic; Laboratory of Plant Genetics, D. Schweizer and K. Riha Graduate School of Agriculture, Kyoto 1Institute of Biophysics, Czech Academy of University, Kyoto 606-8502, Japan Sciences, Brno, Czech Republic; 2Gregor Mendel Institute of Molecular Plant Biology, Austrian Flow cytogenetics is an attractive tool that sim- Academy of Sciences, Vienna, Austria plifies the analysis of complex plant genomes. Flow-sorted chromosomes have been used for the The Mre11 complex, initially described in bud- isolation of molecular markers from targeted ding yeast, is composed of Mre11, Rad50 and genome regions, for physical mapping using PCR Nbs1 proteins, and acts in manifold cellular and FISH, and for the construction of chromo- functions such as DNA checkpoint mechanisms, some-specific DNA libraries. Until now, the double-strand break repair, regular meiotic application of flow cytogenetics in barley has recombination, and telomere maintenance. We been hampered by the inability to sort individual report the outcome of Mre11 deficiency for the chromosomes, except the smallest chromosome stability of mitotic and meiotic chromosomes in 1H and some translocation chromosomes with Arabidopsis. Plants homozygous for the mre11 DNA content different from the remaining chro- mutation exhibit vegetative growth defects and mosomes. However, although the sorted fractions are infertile. Analysis of mitotic chromosomes in of translocation chromosomes have been useful mutant plants revealed a number of anaphase for mapping DNA sequences to subchromosomal bridges during divisions and abundant chromo- regions, their use remains limited. In this work, 15th ICC: Abstracts 83 we demonstrate that wheat-barley telosome addi- sequences. This suggests that AtPot1 is tran- tion lines may be used to sort any of the four- scribed in three splicing variants, and has addi- teen barley chromosome arms. Identity of sorted tional unpredicted exons. Alternative splicing was arms can be verified using chromosome-specific also known to occur in hPot1, and only one spli- markers; the purity in sorted fractions can be cing variant had tissue specificity among five spli- assessed by FISH. Furthermore, we will be able cing variants. Therefore, tissue specificity was to sort subarm chromosomal segments that are investigated also in AtPot1, but all three splicing generated in the wheat-barley chromosome addi- variants were detected in all tissues examined, tion lines by the gametocidal system. These including buds, flowers, leaves, roots, steams, sili- advances make barley flow cytogenetics an ques and cultured cells. Northern blot hybridiza- attractive tool that may greatly facilitate its gen- tion indicated that AtPot1 expresses more in ome mapping. A possibility to purify large quan- meristematic tissues than in vegetative tissues. tities of individual chromosome arms opens Nuclear localization of AtPot1 was confirmed by avenues for targeted isolation of low-copy GFP-fusion experiments, but the telomere locali- sequences, preparation of specific probes for zation was not decided yet. screening EST arrays, HAPPY mapping, and construction of chromosome arm-specific BAC PO:06:94 libraries. Cytogenetic effect of This work has been supported by the Ministry of Education, nitrosomethylurea and its Youth and Sports of the Czech Republic (grants ME527 and modi¢cation by hyperbaric ME528) and the Ministry of Agriculture of the Czech Repub- lic (grant QC1336). oxigenation A. Usatov1, E. Mashkina1 and E. Guskov1 PO:06:93 1Research Institute of Biology, Rostov State Characterization of a telomere end University, Rostov-on-Don, 344090 Russia binding protein in Arabidopsis thaliana Cytogenetic effect of nitrosomethylurea (NMU) 1 2 A. Tani and M. Murata depends on the concentration of mutagen. 1Graduate School of Natural Science and NMU-treatment of sunflower seedlings (0.015% Technology, Okayama University, Okayama NMU) reduces proliferation activity of root mer- 700-8530, Japan; 2Research Institute for isteme сells, leaving the level of chromo- Bioresources, Okayama University, Kurashiki some aberrations unchanged, while 0.03% 710-0046, Japan concentration of NMU increases the level of chromosome aberration as early as 12 hours after Pot1 (protection of telomere) is a G-rich single- the treatment was over. Mutagenesis is a compli- stranded telomeric DNA binding protein, identi- cated process depending on many internal and fied first in Schizosaccharomyces pombe, and external factors, and our research was aimed on shown to play an important role in stabilizing investigation of the possibilities of modification chromosomes. Pot1-like proteins or their encod- of NMU-induced cytogenetic effect by HBO. ing genes have been identified from yeasts to Used in non-inducing chromosome aberrations mammals. Based on the N-terminal amino acid mode, HBO enhances cytogenetic e¡ect of NMU. sequences of fission yeast and human Pot1, two The joint action of NMU and HBO signi¢cantly Pot1-like proteins (AtPot1 and AtPot2) were inhibits the proliferation activity of cells. When identified in Arabidopsis thaliana, but neither of HBO was used before NMU-treatment (0,015%), them was characterized. In this study, we attemp- the level of chromosome aberrations increased ted to amplify full-length cDNAs corresponding reliable as compared not only to control, but to to AtPot1 by RT-PCR, which has higher similar- NMU-treated group as well. Furthermore, HBO ity to human Pot1 (hPot1). As a result, the RT- pretreatment prolongates the cytogenetic e¡ect of PCR products contained three different DNA NMU (0.03%), while HBO post-treatment does 84 15th ICC: Abstracts not increase the level of NMU-induced chromo- SopB molecules, seen as fluorescent foci. some aberrations. SopA, in contrast, behaves in a dynamic man- We suggests that there is speci¢c mechanism ner, oscillating from pole to pole in a spiral for interaction between HBO and NMU. Appar- track, creating a concentration gradient, high- ently, modi¢cation of NMU-e¡ect is associated est at the cell poles and lowest at the cell cen- to formation of mutagenic products of NMU ter. SopA oscillation is absolutely dependent metabolism and their interaction with active on the ATPase activity of SopA. Segregation forms of oxygen. It is possible that during cell of plasmid DNA, is preceded by dissociation metabolism of nitrogen-contained compounds of plasmid DNA from SopB followed by a nitrogen oxide is formed, being capable to redistribution of SopB protein complexes to interact with superoxide to produce high-toxic the cell quarter positions where they act as peroxynitrite. receptors for sister plasmid molecules. We will We suppose that enhancing of NMU-mutagen- discuss the role of the SopA gradient in plas- esis by HBO may be associated with the forma- mid localization and partition. tion of peroxynitrite; the latter can to induce DNA damage directly, to activate free-radical L92 processes and lipid-peroxidation reactions. Visualising chromosome segregation Prokaryote Chromosomes Symposium in vivo and in vitro D. Sherratt1, X. Wang1, C. Possoz1 and S. Filipe1 L91 1Biochemistry Dept, University of Oxford, UK Pole to pole oscillation of an ATPase is required for F plasmid segregation in We analyse the spatial and temporal organisation E.coli of chromosome origins, termini, replication pro- 1 1 teins, recombination proteins, segregation pro- A. Wright and G. Leung teins and cell division apparatus in growing 1Tufts Medical School, Boston, MA 02111, USA E.coli cells. These data are then related to a model for how DNA processing and cell division Segregation of the low copy plasmid, F, in are coordinated. Finally we demonstrate chromo- E.coli, is dependent on plasmid specified parti- some segregation in an in vitro single molecule tion functions which include a centromere like experiment. site (SopC ) and two trans-acting genes, one encoding an ATPase (SopA) the other encod- L93 ing a protein (SopB) which binds to the cen- tromere forming a large nucleoprotein Dual architectural roles of the complex. Interactions between the ATP-bound nucleoid-associated protein HU form of SopA and the SopB-centromere com- from E. coli plex result in stimulation of its ATPase activ- 1 1 2 ity, resulting in conversion of SopA-ATP to J. Van Noort , S. Verbrugge , N. Goosen , 1 3 SopA-ADP and under some conditions, release C. Dekker and R. Dame of SopB from the centromere. We have exam- 1Molecular Biophysics, Department of ined the intracellular distribution of the two Nanoscience, TU Delft, The Netherlands; 2Lab of partition proteins, SopA and SopB and their Molecular Genetics, Universiteit Leiden, The relationship with plasmid DNA by fluorescence Netherlands; 3Physics of Complex Systems, Vrije microscopy, using GFP and its derivatives to Universiteit, Amsterdam, The Netherlands a˜taga¨ each component. Prior to segregation F plasmid DNA is localized at or near the cell Bacterial chromatin is thought to be organized center in complex with a large number of and compacted at least in part by interaction 15th ICC: Abstracts 85 with nucleoid-associated proteins. One of these characterized Min system, which blocks division proteins, HU, has long been thought to act in a in the vicinity of the cell poles, and a hypothe- fashion similar to eukaryotic histones i.e. by tical nucleoid occlusion system that blocks divi- wrapping of DNA around a protein core (1). sion in the vicinity of the nucleoid. The nucleoid Using a combination of single molecule techni- occlusion system would operate to help direct the ques we demonstrate that HU does not employ division machinery to the space between the seg- this type of mechanism to compact DNA. Scan- regating nucleoids. We have identified the key ning force microscopy studies show individual player in nucleoid occlusion in B. subtilis,asa HU molecules that induce sharp bends. Under novel inhibitor of division that is also a non- similar conditions magnetic tweezers studies specific DNA-binding protein. The nucleoid reveal compaction of DNA up to about 50% of occlusion protein, Noc, is closely related to a key its original length. At high concentrations of HU chromosome segregation protein, Spo0J. Under both techniques indicate an increased rigidity of various conditions in which the cell cycle is per- the DNA helix. SFM height images of such rigid turbed Noc prevents the division machinery complexes show that this is due to the formation (FtsZ ring) from assembling in the vicinity of the of an HU filament around DNA with a super- nucleoid. Unexpectedly, cells lacking both of the helical repeat of *16 nm. Taken together these topological inhibitors of division (i.e. Min and data provide firm evidence that DNA compac- Noc) are blocked for division because they estab- tion by HU stems from the (dynamic) induction lish multiple non-productive FtsZ accumulations. of local bends into DNA rather than from DNA The results help to explain how B. subtilis identi- wrapping (2). fies the mid cell site for division and avoids cata- strophic breakage of the chromosome by division through the nucleoid. References

(1) Dame, R.T. and Goosen, N. (2002). FEBS Lett 529, L95 151–156 (2) van Noort. J., Verbrugge, S., Goosen, N., Dekker, C. and Rings and moving helices: the SMC Dame, R.T. Proc Natl Acad Sci USA. In press. complex and actin-like MreB protein are part of the prokaryotic L94 chromosome segregation machinery Coordination of chromosome J. Mascarenhas1, A. Volkov1, H. Defeu Soufo1 segregation and cell division by a and P. Graumann1 nucleoid occlusion protein in Bacillus 1Biochemie, Fachbereich Chemie, subtilis Hans-Meerwein-Straße, Philipps-Universita¨t Marburg, 35043 Marburg, Germany J. Errington1 and L. Wu1 1Sir William Dunn School of Pathology, Bacteria contain an active intracellular segrega- University of Oxford, UK tion machinery that separates sister chromosomes towards opposite cell poles during ongoing repli- Efficient chromosome segregation in bacteria is cation. An important component of this machin- probably achieved by several interacting and par- ery is the SMC complex, which is required for tially redundant processes. Segregation is coordi- complete separation of chromosomes (but not for nated with cell division such that, normally, initial separation of origin regions) and for proper division occurs in the space between the separat- chromosome organization in Bacillus subtilis. ing sister chromosomes. Selection of the correct SMC forms a ternary complex with two proteins, mid cell site for division in rod-shaped bacteria is ScpA and ScpB, that are conserved in prokar- thought to be governed by the combined action yotes, and localizes in two distinct subcellular cen- of two negative regulators comprising the well ters within both cell halves. From there, the 86 15th ICC: Abstracts complex condenses and organizes DNA that is DNA is organized in independent H-NS bridged duplicated through the centrally located DNA DNA loops, which can be disrupted by applying replication machinery. SMC binds to DNA as an an increasing force. unusual ring like structure, condensing DNA by embracing DNA loops. DNA binding is mediated through ATP binding at the head domains, which References leads to dimerization of the heads and thus to ring closure. [1] Dame, R.T.; Wyman, C.; Goosen, N. H-NS mediated We have recently found that the origin regions compaction of DNA visualised by atomic force micro- fail to separate in the absence of B. subtilis actin- scopy. (2000) Nucleic Acids Res 28:3504–10. [2] Dame, R.T.; Wyman, C.; Wurm, R.; Wagner, R.; Goosen, like proteins MreB and Mbl. Both proteins form N. (2002) Structural basis for H-NS mediated trapping of dynamic helical ¢laments that move with a speed RNA polymerase in the open initiation complex at rrnB P1. of 0.1 mm/s underneath the cell membrane, sug- gesting that they could be the motor that pushes PO:08:02 DNA towards opposite poles. MreB and Mbl also a¡ect cell shape, same as the MreC and MreD pro- Spatial and temporal dynamics of teins whose genes are downstream of mreB. Both chromosome segregation in Vibrio MreC and MreD are membrane proteins, which might provide the link between chromosome seg- cholerae regation and formation of rod shaped cells. M. Fogel1 and M. Waldor1 1Departments of Genetics and Molecular PO:08:01 Microbiology, Sackler School of Biomedical Sciences, Tufts University School of Medicine and The mechanism of DNA compaction by Howard Hughes Medical Institute, Boston MA the nucleoid-associated protein H-NS 02111, USA from E. coli Bacterial chromosome segregation has been R. Dame1, M. Noom1, N. Goosen2 and 1 studiedindetailonlyinspecieswithsingle G. Wuite chromosomes. Little is known about chromo- 1Physics of Complex Systems, Vrije Universiteit, some segregation in the growing group of bac- Amsterdam, The Netherlands; 2Lab of Molecular teria with multipartite genomes. The human Genetics, Universiteit Leiden, Leiden, diarrheal pathogen Vibrio cholerae is a gram- The Netherlands negative, gamma-proteobacteria with two cir- cular chromosomes. Using still and time-lapse Bacterial chromatin is thought to be organized fluorescence video-microscopy we have visua- and compacted at least in part by interaction with lized the spatial and temporal dynamics of the nucleoid-associated proteins. Recently we have V. cholerae chromosomes. In all stages of the shown that one of these proteins, H-NS, com- cell cycle, the two origins are localized to dis- pacts DNA by the formation of bridges between tinct sub-cellular locations. In newborn cells, adjacent DNA tracts [1]. This property is exploi- the origin of chromosome 1 (Ori1) is located ted not only for providing a means of compac- near the new pole and the origin of chromo- tion, but is also essential to the regulatory role of some 2 (Ori2) at the cell center. Ori1 segre- H-NS in transcription. Bridging of DNA pro- gates to the poles early in the cell cycle vides an explanation for the recognition of whereas Ori2 segregates close to the time of curved DNA and for the trapping of RNAP in division and repositions rapidly to the new cell the open initiation complex at the rrnB P1 [2]. centers. Staining of the nucleoid with 40,6-dia- In order to further characterize the mechanism midino-2-phenylindole (DAPI) demonstrates underlying DNA compaction by H-NS, we have that both origins are associated with the edge performed DNA stretching experiments with opti- of the nucleoid in new cells and that later in cal tweezers. These experiments suggest that the the cell cycle, Ori1 can be positioned far from 15th ICC: Abstracts 87 the nucleoid mass, a finding consistent with even those with important functions in male models of dynamic positioning of the origin determination and di¡erentiation such as SRY regions. The low frequency of overlap of the and RBMY ^ have partners on the X from which origin foci with the DAPI fluorescence suggests they obviously evolved. Our comparative studies the origins are sequestered away from the rest show that almost all of the original human Y has of the nucleoid. The coordinated dynamics been lost, and the Y was saved from extinction observed would require segregation machinery only by its autosomal addition. that can discriminate between chromosomes How much longer can the human Y hold out and specify their localization. We are currently against the evolutionary forces that degrade it? investigating the roles of the orthologs of plas- mid partitioning proteins present on each L97 chromosome. Features of facultative heterochromatin regulated by Sex Chromosomes Symposium XIST expression C. Brown1, J. Chow1, N. Thorogood1, L. Hall2 L96 and J. Lawrence2 The origin, evolution and future 1Department of Medical Genetics, of human sex chromosomes University of British Columbia, Vancouver, BC 2 1 V6T 1Z3, Canada; University of Massachusetts J. Marshall-Graves Medical Centre, Worcester, Massachusetts 01655, 1Research School of Biological Sciences, The USA Australian National University, Canberra, ACT, Australia Expression of XIST, which encodes a large, heterogeneous, nuclear, non-coding RNA, is In mammals including humans, sex is determined essential for X chromosome inactivation. To by an XY male: XX female system in which a determine which features of the heterochro- male-dominant gene on the Y (SRY) determines matic inactive X chromosome are directly rela- testis, and embryonic testis determines maleness. ted to XIST expression we are studying The X and Y chromosomes evolved from an features generally associated with the inactive ordinary autosome pair as the Y chromosome X after induction of XIST expression. Features was progressively degraded. This must have hap- examined include gene silencing, replication pened within the last 300 million years, since timing, histone modifications, incorporation of birds and reptiles have completely non-homo- macrohistone H2A, and DNA methylation. We logous sex chromosome systems. are utilizing two different somatic cell-based To explore the origin of the human Y chromo- model systems for the induction of XIST some and SRY, we compared X and Y chromo- expression. In the first, human and mouse somes of the three major mammal groups XIST/Xist expression has been induced by 5- (eutherians, marsupials and monotremes). This azacytidine demethylation of active X chromo- allowed us to subdivide both the human X and Y somes in a human/mouse somatic cell hybrid. into a ancient region shared by all other mam- In the second, human XIST expression is mals, and a recently added region that is on the X induced from ectopic sites in human somatic and Y only in placental mammals. The ancient cells. Variable localization of XIST in these and added regions are also separate, and are cells will allow us to address how cell type and further subdivided, in the chicken. integration site influence XIST localization. The human Y chromosome is much smaller Consistent with previous reports, we detect no than the X, and contains only 45 unique and silencing in the mouse/human somatic cell active genes, survivors of the ongoing process of hybrid, even in the presence of Xist localiza- Y degradation. Many or most of these genes ^ tion. However, in human cells features of inac- 88 15th ICC: Abstracts tivation can be observed upon induction of evident. In conclusion: Recruitment of the same XIST. These systems thus provide a means of genes to different functions or evolution of evaluating the role of XIST in establishing the the transcription regulatory systems is an effec- features of facultative heterochromatin in tive mechanism during evolution to higher somatic cells in the presence and absence of complexity. transcriptional silencing. L99 L98 The chicken Z chromosome: A turn on Evolution in the light of genome or a turn off? conservation: Sex chromosomes direct H. McQueen1, L. Bisoni2 and morphological and functional L. Battle-Morera1 diversi¢cation 1ICMB, University of Edinburgh, Scotland; 2 H. Hameister1, M. Kohn1 and Oxford Radcliffe Hospitals Genetics Laboratories, Oxford, UK H. Kehrer-Sawatzki1 1Dept. Human Genetics, University Ulm, Birds undergo genetic sex determination using a D-89070 Germany ZW sex chromosome system. Although the avian mechanisms of neither sex determination The Genome Project has confronted us with the nor dosage compensation are understood, a surprising fact that the human genome contains female specific non-coding RNA (MHM) is less than 25 000 genes and further, that the num- expressed soon after fertilisation from the single ber of genes has remained nearly the same since Z chromosome and is likely to have a role in the appearance of the first vertebrates some 400 one or both processes. We have now discovered million years ago. Nevertheless very different a prominent female specific modification to the species evolved which proves the functional plas- Z chromatin which co-incides with the MHM ticity inherent to this genome. Genome wide gene locus. The same histone modification is highly function tests reveal the special role of sex chro- enriched on the Drosophila melanogaster male X mosomes for the development of new traits. This chromosome and forms an integral part of the contrasts with the fact that comparative gene mechanism for dosage compensation in this spe- mapping data have shown the X chromosome to cies. We suggest that these female sex chromo- be the most conserved gene arrangement. To some specific modifications could be associated study the dominant role or balancing act of sex with the mechanism of sex determination or, chromosomes a comparison of birds and mam- alternatively, dosage compensation in birds mals seems highly informative. Originating from which might operate in a manner analogous to the same ancestral vertebrate genome the birds that seen for the fruitfly. have developed Z and W sex chromosomes, which are genetically different from the X and Y sex chromosomes in mammals. We compare the L100 expression pattern in mouse and chicken of X chromosomal genes. We have chosen X chromo- Ectopic homologous recombination somal mental retardation, MRX, genes which are events in the human Y chromosome human speciation genes, recruited to tele- J. Lange1, H. Skaletsky1, S. Repping2, ncephalic function only recently. It can be shown S. Van daalen2, C. Korver2, L. Brown1, that in the chicken these genes do not serve the 1 1 same telencephalic function. Especially informa- S. Rozen and D. Page tive are those MRX genes which in mammals 1Howard Hughes Medical Institute, Whitehead are also expressed in the testis. Again in chicken Institute and Department of Biology, no testis specific function for these genes is Massachusetts Institute of Technology, 9 15th ICC: Abstracts 89

Cambridge Center, Cambridge, Massachusetts defects compatible with fertility, although sperm 02142, USA; 2Center for Reproductive Medicine, function is impaired in vitro. Department of Obstetrics and Gynecology, We are using microarray analysis of the testis Academic Medical Center, Amsterdam, transcriptome of mice with three di¡erent MSYq The Netherlands de¢ciencies to provide an overview of the tran- scriptional changes that underlie the sperm A prominent feature of the human Y chromo- abnormalities. In addition to the expected down some is the abundance of amplicons strewn regulation of members of the previously chac- throughout the long and short arms. These large, terised multi-copy Ssty gene family, two other tes- nearly identical repeat segments comprise tis-expressed multi-copy MSYq genes were approximately 30% of the male-specific euchro- identi¢ed. ‘Transgene rescue’ strategies using matic sequence, and are present as both direct BAC genomic clones are being used in attempts and indirect repeats. Nine testis-specific gene to identify the relationship between the sperm families, each with multiple copies in the Y chro- abnormalities and speci¢c Y gene de¢ciencies. mosome, are found entirely within the ampli- The other major transcriptional change identi¢ed conic region. It has been proposed that gene was the marked upregulation of a number of X- > conversion has maintained the 99.9% similarity linked genes, some of which are also present in of sequence pairs, evidence that productive multiple copies, suggesting that there are reg- recombination on the Y chromosome is not lim- ulatory interactions between X and Y genes dur- ited to its pseudoautosomal regions. However, ing spermiogenesis. The possible basis of these these amplicons also provide substrates for non- regulatory interactions will be discussed. productive recombination. Several recurrent deletions in the Y chromosome associated with spermatogenic failure have been molecularly L102 characterized, and are due to non-allelic homo- Partial deletions of the AZFc interval logous recombination between amplicons. Our data reveal a novel class of ectopic recombina- of the human Y chromosome and tion events predicted by the ampliconic structure. male infertility N. Machev1, N. Saut1, P. Terriou2, M. Guichaoua3, P. Collignon4, J. Chiaroni5, L101 A. Navarro1, N. Levy1, M. Mattei1 and 1 Multi-copy Y genes and their role M. Mitchell 1 in sperm development Inserm U.491, Faculte´ de Me´decine, 13385 Marseille, France; 2Institut de Me´decine de la P. Burgoyne1, A. Toure1, S. Mahadevaiah1, Reproduction, 13008 Marseille, France; J. Turner1, P. Ellis2, R. Furlong2, 3Laboratoire de Spermiologie, Hopital de la 4 E. Clemente2 and N. Affara2 Conception, 13385 Marseille, France; Service de Ge´ne´tique, Centre Hospitalier de Toulon, 83056 1MRC National Institute for Medical Research, Toulon, France; 5Etablissement Franc¸ais du London, UK; 2Cambridge University Department Sang Alpes-Mediterranee, 13005 Marseille, of Pathology, Cambridge, UK France

The mammalian Y chromosome has an unu- Microdeletions of the AZFc interval of the Y sually high proportion of genes present in multi- chromosome are currently the most common ple copies; in man it has been shown that these known genetic cause of human male infertility. multi-copy genes are organised in large palin- However most AZFc microdeletion are homo- dromic repeats. In the mouse, lack of the male-spe- geneous in extent and smaller deletions must now ci¢c region of the Y long arm (MSYq) leads to be identified and characterised if we are to learn severe sperm abnormalities and consequent infer- how the AZFc genes contribute to human ferti- tility; less extensive deletions result in mild sperm lity. Two partial AZFc deletions (gr/gr and b1/ 90 15th ICC: Abstracts b3) have recently been identified in infertile and somes? Will they degenerate further and gradu- fertile populations that remove some copies of all ally disappear, leading to X0 males, or even to AZFc genes, and an association study indicates extinction of males, resulting in apocalyptic that the resulting reduction in gene dose repre- extinction of many species? This scenario is unli- sents a risk factor for spermatogenic failure. To kely because the efficacy of population genetic determine the incidence of different partial AZFc processes causing genetic degeneration depends deletions and their effect on fertility, we com- on the number of functional genes linked toge- bined quantitative and qualitative analyses of the ther on the non-recombining Y-chromosomes. AZFc interval at the DAZ and CDY1 loci in 300 With only few functional genes left on the mod- infertile men and 399 control men. We detected ern mammalian Y-chromosomes, these forces 34 partial AZFc deletions (32 gr/gr deletions), may be too weak to cause further genetic degen- arising from at least 19 independent deletion eration. Here I report the evidence that, adaptive events, and find gr/gr deletion in 6% of infertile evolution is fairly common in the mammalian Y- and 3.5% of control men (P > 0.05). Our data linked genes, suggesting that genetic degeneration provide evidence for two large AZFc inversion of the mammalian Y-chromosomes does not polymorphisms, and for relative hot and cold actively proceed further. spots of unequal crossing-over within the blocks of homology that mediate gr/gr deletion. Using SFVs (sequence family variants) we discriminate L104 DAZ1/2, DAZ3/4, CDY1a (proximal) and CDY1b (distal) and define four types of DAZ/ Chromosomal radiation of the African CDY1 gr/gr deletion. Only deletions of CDY1a pygmy mice, subgenus Nannomys and DAZ3/4 were not found in our fertile popu- (Rodentia; Muridae): a karyotypic and lation, and show a weak association with inferti- lity (P ¼ 0.042), suggesting that most, but not all, phylogenetic analysis gr/gr deletions are neutral variants. V. Frederic1, C. Josette1, C. Pascale2 and B. Janice1 L103 1Institut des Sciences de l’Evolution (UMR5554), Ge´ne´tique & Environnement, Universite´ The fate of the mammalian Y Montpellier II, Montpellier, France; 2Institut des chromosomes Sciences de l’Evolution (UMR5554), 1 Pale´ontologie, Pale´obiologie et Phyloge´nie, D. Filatov Universite´ Montpellier II, Montpellier, France 1School of Biosciences, University of Birmingham, Edgbaston, Birmingham The African pygmy mice are small-sized rodents B15 2TT, UK widespread throughout sub-Saharan Africa belonging to one of the four subgenera of the Although X- and Y-chromosomes are thought to genus Mus (subgenus Nannomys). Owing to their evolve from a homologous pair of autosomes, highly conserved morphology, diagnostic char- they are extremely different from each other in acters are scarce leading to ambiguous dis- many organisms, including mammals. Actively crimination of the currently recognized 19 recombining X chromosomes are gene-rich, while species. On the contrary, chromosomes have the non-recombining Y-chromosomes have been shown to be useful taxonomic markers, as undergone the process of genetic degeneration several studies have uncovered extensive kar- due to reduced efficacy of adaptive and purifying yotypic evolution within this group involving selection in the non-recombining regions. As a rearrangements such as centric fusions between result, Y-linked genes accumulate deleterious autosomes, between sex chromosomes and auto- mutations, and have a reduced ability to adap- somes, and even deletion of sex chromosomes. tive evolution. What is the further fate of the An analysis of molecular (cytochrome b) and genetically degenerate mammalian Y-chromo- chromosomal markers was performed on speci- 15th ICC: Abstracts 91 mens from 12 countries, to assess the role of their sister order Trichoptera suggest that a com- chromosomal rearrangements in the evolutionary mon ancestor of both orders had a Z/ZZ sex history of this species complex. chromosome system. The W chromosome had The molecular phylogeny supported the mono- evolved later, at a common branch of Tischeriina phyly of this group, and resolved seven major plus Ditrysia. During evolution of Ditrysia, clades, most of them characterized by diagnostic derived sex chromosome systems had evolved chromosomal rearrangements or karyotypes. One such as neo-sex chromosomes, multiple sex chro- of the clades clustered taxa carrying di¡erent mosomes, and secondary W chromosome losses. sex-autosome fusions, in some cases associated Here we summarize our recent results on with partial or total deletions of sex chromo- molecular differentiation of W chromosomes in somes. As these fusions are expected to be highly selected species. Our main research tools were deleterious, they are rarely observed in mammals genomic in situ hybridization (GISH) with and when present, are considered as e⁄cient female-derived genomic probes, comparative reproductive isolating mechanisms. The cluster- genomic hybridization (CGH) with a probe mix- ing of these taxa into one clade, if con¢rmed, ture of differently labelled genomic DNA from would support the occurrence of shared genomic females and males, FISH with probes prepared traits allowing the formation and/or ¢xation of from W chromosome-derived bacterial artificial such rearrangements in taxa within this clade. chromosome (W-BAC) clones of Bombyx mori, The diversity of this type of rearrangement and FISH with W-painting probes prepared by within Nannomys o¡ers the unique opportunity laser microdissection of W chromatin followed to investigate the molecular basis of sex- by DOP-PCR amplification. Results suggest an autosome fusions and the evolutionary conseq- accumulation of two types of repetitive sequences uences on speciation rates and sex determination in W chromosomes: (i) repetitive DNA common mechanisms. to females and males, and (ii) W-specific repeti- tive DNA. Next the lack of cross-hybridization between Bombyx W-BACs and other lepidopter- L105 ans reflects independent evolution of non- Molecular differentiation of W recombining W chromosomes. Finally, a high specificity of W-painting probes denotes the chromosomes in Lepidoptera advanced stage of molecular differentiation of W F. Marec1, M. Vitkova2, I. Fukova2, chromosomes. A. Yoshido3, K. Sahara3 and W. Traut4 1Institute of Entomology ASCR, CZ-370 05 Ceske Budejovice, Czech Republic; 2Faculty of L106 Biological Sciences, University of South Bohemia, In platypus a meiotic chain with ¢ve X CZ-370 05 Ceske Budejovice, Czech Republic; 3Graduate School of Agriculture, Hokkaido and ¢ve Y chromosomes links bird and University, Sapporo 060-8589, Japan; 4Institute of mammal sex determination Biology, Medical University, D-23538 Lubeck, 1 2 1 Germany F. Gru¨tzner , W. Rens , E. Tsend-Ayush , N. El-Mogharbel1, P. O’Brien2, 2 3 In Lepidoptera (moths and butterflies), the major- M. Ferguson-Smith , R. Jones and ity of species with identified sex chromosomes J. Marshall Graves1 possess a WZ/ZZ (female/male) system or its 1Research School of Biological Sciences, numerical variations. The W and Z chromosomes Australian National University, ACT 2601, show various degrees of structural differentiation Australia; 2Centre for Veterinary Science, from undetectable to obviously non-homologous. Department of Clinical Veterinary Medicine, In most species, W chromosomes are largely University of Cambridge, UK; 3Department of composed of heterochromatin. Data on the evo- Biological Sciences, The University of Newcastle, lution of sex chromosomes in Lepidoptera and New South Wales 2308, Australia 92 15th ICC: Abstracts

Two centuries after the discovery of the platypus, signal from background noise, whereas in com- monotreme chromosome systems remain deeply parisons between vertebrate classes (eg human puzzling. Karyotypes of male or both sexes were and chicken *310 MY) signal may be lost. Kan- claimed to contain unpaired chromosomes garoos (180 MY) and platypus (210 MY) occupy (including the X) that form a multivalent chain useful middle ground. Sequence has diverged suf- at meiosis. Other systems of translocation hetero- ficiently for stringent detection of homologies zygosity are known in plants and insects but not that can reveal coding regions and regulatory sig- in vertebrates. nals. Even more importantly, because marsupials How the platypus chromosome system works and monotremes are mammals, they share with to determine sex, and how balanced gametes and humans many mammal-specific developmental viable embryos are produced, has been con- pathways and regulatory systems such as sex troversial for 30 years. In order to decipher the determination and X chromosome inactivation. complex chromosome system in platypus we gen- The major Australian research groups working erated chromosome paints for platypus chromo- on marsupial genetics have established a Centre somes and hybridised them onto male and female for Kangaroo Genomics to gather resources, metaphase chromosomes, meiotic cells and develop expertise and foster Australian and inter- sperm. This showed that platypus has ¢ve male- national collaborations for a serious onslaught on speci¢c chromosomes (Y), as well as ¢ve chromo- the genome of the model Australian kangaroo somes present in one copy in males and two Macropus eugenii (the tammar wallaby). We are copies in females (X). These ten chromosomes developing detailed physical and linkage maps of form a multivalent chain at male meiosis, in the genome to complement sequencing, and will which X and Y chromosomes alternate. the X and prepare and array cDNAs for functional studies, Y chromosomes segregate into two di¡erent types especially of reproduction and development. At of sperm that determine female and male embryos the same time, the Brazilian short-tailed opossum respectively. An X chromosome with homology Monodelphis domestica is being fully sequenced by to the human X lies at one end of the chain, and a the NIH, so that it is possible to compare two chromosome bearing the platypus homologue of very distantly related marsupials. We have also DMRT1, the putative male determining gene in submitted a proposal to sequence the genome of chickens, at the other. the platypus, Ornithorhynchus anatinus,andthis This is the ¢rst evidence of a link between the has received high priority for full sequencing by mammalian and avian sex chromosome systems, the NIH. whichwerepreviouslythoughttohaveevolved independently. PO:08:03 L107 A post sex mismatched bone marrow Exploring the kangaroo and FISH analysis showing an XXY, platypus genomes XXYY clone J. Marshall-Graves1 S. Engelbrecht1, M. Loubser2, J. Wakefield1, 1 1 3 1Research School of Biological Sciences, The S. Kolia , L. Mogatusi and P. Willem Australian National University, Canberra, ACT, 1National Health Laboratory Service; 2Private Australia Practice at Olivedale Clinic Johannesburg; 3National health Laboratory Service and It makes excellent sense to include marsupial and University of the Witwatersrand, South Africa monotreme species among mammal and other ver- tebrate species lined up for exhaustive genomic An eight-year old boy was diagnosed with the study. Comparisons between the more closely rela- rare autosomal recessive disorder, mucpoly- ted eutherian species (eg human and mouse *70 saccharidosis type VI. Bone marrow transplant MY) often suffer because it is hard to distinguish (BMT) is a commonly accepted treatment for 15th ICC: Abstracts 93 this syndrome. The patient received his first BMT about W chromosomes, and data about their in 1997, which was subsequently rejected. In molecular composition and actual role in sex August 2002 he received his second BMT from a determination are scarce. Here we present a blood type matched unknown female donor. A detailed analysis of the W chromosome in the month post BMT the patient had Epstein-Barr codling moth, Cydia pomonella (Tortricidae), the virus (EBV) infection, which was treated with key pest of pome fruit in temperate regions of Retuximab. After an initial allergic reaction the the world. The karyotype of codling moth con- patient recovered and was PCR negative for EBV sists of 2n ¼ 56 chromosomes. In female meiosis, infection. Fluorescent in situ hybridisation (FISH) the W and Z chromosomes pair regularly in spite was performed to determine chimerism following of the lack of apparent homology. The W con- the sex mismatched BMT. The first FISH analysis sists largely of heterochromatin and is deeply using the commercially available probes from stained with DAPI, indicating a high content of Vysis (CEP X, Xp11.1-q11.1 and CEP Y, Yq12) AT-base pairs. We studied molecular differentia- was performed by ourselves in March 2003. The tion of the sex chromosomes by genomic in situ analysis revealed 5% of cells displaying an XXY hybridization (GISH) and comparative genomic pattern. The same clone was still present in July hybridization (CGH). GISH detected the W 2003, along with an XXYY pattern. A full per- chromosome by strong binding of the Cy3-label- ipheral blood cytogenetic analysis was required led, female-derived DNA probe. With CGH, and revealed a normal female karyotype. FISH both the Cy3-labelled female-derived probe and analysis on the same specimen’s metaphases Fluor-X labelled male-derived probe evenly showed the presence of Y-chromosome sequences bound to the W. This suggested that the W is on the telomeric end of the p-arm of the two X- composed predominantly of repetitive DNA chromosomes. In the latest FISH study on the sequences occurring scattered in other chromo- patient’s BM the clone was not present, but had somes but accumulated in the W. Finally, we 89.25% donor cells. We hope to conduct studies prepared W-specific probes by laser microdissec- on the BM donor to establish if the XXY, XXYY tion of the W chromatin followed by DOP-PCR cells are present. If this is not the case we postu- and PCR labelling. The probes stained the W late that the EBV infection might have played a with a high specificity in a chromosome-painting role in the appearance of the aberrant clones. manner. DNA fragments of the microdissected W chromatin were cloned and sequenced. The W-sequence analysis revealed no homology to PO:08:04 any DNA sequenced so far. Molecular analysis of microdissected W chromosomes of the codling moth, PO:08:05 Cydia pomonella The structure and evolution of sex 1 1 2 I. Fukova , M. Vitkova , W. Traut , chromosomes in Silene latifolia S. Kubickova3 and F. Marec4 R. Hobza1, M. Lengerova2, E. Kejnovsky1, 1 Faculty of Biological Sciences, University of J. Siroky1, J. Macas3 and B. Vyskot1 South Bohemia, CZ-370 05 Ceske Budejovice, Czech Republic; 2Institute of Biology, Medical 1Institute of Biophysics, Czech Academy of University, D-23538 Lubeck, Germany; Sciences, Brno, Czech Republic; 2Children’s 3Veterinary Research Institute, CZ-621 32 Brno, Medical Center, Brno, Czech Republic; 3Institute Czech Republic; 4Institute of Entomology ASCR, of Plant Molecular Biology, Czech Academy of CZ-370 05 Ceske Budejovice, Czech Republic Science, Ceske Budejovice, Czech Republic

The sex chromosome system in Lepidoptera is of Silene latifolia is the basic plant model to study the WZ type, and females are the heterogametic sex determination and sex chromosome evolu- sex with a WZ sex-chromosome pair. Unlike Y tion. Here we present an improved FISH stra- chromosomes in XY systems very little is known tegy for differentiating the sex chromosomes of 94 15th ICC: Abstracts

Silene latifolia by chromosome painting. The X physician, while performing US examination, or Y chromosomes were isolated using nitrogen found extended nuchal translucency. Having laser beam microdissection, catapulted by laser consulted that observation with an obstetrician, pressure, and amplified by DOP-PCR. We used a the parents discontinued further prenatal diag- modified FAST-FISH protocol based on a short nostics of the foetus. In delivery, oedema of feet hybridization time combined with a low con- and palms was noted. The psychomotor develop- centration of probe. The success of these experi- ment of the child was normal. Periodical exam- ments is demonstrated by the differential labeling ination after the 2nd year of life did not reveal of the X and Y chromosomes. This new approach any major abnormalities, except a slight skrzy- represents a quick tool to compare organization dlowatosc of the neck and clearly visible of plant genomes. We have also generated new podpaliczkowe areas in form of a˜pouchesa¨. Pre- markers by constructing and screening a sample liminary cytogenetic examination, performed at a bacterial artificial chromosome (BAC) library for regional centre, revealed 46,X þ mar karyotype. appropriate FISH probes. Five newly isolated Another examination of the karyotype, per- BAC clones yielded discrete signals on the chro- formed at our Laboratory, verified the karyotype mosomes: two were specific for one autosome type into 46,XY. FISH technique with an SRY pair and three hybridized preferentially to the sex gene-specific probe enabled us to find deletion in chromosomes. We present the FISH hybridiza- the short arm of chromosome Y, involving the SRY tion patterns of these five BAC inserts together gene. In molecular examination, employing PCR with previously described repetitive sequences technique, aiming at determination of the extent (X-43.1, 25S rDNA and 5S rDNA) and use them of the described Yp deletion, it was found that to analyze the S. latifolia karyotype. Using one in the patientaˆs karyotype, except the SRY gene, BAC insert and the three repetitive sequences, neither PABY nor ZFY loci occur, what we have constructed a standard FISH karyotype cytogenetically confirms the loss of Yp11.3 band. that can be used to distinguish all autosome pairs. We have analyzed in detail the hybridization patterns of these sequences on the sex chromo- PO:08:07 somes. The results show that divergence of the A novel structure associated with the sex chromosomes of S. latifolia is already in pro- chicken ZW lampbrush bivalent cess and degeneration of the Y chromosome by 1 1 accumulation of specific sequences has begun. A. Krasikova , A. Saifitdinova , S. Derjusheva1, S. Mizuno2 and E. Gaginskaya1 PO:08:06 1Biological Research Institute of Saint-Petersburg University, 198504, Russia; 2Department of Yp terminal deletion in a 3-year old Agricultural and Biological Chemistry, College female patient with some stigmata of Bioresource Sciences, Nihon University, of Turner’s syndrome Fujisawa, Japan 1 1 B. Kaluzewski , M. Constantinou , In avian oocytes, sex chromosomes Z and W form 1 1 2 Z. Helszer , I. Plowas and L. Korniszewski an asymmetrical lampbrush bivalent with one 1Department of Medical Genetics, Medical chiasma, The W chromosome is strongly hetero- University, Lodz, Poland; 2Department of chromatic. Here we describe a spherical entity Pediatrics, Section of Diabetology and Birth that forms in chicken germinal vesicles (GVs) in Defects, Warsaw Medical Academy, Warsaw, association with one of heterochromatic regions Poland of W chromosome. By morphology, it resembles centromeric protein bodies (PBs) attached to A child, delivered spontaneously from the first lampbrush chromosome (LBCs) axis in Passerine pregnancy on the 40th week from young unre- birds and pigeons. In GVs, chicken W chromo- lated parents. On the 12th week of pregnancy, a some is known to consist of 7 chromomeres and 15th ICC: Abstracts 95 to comprise XhoI, EcoRI and SspI highly repe- and recombination disruptions are supposed to ated DNA sequences. Using FISH, we found be the major factors. that the spherical body associates with SspI Sex chromosomes represent good examples not repeat sequence, which localizes in non-cen- only of repetitive sequence accumulation sites, tromeric chromomere 6 of W chromosome. In but also of systems with an almost complete lack chaffinch GVs, PBs are known to form in asso- of recombination, and as such, they are ideal can- ciation with centromeric FCP repeat. Using RT- didates for subfamily formation. PCR we have verified that both SspI and FCP Speci¢cally, in this study we analysed the sex- repeats transcribe during oogenesis, while EcoRI chromosome system of the dioecious species and XhoI repeats of chicken W chromosome do Rumex acetosa (Polygonaceae) paying special not. The sphere on chicken ZW bivalent does not attention to a Y-speci¢c satellite DNA family, stain by antibodies against phosphorilated form RAYSI. Rumex acetosa is a classical model in of RNA-polymerase II, while simple loops on Z sex-determining mechanisms research due to the lampbrush chromosome and small loops on the presence of a complex sex system comprising of W brightly stain. Splicing snRNPs and SR-pro- females 2n ¼ 14 (XX þ 12 autosomes) and males tein SC35 were not revealed in the sphere either. 2n ¼ 15 (XY1Y2 þ 12 autosomes). The two Y Since similar data are known for centromeric chromosomes appear not to recombine during PBs on chaffinch and pigeon LBCs, the sphere male meiosis and both of them will only pair with on chicken W was identified with a kind of PBs. the ends of each X chromosome arm. In this Unlike PBs in chaffinch and pigeon oocytes, the study, by means of molecular as well as £uor- PB on chicken W does not stain by monoclonal escent in situ hibridization techniques, we demon- antibodies 4A6 against DNA-topoisomerase II. strate the existence of two subfamilies of the RAYSI family (called RAYSI-S and RAYSI-J) Supported by RFBR (02-04-49116), Russian Ministry of looking at both ¢xed positions (homogenizated Education (PD02-1,4-291) and Science (MK-2655.2003.04). positions) and also their di¡erential chromosome location. Causes and implications are discussed as well. PO:08:08 Analysis of two different satellite DNA subfamilies in the complex PO:08:09 sex-chromosome system of Rumex Reduced rates of sequence evolution acetosa (Polygonaceae) of Y-linked satellite DNA in Rumex R. Navajas-Pe´rez1, T. Schwarzacher2, (Polygonaceae) R. De La Herra´n1, C. Ruiz Rejo´n1, R. Navajas-Pe´rez1, R. De la Herra´n1, M. Ruiz Rejo´n1 and M. Garrido-Ramos1 M. Jamilena2, R. Lozano2, C. Ruiz Rejo´n1, ´ 1 1 1Departamento de Gene´tica, Facultad de M. Ruiz Rejon and M. Garrido-Ramos Ciencias, Universidad de Granada, Campus de 1Departamento de Gene´tica, Facultad de Fuentenueva s/n, 18071, Granada, Spain; Ciencias, Universidad de Granada, 18071. 2 Department of Biology, University of Leicester. Granada, SPAIN; 2Departamento de Biologia LE1 7RH, UK Aplicada, Escuela Te´cnica Superior de Ingenieros Agro´nomos, Universidad de Almeria, Almeria, Although satellite DNAs normally evolve con- Spain certedly, resulting in intra-specific sequence homogenization and inter-specific divergence, the One characteristic feature of the sex chromo- existence of several mechanisms leading to satel- somes is the accumulation of a sort of repetitive lite DNA subfamilies formation has been largely DNA sequences in the Y chromosomes. How- reported. Among the proposed causes, the exis- ever, little is known about how this occurs and tence of higher-order repeats, the population size about how the absence of recombination affects 96 15th ICC: Abstracts the subsequent evolutionary fate of the repetitive analysis using lymphocyte culture, PCR and sequences in the Y chromosome. Here, we Southern technique, were done. The PCR method compare the evolutionary pathways leading to provided rapid and reliable results for the the accumulation of three families of satellite identi¢cation of fragile X negative and positive DNA sequences within the genomes of Rumex patients. The PCR-positive case was con¢rmed by acetosa and Rumex papillaris, two dioecious the CGG repeat expansion on Southern blot ana- plant species with a complex XX/XY1Y2 sex- lysis with a positive cytogenetic result. Our con- chromosome system. We have found that two of clusions are that cytogenetic and molecular these families, one autosomic and one Y-linked, genetic techniques improve diagnostic accuracy shared a common origin. Conversely, they are and allow veri¢cation of the normal condition of not related in origin with the third one, also FRAXA locus, identi¢cation of carriers and detec- located in the Y chromosomes. However, we tion of complete mutations in fragile X syndrome have demonstrated that the two satellite DNA and, ultimately, proper genetic counselling in fra- families in the Y chromosomes of these species gile X families. With fragile X syndrome being have reduced rates of evolution and sequence such a complex condition, national screening homogenization in relation to those found for would need to be considered for families before a the satellite DNA in autosomes. child is conceived so that an informed choice can be made. PO:08:10

Cytogenetic and moleculargenetic PO:08:11 screening of fragile X syndrome 1 1 Nuclear spacing in the syncytial S. Padma , K. R. Manjunatha , blastoderm of Drosophila melanogaster G. K. Chetan1, S. R. Girimaji*1, S. Roy1, E. Venkataswamy1, S. Balu1 and embryos H. N. Venkatesh1 S. Pichler1, T. Gregor2 and D. Glover1 1Departments of Human Genetics & Psychiatry*, 1Department of Genetics, Cambridge University, National Institute of Mental Health & Cambridge CB2 3EH, UK; 2Department of Neurosciences (NIMHANS), Bangalore 560029, Molecular Biology, Princeton University, India Princeton, New Jersey, 08544, USA

Fragile X syndrome represents the most common In D. melanogaster, inhomogeneities in the spa- inherited cause of mental retardation. The key cing of nuclei along the A-P axis are first visible clinical features of the fragile X-syndrome in in cycle 11 of the syncytial blastoderm embryo males are mental retardation, elongated trian- (Blankenship and Wieschaus, 2001). In the ante- gular face, lop ears and macro-orchidism. The rior domain, spaces between nuclei are expanded, fragile X syndrome was the first disease shown to in the pre-cephalic furrow domain, nuclear dis- be associated with ‘dynamic mutations’, caused tances are the smallest, and in the posterior by an amplification of an unstable CGG repeat domain, nuclear distances are intermediate. sequence located at the 50 untranslated region of Nuclear spacing represents the earliest manifesta- FMR1 gene. A fragile site at the distal long arm tion of zygotic transcription mediated by bicoid. of the X chromosome (FraXq 27.3) is the hall- Alpha-amanitin injections inhibit zygotic tran- mark cytogenetic feature of the syndrome. AIM:- scription and nuclear spacing is established in This study was aimed to facilitate both uniform manner along the A-P axis. We have cytogenetic and molecular screening of fragile X developed Mathlab programmes which measure syndrome in Indian population for the analysis nuclear distances and densities to identify the of the mutation. genes and mechanisms that regulate nuclear spa- Fifty subjects with fragile X syndrome clinical cing before cellularization. Embryos lacking the features, were studied. Cytogenetic and molecular X-chromosome do not form the anterior domain 15th ICC: Abstracts 97 with expanded nuclear spacing. Genetic mapping This clinical report could be helpful in de¢ning places the gene required for nuclear spacing in the phenotypic range associated with mosaic Y region 7A-7B. I am genetically fine-mapping the isodicentrics. region and setting up RNAi experiments to iden- tify the gene on the X-chromosome involved in nuclear spacing. PO:08:13 I am investigating nuclear spacing in bcd;nos;tl, Markers for uncovering the evolution nos;bcd and osk embryos to investigate a poten- of sex chromosomes in Lepidoptera tial function of maternal protein gradients in nuclear spacing and in mutants of the gap genes M. Vitkova1, I. Fukova2 and F. Marec2 giant, knirps and even-skipped. 1Faculty of Biological Sciences, Univerzity of South Bohemia, CZ-370 05 Ceske Budejovice, Czech Republic; 2Institute of Entomology ASCR, CZ-370 05 Ceske Budejovice, Czech Republic PO:08:12 Most insects possess Drosophila/mammalian-type Cytogenetic and molecular sex chromosome systems, XX/XY or derived characterization of an isodicentric variants, with males being the heterogametic sex. Y-chromosome in an infertile man Female heterogamety occurs only in two insect 1 1 2 orders, the Trichoptera (caddis flies) and A. Veble , K. Writzl , B. Zorn and Lepidoptera (moths and butterflies), both orders 1 B. Peterlin forming a common clade, the superorder 1Division of Medical Genetics, Department of Amphiesmenoptera. Trichoptera and primitive Obstetrics and Gynecology, UMC Ljubljana; Lepidoptera possess a Z/ZZ system. Whereas a Ljubljana, Slovenia; 2Andrology Centre, WZ/ZZ system is common in advanced Lepi- Department of Obstetrics and Gynecology, UMC doptera, and numerical variations of the WZ Ljubljana; Ljubljana, Slovenia type are known as well. Here we examined sev- eral lepidopteran species from primitive ones, One of the most common structural changes of which lack the W chromosome, to advanced ones the Y chromosome is dicentrism. The phenotype with well-differentiated W, in order to uncover of patients with a karyotype 45,X/46,X,idic(Y) the evolutionary history of their sex chromo- range from almost normal males through mixed somes. The use of genomic in situ hybridization gonadal dysgenesis to females with Turner (GISH) and comparative genomic hybridization syndrome phenotype. (CGH) enabled us to identify the female-deter- We present a 29-year-old man who was refer- mining W chromosomes and define the level of red to cytogenetic assessment because of azoos- their molecular differentiation from the Z chro- permia. He had testicular atrophy (left and right mosomes. Potential homology between sex chro- testicular volumes were 7 ml); histopathologic mosomes of different species was tested with the analysis of testis biopsy demonstrated maturation help of several sex-chromosome markers such as, arrest. The GTG- and CBG-banded karyotype for example, the MBSAT1 satellite recently dis- showed two cell lines, one of them contained isodi- covered in Mamestra brassicae, perZ gene from centric Y [idic(Yp)] chromosome, which was seen the Z chromosome in Antheraea pernyi,and in 76% of metaphases analysed, and a 45,X cell Bmkettin gene found on the Z chromosome of line (24%). This was con¢rmed by FISH analysis Bombyx mori. The presence of selected markers with CEP Y, ptel Y and qtel Y probes. For pre- in the genome of a particular species was exam- cise characterisation of the Yq breakpoint, PCR ined by Southern hybridization. Then the mar- analysis using primers for six markers loci (sY84, kers were localized on chromosomes by FISH. sY127, RBMY1, sY152, sY147 and sY254) was Results achieved enabled us to restate the performed. The deletion of AZFb and AZFc hypothesis on the evolution of lepidopteran sex region was con¢rmed. chromosomes. 98 15th ICC: Abstracts

PO:08:14 W-heterochromatin segment and two segments translocated onto the W chromosome from auto- Detection of sex chromosome somes. The sex chromosome evolution in Samia constitution by GISH and cynthia is discussed. telomere-FISH in some Lepidoptera 1 1 2 A. Yoshido , K. Sahara , Y. Yasukochi Chromosome Transfer Symposium and F. Marec3 1Graduate School of Agriculture, Hokkaido L108 University, Japan; 2Genome Research Department, National Institute of Tumorigenicity suppressor genes ^ Agrobiological Sciences, Japan; 3Institute of their history as a prime example of bias Entomology, Czech Academy of Sciences, Czech Republic in science H. Klinger1 Sex chromosomes are designated X and Y in sys- 1Albert Einstein College of Medicine, NY, USA tems with male heterogamety and W and Z in those with female heterogamety. In species with No abstract was submitted for this talk. multiple sex chromosomes, it is sometimes diffi- cult to disclose the actual sex chromosome con- stitution. Examination of several specimens with L109 various chromosome stages is inevitable to get the definitive conclusion in such cases. One must Chromosome engineering using conventionally compare the number of chromo- chromosome transfer for functional somes between females and males, and analyze analyses chromosome pairing, the synaptonemal complex 1 formation and/or chiasma formation, if adequate M. Oshimura molecular markers of sex chromosomes are not 1Department of Biomedical Science, Institute of available. Regenerative Medicine and Biofunction, Moths and butter£ies (Lepidoptera) exemplify Graduate School of Medical Science, Tottori the female heterogamety with a WZ/ZZ sex chro- University, Yonago, Tottori 683-8503, Japan mosome system or its numerical variations (e.g., Z/ZZ, W1W2Z/ZZ, WZ1Z2/Z1Z1Z2Z2). By The method of microcell-mediated chromosome using combination of GISH and telomere-FISH, transfer is important for mapping genes to spe- we have developed a simple method to identify cific chromosome when the gene has a specific the sex chromosome constitution in Lepidoptera. function. For example, we have used this method GISH highlighted the heterochromatin of W chro- to map genes involved in tumor suppression, cel- mosomes and PCR-generated telomeric repeat lular aging, metastasis, DNA repair and genomic probes, (TTAGG)n, detected chromosome ends. imprinting. More recently, we have established a This approach enabled us to acquire gross but reli- novel procedure to introduce foreign genetic mate- able information about sex chromosomes from a rial into mice by using the chromosome itself as a single-specimen preparation of pachytene vector and provided the first examples of trans- oocytes. Another merit of this method consists in chromosomic mice containing a heritable foreign its universal use for any species with molecularly chromosome. Application of this procedure was di¡erentiated sex chromosomes. With this demonstrated on creations of the mice expressing method we showed that Samia cynthia pryeri has fully human antibodies and Down syndrome a WZ1Z2 sex chromosome constitution, whereas model mice with an extra human chromosome 21. the subspecies S. c. ricini has a Z/ZZ sex chromo- For more precise mapping, functional studies, some system. The W chromosome in S. c. pryeri making TC mice and human artificial chromo- presumably consists of 3 parts, an original some, we are currently engineering human 15th ICC: Abstracts 99 chromosomes in the chicken DT40 cells with a genic than the parental UACC-903 cell and con- high homologous recombination rate. For this trol hybrids of chromosomes 9(q-arm), 5 and 15, pourpose, the human chromosome containing a as judged by their ability to form colonies in soft locus of interest is modified by chromosome agar and tumors in athymic female nu/nu mice. truncation at the integration site of the telomeric The data presented here defines additional repeat or by the integration of a target construct TSG(s) on chromosome 9 independent of the by homologous recombination. In this talk, p16/p15 tumor suppressor genes and provides lessons learned from our studies using the strong evidence for the presence of a TSG(s) cen- chromosome engineering will be introduced. tral to CMM development on chromosome 10.

References L111 The role of the nuclear envelope in 1) Tomizuka et al. Functional expression and germline transmission of a human chromosome in chimeric mice. chromosome positioning: using Nature Genet., 16:133, 1997 monochromosome hybrids cells as 2) Kuroiwa et al., Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts. a model system Nat. Biotechnol., 18:1086, 2000. K. Meaburn1, H. Cox1, J. Ellis2, R. Newbold3 3) Horike et al., Targeted disruption of the human LIT1 locus 1 defines a putative imprinting control element playing an and J. Bridger essential role in Beckwith–Wiedemann syndrome. Hum. 1Lab. of Nuclear and Genomic Health, Cell and Mol. Genet., 9:2075, 2000. Chromosome Biology Group, Dept Biological Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH, UK; 2Randall Centre for the L110 Molecular Mechanism of Cell Function, Kings Tumour suppressor genes in malignant College, London University, Guys Campus, London, SE1 1UL, UK; 3Institute of Cancer melanoma Genetics and Pharmacogenomics, Dept Biological C. Parris Sciences, Brunel University, Uxbridge, Middlesex, UB8 3PH, UK Brunel University, UK Specific human chromosomes are found repro- Deletions involving regions of chromosomes 1, 6, ducibly at the nuclear periphery in proliferating 9 and 10 are frequent events in the development cells. There are a number of inner nuclear and progression of cutaneous malignant mela- envelope (INE) proteins that may have roles in noma (CMM). To investigate the possibility that genome organisation and location in interphase these chromosomes encode important tumor sup- nuclei; examples of such proteins are emerin pressor genes, we introduced normal copies of and A-type lamins. Mutations in the genes of chromosomes 1, 6 and 10 to the tumorigenic either can result in X-linked EDMD or a wide human metastatic melanoma cell line UACC-903 range of diseases collectively termed lamino- using microcell mediated chromosome transfer. pathies, respectively. We find that specific chro- In addition, two chromosome 9 variants micro- mosomes normally located at the nuclear deleted for the p16 and p15 loci were also trans- periphery, in normal proliferating human dermal ferred to determine whether an additional fibroblasts (HDF) are located more internally in melanoma tumor suppressor gene (TSG) resides proliferating HDF derived from patients with on chromosome 9 independent of these loci. Chro- mutations in the A-type lamin or emerin gene. mosome 1 hybrids failed to show consistent sup- The emerin mutant cell line we have studied pression of growth in soft agar or reduced tumour lacks both emerin and lamin C. development in nude mice. The resulting micro- In order to further study the role of INE pro- cell hybrids of the chromosomes 6,9Dp16/p15 teins in targeting chromosomes to the nuclear per- variants and 10 were significantly less tumori- iphery, we have employed a model cell system, in 100 15th ICC: Abstracts which stable hybrid cell lines carry a single, intact 46,XY,t (3;15) (p21;q24)pat ; 46,XX,t (3;13) human chromosome in immortalised mouse ¢bro- (q23;q22)mat. The parents decided to continue blasts. We have found that the position of most the pregnancies in all the cases. The clinical sig- human chromosomes normally found at the ni¢cance of de novo reciprocal translocation is a nuclear periphery has altered. We are testing the major problem in prenatal counselling. Molecular role of various human INE proteins in the posi- cytogenetics tools such as FISH are indispensable tioning of selected human chromosomes by stable for characterizing the breakpoints involved in the transfection of INE containing constructs into reciprocal translocation. these cells. Our data so far suggests that, in this hybrid system, Lamin A alone is not critical for positioning chromosomes at the nuclear peri- PO:08:16 phery. Taken together, these data imply a role for lamin C in the positioning of human chro- De novo partial duplication of mosomes to the nuclear periphery. chromosome 7p at a prenatal diagnosis M. Souto1, R. Pinto Leite1, B. Carvalho1 1 PO:08:15 and E. Ribeiro 1 Reciprocal translocations detected at Centro Hospitalar de Vila Real-Peso da Re´gua, SA prenatal diagnosis: results from 5 years experience Partial duplications of the short arm of chromo- some 7 are a rare occurrence. The most common R. Pinto Leite1, B. Carvalho1, M. Souto1 1 mechanism leading to 7p duplication is and E. Ribeiro unbalanced segregation of a parental reciprocal 1Centro Hospitalar de Vila Real-Peso translocation at meiosis. da Re´gua, SA We report a case, detected in a prenatal diag- nosis that has an extra segment on the proximal Reciprocal translocations are not uncommon, short arm of chromosome 7. arise 1 in 625 new born of the general population A 34 year-old pregnant woman was referred for and once in every 1000 amniocentesis. It is amniocentesis in the 16th week of pregnancy urgent to do parental chromosomal studies, to because of ultrasound anomalies. The couple had distinguish between a familiar or a de novo rear- an obstetric history of 5 miscarriages and 3 rangement in the fetus, to predict the risk for healthy children. physical malformation and mental deficiency; The GTG analysis revealed an extra segment that is approximately 10 percent when it is a de on the proximal short arm of chromosome 7 in novo situation. two di¡erent cultures. Parent’s karyotypes were A total of ¢ve foetuses with reciprocal translo- normal. In order to clarify the chromosome ori- cations, 1 de novo and 4 familiar, were detected at gin of this extra segment, chromosome painting prenatal diagnosis, during July 1998 through was done. Fluoresecence in situ hybridization December 2003 at the Centro de Diagnostico Pre- (FISH) using a whole chromosome 7 DNA probe natal of the Centro Hospitalar de Vila Real-Peso (Cytocell), showed that the extra chromosome da Re¤ gua, S.A. Using conventional cytogenetics material is derived from chromosome 7, con¢rm- and £uorescence in situ hybridization (FISH) we ing that the foetus is partially trisomic for chromo- characterized the rearrangements. Amniocenteses some 7. The exclusion of a balanced reciprocal were performed because of maternal age (2 cases); translocation at one of the parents suggests the positive biochemical screening for trisomy 21 presence of a gonadal mosaicism. (2 cases) and one familiar case of reciprocal When the couple came to prenatal counselling, translocation. The karyotypes were interpreted to explain the implications of this result, the ultra- as 46,XX,t (3;5) (q25;q13) ; 46,XY,t (4;7) sound detected in utero death. (q21;p15)pat ; 46,XX,t (2;14) (p25.1;q13.1)mat ; A bibliographic review is presented. 15th ICC: Abstracts 101

Gene Therapy Symposium 1School of Biological Science, Royal Holloway – University of London, Egham, Surrey, TW20 L112 0EX, UK Mammalian arti¢cial chromosomes A number of approaches are under development (MACs) as gene therapy vectors to provide genetic therapies based upon the con- 1 1 1 cepts of (i) gene addition to complement inher- C. Huxley , G. Kotzamanis , W. Cheung , ited genetic deficiencies, and (ii) gene correction 1 1 H. Abdulrazzak and S. Perez-Luz to repair endogenous mutations. In the case of 1Imperial College, London, UK gene addition therapy, a range recombinant viral and non-viral vector systems have been adapted A Mammalian Artificial Chromosome (MAC) to transfer genes to human and animal cells. vector should carry all the functional elements of Conversely, for gene correction strategies, the a chromosome (centromere, telomeres and repli- most successful approaches have involved the use cation origins) to allow the DNA to be main- of synthetic and chemically-modified oligonuceo- tained as an independent replicating and tides to either target base changes in specific segregating molecule in mammalian cells. MACs genes, or to modulate gene expression at tran- carrying large genomic regions are potentially scriptional and post-transcriptional levels. Recent useful gene therapy vectors as they should com- advances in both areas will be described in the bine long-term stable maintenance with full levels context of two disease scenarios: firstly the rare of tissue specific and controlled expression from the inherited neuromuscular disease, Duchenne mus- large DNA inserts. As MACs carry no viral DNA cular dystrophy, and secondly in the context of there should be few immunological problems and atherosclerosis, a common disorder which repre- as they are maintained as episomes there is little sents a healthcare issue of virtual epidemic pro- danger of mutagenesis due to integration. portions, and for which no effective non-invasive We have utilized homologous recombination in therapy exists. In particular, specific case studies E. coli using the Red genes to recombine BACs car- will be presented in relation to preclinical investi- rying large genomic regions and alphoid DNA gations with experimental animal models. into a single BAC vector which can form MACs when transfected into HT1080 cells. We have also L114 developed a system using Red recombination which allows one to link overlapping BACs and DNA and chromatin structures have used this system to recombine three over- required for de novo formation of a lapping BACs covering the entire CFTR gene with human arti¢cial chromosome all its regulatory elements into a single BAC clone, H. Masumoto1, H. Nakashima2, M. Nakano1 and also to recombine two overlapping BACs cov- 1 ering the entire Factor VIII gene into a single and J. Ohzeki BAC. We are using these BAC/MAC vectors to 1LBC, NCI/NIH, Bldg37, Rm5040, 9000 study methods for delivery of large DNA into cells Rockville Pike, Bethesda, MD 20892, USA; in vitro and in vivo, MAC formation in a variety of 2Graduate School of Science, Nagoya Univ., cell lines and gene expression from MAC vectors. Nagoya 464-8602, Japan

L113 HAC formation assays with mutation analyses indicated an obvious requirement of alpha- Approaches to genetic therapy for satellite (alphoid) DNA and functional CENP- neuromuscular and cardiovascular B boxes reflecting human normal centromere diseases: gene addition and gene DNA configurations for de novo centromere correction strategies assembly. However, this de novo-created HAC has some unsolved problems. All the alphoid G. Dickson1 YAC/BAC DNA introduced into cultured 102 15th ICC: Abstracts human cells were multimerized. A strong tran- Artificial chromosomes containing the HPRT scriptional activity from a gene on the YAC gene, were used successfully in our group to comp- arms was conflicted with the HAC formation. lement the defect in human HT1080 HPRT- defi- Before applying the HAC as a therapeutic gene ciency cells. We are investigating a system for delivery vector, it is necessary to overcome efficient delivery of HAC vectors to other cell these unsolved problems. The first-generation types based on the Herpes Simplex Virus-1 ampli- HACs still have a crucial advantage for identi- con technology. We are also establishing artificial fying the important as yet undefined property chromosomes in murine cells to develop novel or structure required for a stable human chro- gene expression vectors which will be important mosome because they consist entirely of intro- for investigation of human transgene expression duced DNA molecules. Our data obtained from in murine models of human genetic diseases. analyses of the chromatin structures on the stable HAC and on ectopic alphoid YAC inte- gration sites indicate that the active state of the L116 centromere is generated and/or maintained in Assaying centromere assembly using association with an open chromatin structure on the alphoid YAC multimer, and the inactive engineered mini-chromosomes and state of centromere is rather linked with a silent novel site-speci¢c recombinases and suppressed structure like heterochromatin. W. Brown1, F. Dafhnis-Calas1, S. Malla1, And, either an active or inactive state of the Z. Xu1 and M. Smith2 centromerecanbeconvertedbyastructuralchange in the chromatin on type-I alphoid DNA. I 1Institute of Genetics, Queens Medical Centre, also discuss the effect of the strong transcription. Nottingham University, NG7 2UH, UK; 2 For the efficient de novo centromere assembly Institute of Medical Sciences, University of and HAC formation it might be necessary to Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, control such a delicate balance between open Scotland, UK chromatin formation on the introduced naked type-I alphoid DNA and heterochromatiniza- We discovered that engineered human mini-chro- tion pressure on the naked DNA. mosomes with limiting amounts of centromeric DNA assemble centromeres in chicken and human cells but fail to do so in mouse cells where L115 consequently they segregate inaccurately. This Establishing arti¢cial chromosomes observation is important because it provides the basis of an assay for the ability of a piece of DNA in human and murine cells to be assembled into a centromere. In order to Z. Larin Monaco1 exploit this potential we have developed a new set 1 of techniques and reagents that allow us to build Weatherall Institute of Molecular Medicine, and swap centromeres on mini-chromosomes. University of Oxford, John Radcliffe Hospital, Our techniques enable the assembly and replace- Oxford OX3 9DS, UK ment of long tracts (>150 kb) of DNA of defined sequence organization on mini-chromosomes. Human artificial chromosomes (HACs) are auton- omous molecules that can function and segregate as normal chromosomes in human cells. They are L117 generated as newly formed (de novo) artificial chromosomes in human cells following the intro- Characterization of euchromatin and duction of vectors containing human alpha satel- heterochromatin on human arti¢cial lite (alphoid) DNA, which is the primary chromosomes sequence present at all human centromeres. We 1 2 3 are developing artificial chromosomes for gene B. Grimes , J. Babcock , M. Rudd , expression studies in human and murine cells. B. Chadwick3 and H. Willard3 15th ICC: Abstracts 103

1Department of Medical and Molecular Genetics, 1Division of Biomedical Sciences and MRC Indiana University School of Medicine, 975 W. Clinical Sciences Centre, Imperial College, Walnut St, IB130, Indianapolis, IN 46202, USA.; London, UK 2Department of Genetics, Center for Human Genetics, Case Western Reserve University For gene therapy to be effective in the treatment School of Medicine and University Hospitals of 3 of human diseases, vectors with sustained levels Cleveland, Cleveland, OH 44106, USA.; Institute of gene expression over long periods of time are for Genome Sciences & Policy and Department of required. The aim here is to develop vectors car- Molecular Genetics and Microbiology, Duke rying large genomic loci that are maintained as University, Durham, NC 27710, USA episomes in cells. Entire genomic loci possessing all the long-range controlling elements of a gene Formation of euchromatin on artificial chromo- should confer tissue-specific and regulated somes is considered important for creating a sui- expression. Episomal vectors should allow long- table chromosome environment for gene term maintenance of the DNA, even in dividing expression in gene transfer applications. In addi- cells, without the hazard of integration. tion, deposition of heterochromatin on artificial chromosomes may be important for centromere The oriP and EBNA-1 components from function. To evaluate the chromatin composition Epstein-Barr virus allow e⁄cient maintenance of of human artificial chromosomes, we have used DNA as multicopy episomes in human and mouse indirect immunofluorescence on metaphase cells. A 170-kb BAC vector carrying the intact spreads to characterize markers of either euchro- human HPRT genomic locus and oriP/EBNA-1 matin (H3DimK4 modified nucleosomes) or hetero- was transfected into mouse B16F10 cells and stable chromatin (H3MeK9 modified nucleosomes and cell lines established. The BAC was maintained as HP1-alpha) on a panel of human artificial chromo- an intact, unrearranged episome in about half of the somes derived from higher-order repeat alpha satel- cell lines and expression of human HPRT was lar- lite from chromosomes X or 17. Whilst all artificial gely proportional to copy number. chromosomes assembled euchromatin markers, Alternatively, vectors with centromeric alphoid deposition of heterochromatin was variable. Lar- DNA from mammalian chromosomes can be ger artificial chromosomes (3–10 Mb) were enri- maintained as arti¢cial chromosomes following de novo ched for heterochromatin and replicated later in S centromere formation. A BAC carrying phase while smaller artificial chromosomes the entire HPRT locus and 70 kb of alphoid DNA (<3 Mb) were depleted for heterochromatin, lar- was transfected into HPRT-negative HT1080 de novo gely euchromatic and replicated earlier in S phase. cells and formed a mammalian arti¢cial chromosome in 2 of the 14 cell lines analysed. Since these data suggest that the chromatin envir- onment on arti¢cial chromosomes is likely to be Both cell lines expressed HPRT and grew in the presence of HAT selection. broadly permissive for gene expression, they lend We are continuing to investigate the relative further support for the proposed development of merits of oriP/EBNA-1 and MAC vectors carry- arti¢cial chromosomes as gene delivery vectors. ing large genomic loci as gene therapy vectors. Further, it appears that heterochromatin can be entirely dispensible or required only in small amounts for human centromere function. L119 The ACE system: a chromosome engineering and delivery technology L118 for cell-based gene therapy Development of episomal vectors C. Perez1, S. Vanderbyl, S. Stewart, carrying large genomic regions for N. Macdonald, T. Stodola, K. Mills, E. Lee, gene therapy G. Lee, A. Telenius and H. Ledebur, Jr. 1 1 1 W. Cheung , H. Abdulrazzak , G. Kotzamanis 1Chromos Molecular Systems, Inc., Burnaby, BC, and C. Huxley1 Canada V5A 1W9 104 15th ICC: Abstracts

The Artificial Chromosome Expression [ACE] Mammalian artificial chromosomes (MACs) are System is a versatile, reliable technology for the useful not only for allowing basic research into rapid generation of mammalian cells/cell lines the requirements for mammalian chromosome expressing heterologous genes. These cells are function and stability during cell division but candidates for cell-based gene therapy and/or may serve to provide a vector for the introduc- recombinant protein production at industry- tion of individual genes or a cluster of genes into relevant levels. The ACE System consists of a recipient cells. It has been shown that the intro- mammalian artificial chromosome engineered to duction of cloned human alphoid DNA into the contain multiple integration sequences for site- human fibrosarcoma cell line HT1080 is sufficient specific recombination, a targeting vector that for formation of de novo MACs. recombines onto the chromosome at the integra- HPRT-alphoidNELBAC is a construct that we tion sites to load the gene of interest, and a generated which contains the HPRT gene, 70 kb proprietary plasmid-encoded integrase that is co- of alphoid DNA as well as a neomycin resistance transfected to catalyze integration. ACE chromo- and enhanced £uorescent green protein genes. somes [ACEs] are stably maintained, non-inte- The construct was transfected into HPRT- grating, autonomously replicating, and easily negative HT1080 cells and formed a de novo MAC isolated by flow cytometry. They have been trans- in 2 of the 14 cell lines analysed. The construct was fected with standard reagents into a variety of cell also introduced into the mouse cell line, B16F10. types, including adult human mesenchymal stem Nine drug resistant clones were analysed and cells (hMSCs), and have been microinjected into found to contain genomic integration of the DNA. fertilized oocytes to generate transgenic animals. In one of these clones, the construct integrated in Using the ACE system we generated CHO and a small percentage of cells. The remainder of the LMtk- cells secreting human erythropoietin [EPO], cells contained multiple episomally maintained each of which were subcutaneously implanted into structures. These were rapidly lost upon propaga- SCID mice. Hematocrits were measured for 2^3 tion of the cells in the absence of selection. weeks post-implantation, and demonstrated ther- A second construct (70alphoidNELBAC) has apeutically signi¢cant increases in 8 mice relative been generated through partial digestion which to mice with control cells carrying unloaded lacks the HPRT gene but retains the other ele- ACEs. Recently we have engineered an ACE ments. This construct will be introduced into encoding both green £uorescent protein [GFP] and HPRT-negative HT1080 in parallel with HPRT- EPO. It was puri¢ed and transfected into hMSCs. alphoidNELBAC to assess the in£uence of the The resulting population was enriched to 20% HPRT gene on MAC formation. The construct GFP-positive cells, which secreted EPO at a rate will also be introduced into a variety of mamma- of 50^100 IU/million cells/day. EPO expression at lian cells to assess their ability to form de novo this level is comparable to what can be attained in MACs. hMSCs with retroviral vectors, suggesting that the ACE System is a novel versatile technology for PO:08:18 implementing cell˛based gene therapy. Construction of a novel human PO:08:17 arti¢cial chromosome (HAC) and its use for gene delivery The formation of de novo arti¢cial 1 2 3 chromosomes in mammalian cells M. Katoh , F. Ayabe , S. Norikane , H. Hoshiya2, X. Ren2, T. Suda1, C. Okita2 1 1 1 H. Abdulrazzak , W. Chung , G. Kotzamanis and M. Oshimura2 and C. Huxley 1Department of Human Genome Science, 1Division of Biomedical Sciences and MRC Graduate School of Medical Science, Tottori Clinical Sciences Centre, Imperial College University, Japan; 2Department of Biomedical School of Science Technology and Medicine, Science, Institute of Regenerative Medicine and London, UK Biofunction, Graduate School of Medical Science, 15th ICC: Abstracts 105

Tottori University, Japan; 3Department of Mammalian Artificial Chromosomes (MACs) can Molecular and Cell Genetics, Graduate School of be made by transfecting large (>50 kb) arrays of Medical Science, Tottori University, Japan human centromeric (alphoid) DNA into human HT1080 cells in tissue culture. The transfected Ex vivo gene delivery and auto transplantation is DNA forms stable, single-copy mini chromo- a way for gene therapy. Prerequisites for vectors somes de novo. These MACs constitute a poten- are 1) long term stable maintenance in host cells, tially useful tool for gene therapy as intact genes 2) no risk for disrupting host genome by integra- with all their regulatory elements can be cloned tion and 3) being under physiological regulation into them and they should provide long term of host cells. Human chromosome fragment expression without the dangers of mutagenesis (hCF) is transferable from donor to recipient due to integration. cells by microcell fusion in vitro. Transfer of A method for adding large sequences onto hCFs to ES cells successfully achieved delivery BACs using homologous recombination based on and functional expression of human immunoglo- the Red system has been developed. This method bulin genes in mice, suggesting the potential of was used to add a 70-kb array of alphoid DNA chromosome vector. Thus we are prompted to (which has already been shown to be able to form address the feasibility of constructing chromo- de novo centromeres) to a 150-kb BAC carrying some-based ex vivo gene delivery system. the intact human HPRT gene. The potential use- We aimed at developing a structurally de¢ned fulness of this BAC as a MAC vector for gene HAC into which circular DNA can be inserted by therapy of the Lesch-Nyhan syndrome was Cre/loxP system. The sequence de¢ned human demonstrated by de novo formation of MACs chromosome 21 was manipulated in chicken expressing the HPRT gene in HT1080 cells. DT40 cells, i.e. truncation of distal q- and p-arm MACs could also be useful vectors for gene ther- by telomere seeding and targeted introduction of apy of cystic ¢brosis. However, the intact CFTR a loxP site. These 21HAC vectors were success- gene (which spans 200-kb of genomic DNA) was fully transferred and stably maintained in human not available in a single useful vector, though it HT1080 and mouse ES cells. The GFP gene inser- was contained in three contiguous overlapping ted in the HAC expressed persistently in the BACs which have been sequenced. A method for HT1080 hybrids. These results indicated that the combining inserts of overlapping BACs using Red 21HAC vector might be useful for functional recombination has been developed. This method study of transgene in cells. wasusedtoclonethecompleteCFTRgeneinone Progress in testing the performance of the HAC BAC. This BAC is the basis for the construction vector will be also reported; 1) hepatocyte speci¢c of a MAC vector for CFTR expression. expression of reporter gene under regulation of albumin promoter, 2) induction of tissue speci¢c expression of reporter gene accompanying in vitro di¡erentiation of human Mesenchymal Stem PO:08:20 Cells and 3) construction of non-beta cells in Delivery of arti¢cial chromosomes to which insulin expression is inducible. (Correspon- human cells using HSV-1 amplicons denceto [email protected]) D. Moralli1, K. Simpson1, R. Wade-Martins2, PO:08:19 M. Assenberg1 and Z. Larin-Monaco1 Construction of mammalian arti¢cial 1Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, chromosome vectors for the expression Oxford, OX3 9DS, UK; 2The Wellcome Trust of therapeutic genes by recombineering Centre for Human Genetics, University of G. Kotzamanis1, W. Cheung1, H. Abdulrazzak1, Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK J. Gifford-garner1 and C. Huxley1 We are investigating an efficient method for 1Imperial College, London, UK delivery of HACs into human cells using a 106 15th ICC: Abstracts disabled Herpes Simplex Virus 1 system. The FVIII from cDNA minigenes due to the large HSV-1 technology includes generating infectious size of the gene (180 kb). HSV-1 amplicons in a helper virus free system According to the Ensemble database, the BAC where the HSV-1 genome lacks essential compo- RP11-524G17, belonging to the RPCI human nents for viral propagation. In this way BAC BAC library, contains the whole gene. However, vectors are packaged into an HSV-1 capsid in a after checking this BAC by restriction analysis specific cell line (African Green Monkey cells), and PCR we found that a 70-kb NotI fragment is while HSV-1 viral particles themselves cannot be not present. maintained or replicated. The amplicons which In order to make a BAC carrying the intact are generated can then transduce mammalian factor VIII gene, we have used homologous cells. We have prepared HSV-1 amplicons with recombination (the Red system) in E. coli to HAC vectors containing alpha satellite DNA and recombine together two overlapping BACs, each infected several different human cell types includ- containing part of the factor VIII gene. We now ing HT1080 (fibrosarcoma), G16-9 (glioma), have a 230-kb BAC containing not only all the MRC5V2 (lung fibroblast), 293 (kidney) and exons of the gene, but also about 50 kb upstream HaCAT (keratinocyte) cells. GFP expression was of the ¢rst exon and 30 kb downstream of the last monitored after 24 hours, and neomycin resistant exon. Hopefully this BAC includes all the reg- clones in HT1080, MRC5V2, 293 and G16-9 ulatory elements needed for full levels of tissue cells were isolated after 14 days following selec- speci¢c and controlled expression of the gene. tion with G418. We determined that the effi- In a second step we have introduced onto this ciency of transient transfection of HAC vectors BAC either oriP/EBNA-1 which confers multi- following HSV transduction is several orders of copy episomal maintenance, or alphoid DNA magnitude better than using lipofection as which is able to form mammalian arti¢cial chro- the delivery method. Positive clones have been mosomes (MACs). These constructs will be intro- selected and are being screened by FISH with duced into di¡erent human and mouse cell lines specific alpha satellite probes for the presence of and expression of factor VIII will be analysed. HACs and by immunofluorescence with CREST antisera for HACs with active centromeres. PO:08:22 The behaviour of human X PO:08:21 centromere-based minichromosomes in Construction of episomal vectors human, mouse and chicken cell lines 1 1 1 carrying the intact human factor VIII J. Spence , W. Mills and C. Farr gene for gene therapy 1Department of Genetics, University of 1 1 Cambridge, Downing St, Cambridge, S. Perez-Luz and C. Huxley CB2 3EH, UK 1Division of Biomedical Sciences and MRC Clinical Sciences Centre, Imperial College A series of linear minichromosomes (ranging in School of Science Technology and Medicine, size from several Mbs down to 450 kb) has been London, UK generated through the targeted truncation of a human X chromosome maintained in chicken Haemophilia A is a disorder caused by deficiency DT40 cells. In DT40 human minichromosomes in the activity of factor VIII. Two aspects that as small as 1 Mb are maintained very stably make this disease attractive for gene therapy are (loss rates/gen. <0.5%). The 450 kb chromosome that it is caused by mutations in just one gene has a loss rate of *4%, but has immuno- and that low levels of expression are expected to fluorescence(IF)-detectable CENP-A and -C, have a therapeutic effect. So far, all gene therapy indicating the presence of at least a partially func- approaches have been based on expression of tional centromere. In human HT1080 cells linear 15th ICC: Abstracts 107 minichromosomes of 2–3 Mb exhibit loss rates of Recombination in female mammals occurs during <0.1% and good copy number control. Struc- prenatal development, thus, direct studies of recom- tures below 1.8 Mb show increasingly higher loss bination in humans have been largely limited to the rates, with a 850 kb chromosome being lost at male. To study the factors that influence the num- *3% per gen. (again CENP-C is detectable). In ber and placement of exchange events in mammals, mouse LA-9 cells a 2.7 Mb human minichromo- we have utilized a variety of mouse models. Natu- some (with >2 Mb of DXZ1 alpha-satellite rally occurring and targeted gene mutations in mice DNA) behaves acentrically: loss rate of 5–10% are not only providing insight to the role of indivi- per gen., no IF-detectable CENP-C and a hyper- dual genes in the meiotic process but also contribut- variable copy number on selection (0 – >50/ cell). ing to our understanding of sex-specific differences. Larger human chromosomes based on the same In addition, rodent studies also suggest that envir- *3 Mb DXZ1 array have been transferred into onmental exposures have the ability to influence LA-9. A *140 Mb human X chromosome and a meiotic recombination in both males and females. 50 Mb derivative are both very stable, bind The combined results of these studies demonstrate CENP-C and show good copy number control. the complexity of the meiotic process in mammals A *10 Mb derivative, however, has low mitotic and raise a host of new and urgent questions. stability although it does show better copy Importantly, they suggest that understanding and number control than the 2.7 Mb minichromo- preserving human fertility will require the combined some, with CENP-C detectable on some chromo- efforts of reproductive biologists, geneticists, and somes. The sharply differing mitotic behaviour of toxicologists. human X centromere-based chromosomes in chicken, human and mouse cells suggests that species differences in centromere function exist. L121 How do cells hold sister chromatids Meiosis Symposium together during mitosis and meiosis? K. Nasmyth1 1 L120 IMP (Research Institute of Molecular Pathology), Dr. Bohr-Gasse 7, A-1030 Vienna, Factors in£uencing recombination Austria levels in mammals 1 There are two fundamental problems facing any cell P. Hunt trying to segregate sister DNA molecules. The first 1Department of Genetics and Center for Human is how to disentangle them and package them into a Genetics, Case Western Reserve University and confined space and the second is how to ensure that University Hospitals of Cleveland, Cleveland, sister DNA molecules are placed in opposite sides of Ohio 44106-4955 the cell. The former depends on the condensin com- plex whereas the latter relies on amphitelic attach- The production of haploid gametes requires ment of sister kinetochores to microtubules with unique modifications to the cell division process. opposite orientations. Because sister DNA mole- The most complex is the establishment of physical cules produced by replication are held together by associations between homologous chromosomes the cohesin complex, amphiletic attachment results during prophase. Studies in yeast, flies and mam- in tension. Syntelic attachments, which do not result mals have demonstrated that this physical tethering in tension, are selectively destabilized through the serves an essential function in facilitating homolog action of the Ipl1/Aurora B kinase. When and only segregation at the first meiotic division. Moreover, when all chromatid pairs have come under tension is recent studies in humans have demonstrated that not a cysteine protease called separase activated only the number of recombination events but their through the removal of its inhibitory chaperone, placement along the length of the chromosome securin, by a ubiquitin protein ligase called the influences the fidelity of chromosome segregation. Anaphase-promoting complex. Separase triggers 108 15th ICC: Abstracts disjunction of sister molecules at anaphase onset by In addition, crossover-associated recombina- cleaving cohesin’s Scc1 subunit. Both cohesin and tion foci are absent or reduced, and meiosis- condensin contain SMC proteins, dimers of which specific perinuclear telomere arrangements are form V shaped molecules with 50 nm long arms impaired. Thus, SMC1b plays a key role in with ABC-like ATPases at each head. Cohesin’s meiotic cohesion, assembly of AEs, synapsis, Scc1 subunit links its two SMC heads and thereby recombination, and chromosome movements. forms a gigantic ring structure with the potential for trapping DNA strands inside. Scc1 belongs to a superfamily of related proteins (kleisins), some of L123 which bind to SMC proteins in bacteria while oth- Establishing a molecular time scale ers bind to SMC proteins in condensin. for meiosis in human fetal oocytes P. Cohen1 and M. Lenzi1 L122 1Albert Einstein College of Medicine, SMC1b is required for mammalian Dept. of Molecular Genetics, chromosome dynamics, sister 1300 Morris Park Avenue, Bronx, New York chromatid cohesion, and DNA 10461, U.S.A. recombination in meiosis Successful meiosis is dependent on three rela- E. Revenkova1, M. Eijpe2, C. Heyting2, ted processes during prophase I: the pairing C. Hodges3, P. Hunt3, B. Liebe4, and synapsis of homologous chromosomes, H. Scherthan4 and R. Jessberger1 formation of the synaptonemal complex, and 1 the initiation and resolution of recombination Center for Gene Therapy and Molecular between homologs. The molecular mechanisms Medicine, Mount Sinai School of Medicine, underlying these events have been well studied New York, NY 10029, USA; 2 in many organisms, aided by the recent advent Molecular Genetics Group, Wageningen University, 6703 BD Wageningen, The of meiotic mutant phenotypes in mice. How- Netherlands; 3Department of Genetics, ever, our knowledge of human meiosis, parti- Case Western Reserve University, Cleveland, cularly females, remains limited because of the OH 44106; 4Max Planck Institute for Molecular difficulties associated with collection of meiotic Genetics, Berlin, Germany cells from human embryos. The importance of such studies is underscored by the fact that Sister chromatid cohesion ensures faithful seg- human female meiosis is particularly suscep- regation of chromosomes in mitosis and meio- tible to non-disjunction, with *50% of con- sis. For meiosis it has been specifically adapted ceptuses being chromosomally aneuploid as a to facilitate both meiotic divisions. Meiosis- result of errors in maternal meiosis I. Thus, specific components of the cohesin complex, the studies presented herein were aimed at including the recently described vertebrate SMC1 establishing a molecular timeframe for recom- isoform SMC1b, were suggested to be required bination in human fetal oocytes obtained from for meiotic sister chromatid cohesion and patients undergoing pregnancy terminations DNA recombination. We generated SMC1b- between 14–24 weeks gestation. deficient mice to assess the role off SMC1b. Chromosome spreads were stained for key Mice of both sexes are sterile. Male meiosis is recombination proteins, including RAD51, blocked in pachytene, female meiosis is highly RPA, and the mismatch repair proteins, MSH4, error-prone but continues until metaphase II. MSH5, MLH1 and MLH3. These latter two pro- Prophase axial elements (AEs) are dramatically teins are now accepted to be cytological markers shortened, chromatin extends further from forcrossovereventsand,inmice,thenumberof the AEs, chromosome synapsis is incomplete, MLH1/3 foci on meiotic chromosomes at pro- and sister chromatid cohesion in chromosome phase I is remarkably consistent, corresponding arms and at centromeres is prematurely lost. to the exact number of crossovers observed at 15th ICC: Abstracts 109 metaphase I. However, we show here that there wide variety of phenotypic traits of DS in humans. is much variability in MLH1/3 focus frequency Furthermore, the ES cells led to the disturbance in human fetal oocytes. These results suggest of cardiogenesis and the elevated apoptosis in that the mechanisms of recombination are pre-mature neurons in vitro. poorly regulated in human oocytes and might in Two-dimensional electrophoresis revealed that part explain the high rate of aneuploidy the endogenous mouse mlc2a and PEBP proteins observed in human oocytes. were remarkably downregulated in hearts of the chimeric mice. Further, we truncated the Chr 21 at ETS2 loci (Chr 21E) in homologous recombina- L124 tion pro¢cient chicken DT40 cells and made chi- Plasticity of histone methylation states meric mice containing the Chr 21E. Expression of PEBP and mlc2a was not repressed in Chr 21E chi- during mammalian meiosis meras, suggesting that gene(s) from ETS2 to the A. Peters1 21qter control expression of these proteins in DS. 1 However, each chimeric mouse has a di¡erent Novartis Research Foundation, Institute of pattern of trisomic and euploid cells, compli- Molecular Pathology, Vienna, Austria cating analysis. We are developing mice that have Chr 21 and de¢ned fragments of it translocated to No abstract was submitted for this talk. a mouse chromosome, using a combination of tel- omere-directed chromosome truncation in DT40 L125 cells and Cre-loxP mediated chromosome translo- cation in ES cells. These mice are expected to Towards production of a new mouse allow stable germ-line transmission of the de¢ned model for Down syndrome Chr 21, circumventing the inherent di⁄culties in Y. Kazuki1, M. Kimura2, Y. Kai1, S. Abe2, analysis of chimeras. The current progress and les- C. Okita2, Y. Shirayoshi1, T. Wakayama3, sons learned from the project will be introduced. K. Hanaoka4, K. Tomizuka5 and M. Oshimura2 L126 1Department of Molecular and Cell Genetics, Recurrent trisomy 21: four cases in Graduate School of Medical Science, Tottori three generations University, Japan; 2Department of Biomedical Science, Institute of Regenerative Medicine and J. Gair1, R. Rupps1, L. Arbour1, R. Jiang1, Biofunction, Graduate School of Medical Science, H. Bruyere2 and W. Robinson1 Tottori University, Japan; 3Laboratory for 1 Genome Reprogramming, RIKEN Center for Department of Medical Genetics, University 2 Developmental Biology, Japan; 4Laboratory of of British Columbia, Canada; Department Molecular Embryology, Department of of Pathology, University of British Bioscience, Kitasato University School of Science, Columbia,Vancouver BC, Canada Japan; 5Pharmaceutical Research Laboratory, KIRIN Brewery Co., Ltd., Japan While gonadal mosaicism can lead to recurrence of trisomy 21 for a single couple, recurrence of Trisomy 21 is the most common live-born human free trisomy 21 in multiple members of a single aneuploidy. It results in a constellation of fea- pedigree has rarely been reported. We hereby pre- tures known as Down syndrome (DS). To inves- sent an unusual pedigree with four cases of Down tigate the gene dosage effects of an extra copy of syndrome (DS) born to four separate women rela- human chromosome 21 (Chr 21) on various DS ted through three generations of one family. The phenotypes, we generated chimeric mice carrying mothers were aged 18, 21, 29 and *30 at the an intact Chr 21 via microcell-mediated chromo- time of the births. G-banded karyotypes in two of some transfer (MMCT) into mouse embryonic the mothers of DS individuals were normal and stem (ES) cells. These chimeric mice showed a karyotypes of all of the DS individuals showed 110 15th ICC: Abstracts free trisomy 21. Microsatellites spanning chromo- A semen sample was obtained and processed for some 21 were typed in this family to determine if meiotic studies. Prior to the application of any regions were shared in common among the M-FISH protocol (determination of metaphase I mothers of the DS children. The centromeric and con¢guration and segregation), MI and MII sper- telomeric regions of chromosome 21 could be matocytes images were captured and coordinates excluded from being shared, and of those tested, were noted. M-FISH was subsequently applied only the markers D21S215 and D21S258 and analysis was performed using a Cytovision (21q11.2) and D21S1440 (21q22.3) were poten- 2.7 system (Applied Imaging Corporation). FISH tially shared. Two members of the pedigree techniques were used to determine the incidence including one DS mother carried supernumerary of normal/balanced sperm. Embryos obtained alleles at markers 2503J9TG, D21S369 and from two IVF-PGD cycles were also screened. D21S215, which span the region from 21p– Chromosome content and segregation patterns 21q11.1. FISH studies with probes for the chro- were inferred from FISH signal evaluation. Inter- mosome 13/21 centromere and 21q22 showed an chromosomal e¡ects (ICE) over chromosomes 18, enlarged signal at the centromere and a dimin- 21, 22, X and Y was evaluated (Multi-FISH; ished signal at 21q22, compared to a control aneuploidy screening in sperm and embryos). slide, on one of the chromosomes. The level of Chain con¢guration was the most frequent meiotic recombination on chromosome 21 was ¢gure observed in MI spermatocytes (77.27%). unusually high in this family as well. We present Normal/balanced sperm and embryos were pre- a model (currently being tested) whereby a cryptic sent in 86.48% and 61.54% of the analyzed cells inversion with breakpoints in 21q11.2 and 21q22.1 respectively. Screening of aneuploidies in sperm explains the unusual findings in this family. revealed negative ICE. Overall, data obtained resulted in an exhaustive cytogenetic evaluation of the patient addressed to PO:08:23 an accurate reproductive counseling. Spermatocytes, spermatozoa and Acknowledgments: Project-SAF2003-04312 (Spain) embryos: FISH study of a Robertsonian translocation t(13;14)(q10;q10) PO:08:24 J. Blanco1, Z. Sarrate1, E. Anton1, Differing roles of the Rad51 M. Parriego1, J. Santalo1, J. Egozcue1 1 paralogues in mitotic and meiotic and F. Vidal homologous recombination. 1 Unitat de Biologia Celular. Facultat de Ciencies. 1 1 1 Universitat Autonoma de Barcelona, Spain J. Bleuyard , M. Gallego and C. White 1CNRS UMR6547, Universite´ Blaise Pascal, 24, As a consequence of the meiotic behavior of avenue des Landais, 63177 Aubiere, France translocated chromosomes, Robertsonian hetero- zygous carriers produce high numbers of unba- Since its identification as the eukaryotic homo- lanced sperm. An integral meiotic study, from logue of the bacterial recombinase RecA, the spermatocytes to spermatozoa, could be useful to Rad51 protein has been the object of consider- understand the segregation pattern of the reorga- able interest. Rad51 catalyses the strand nized chromosomes and to determine the risk of exchange reaction, which is the motor of homo- transmission to offspring. Moreover, in carrier logous recombination (HR) events. Rad51 is thus couples, embryos resulting from IVF cycles can involved in a number of vital cellular processes be analyzed through the application of PGD including recombination, chromosome main- techniques. tenance and meiosis. We report the meiotic behavior of a Robert- In addition to Rad51, ¢ve paralogues of sonian translocation carrier t(13;14)(q10;q10). Rad51 have been shown to play roles in mitotic 15th ICC: Abstracts 111 recombination, DNA repair and chromosome sta- localised on the XY body at pachynema. In order bility. These proteins are essential for embryonic to analyse the potential effect of radiation- viability in vertebrates and this fact has meant induced DSBs during meiosis on meiotic recombi- that understanding of their roles in meiosis is rela- nation, we performed immunodetection of DSBs tively limited. We have recently reported that the and crossovers on mouse spermatocyte chromo- Arabidopsis homologue of one of these Rad51 some spreads with gamma-H2AX and MLH1 paralogues (ATXRCC3) is involved in DNA antibodies, respectively. In non-irradiated pachy- repair and that its absence leads to a sterility phe- nema, numerous small gamma-H2AX foci were notype due to extensive chromosome fragmenta- observed along the pairing region of homologous tion during meiosis, ¢rst visible in diplotene of chromosomes (synaptonemal complex SC). meioticprophase(1).Wehavenowidenti¢edAra- Gamma-radiation exposure at pachynema bidopsis mutants of three other Rad51 paralogues induced large gamma-H2AX foci assumed to deco- (ATRAD51B, ATRAD51C and ATXRCC2). rate DNA loops attached to the SC. The number While disruption of any of the paralogues confers of MLH1 foci was unchanged, which suggests hypersensitivity to DNA damaging treatments, that these DSBs were repaired by gene conver- the atrad51c mutant is the only one to show dra- sion or alternative recombination pathways. By con- matic meiosis defects. Our results point to di¡er- trast, after a low dose of gamma-radiation given ing roles for Rad51 paralogues in mitotic and at preleptotene/leptotene stage, a significant ele- meiotic HR. While the Rad51C/Xrcc3 complex is vated rate of gamma-H2AX foci was observed involved for both mitotic and meiotic HR, the along the SC at early pachynema. An increased Rad51B/C/D/Xrcc2 complex is only required for rate of MLH1 foci was also found at late pachy- mitotic HR. nema and we propose that some radiation- induced DSBs were repaired by the homologous (1) Bleuyard et al (2004) EMBO J 23: 439–449 recombination pathway. All together, these results will permit to better understand the repair PO:08:25 process of radiation-induced DSBs in meiosis. In situ analysis of radiation-induced DNA double-strand breaks and their PO:08:26 repair by homologous recombination Chromosome synapsis under the effect during murine male meiosis of colchicine in wheat A. Chicheportiche1, J. Bernardino-Sgherri1 E. Corredor1 and T. Naranjo1 and B. Dutrillaux1 1Departamento de Gene´tica, Facultad de 1Unite´ Mixte de Game´togenise et Ge´notoxicite´, Biologia, Universidad Complutense, 28040 INSERM U566-CEA-Paris VII, BP Ni6 F-92260 Madrid, Spain Fontenay-aux-roses, France Colchicine has been described as an agent able to DNA double strand breaks (DSBs) are the major affect chromosome pairing at metaphase I in cytotoxic lesions induced by ionising radiation bread wheat when applied at premeiotic inter- leading to chromosomal aberrations. During pro- phase. With the aim of investigating the effect of phase I of meiosis, programmed DSBs are gener- colchicine on chromosome synapsis, immature ated by the transesterase SPO11 to initiate spikes of wheat carrying two rye telosomes were homologous recombination involved in crossing- injected with colchicine. The behaviour of the rye overs. These events are required for normal hap- homologous chromosomes was studied using loid gamete production. Phosphorylated H2AX multiple FISH with telomere, a rye-specific cen- histone (gamma-H2AX) has been shown to point tromere and rye genomic DNA probes performed out DSBs and participate in their repair. During on spread cells of anthers collected between two meiosis, it is associated with Spo11-dependent and four days after colchicine injection. In meio- DSBs at leptonema and zygonema and is cytes of the control plants, chromosome synapsis 112 15th ICC: Abstracts starts at the bouquet stage, by both the telomeric collected two or three days after colchicine treat- and the centromeric chromosome ends, provided ment and the centromere and telomere position the centromeric end is incorporated to the revealed by two colours fluorescence in situ telomeres cluster. Synapsis progress quickly and hybridisation with centromere and telomere can be completed before the bouquet is dis- DNA probes. At premeiotic interphase the num- organized. Meiocytes at early prophase I from ber of centromere signals is close to the haploid anthers collected two days after colchicine appli- number. At the early bouquet stage a number of cation showed a similar behaviour to that of centromeres associate in multimeric structures untreated material. However, meiocytes at pro- but other remain associated in pairs or separated phase I of anthers collected three or four days as deduced from the observation of both big and after colchicine application showed a delay in the small fluorescence signals. Complex centromere initiation and development of chromosome clusters get resolved in centromere pairs before synapsis. In these cells, with rye chromosomes the bouquet is disorganized. Colchicine does not completely separated in some cases, the level of affect centromere association. By contrast, telo- synapsis between rye chromosomes was lower mere grouping is severely affected by the colchi- than in the control material. Meiocytes affected cine action. Nuclei that started meiosis in the by colchicine treatment showed a preference for presence of colchicine show multiple telomere the initiation of synapsis at the telomeric end of clusters scattered throughout the nuclear mem- the rye telosomes. At diplotene-diakinesis, rye brane in the hemisphere opposite the centro- univalents were observed in all treated plants, meres. Migration of dispersed telomere clusters prompting a possible effect of colchicine on to the telomere site is impeded by the colchicine chiasma formation in addition to that on the action. This arrangement may represent an inter- initiation of synapsis. mediate stage of the bouquet formation.

PO:08:28 PO:08:27 Cytogenetic processes of Association and migration of telomeres Secalotriticum genome formation are two steps of the bouquet I. Gordei1 and O. Lyusikov1 organization in hexaploid wheat 1 1 1 Institute of Genetics and Cytology at National E. Corredor and T. Naranjo Academy of Sciences of Belarus 1Departamento de Gene´tica, Facultad de Biologia, Universidad Complutense, 28040 Investigations on development of secalotriticum– Madrid, Spain rye-wheat amphidiploids with rye cytoplasm were carried out with a view to intensify rye genome Chromosomes adopt the bouquet configuration expression for increasing ecological resistance of at the onset of meiosis in polyploid wheats. Con- remote rye x wheat hybrids (Inventor’s certificate comitant to the congregation of telomeres, cen- 1734602). tromeres form complex multimeric structures in Genome and chromosome composition of rye- the opposite pole. Earlier works demonstrated triticale pentaploids of F1 (RRABR, 5/35) the existence of a premeiotic interphase stage de¢nes meiosis distinctions and is a key stage in sensible to the colchicine action. Application of secalotriticum genome formation. A high level of colchicine at this stage causes irregular chromo- homeologous chromosome pairing of A- and B- some pairing at metaphase I. Whether colchicine genomes was observed in hybrid diakinesis. It was action is related to centromere association and related with inhibition of wheat Ph-system with a chromosome movements produced during the triple dose of rye genes under conditions of rye bouquet formation has been studied in meiotic cytoplasm (with 21 univalents expected, on the cells isolated from immature spikes injected with average 11,4I þ 6,3IIring þ 4,4IIrod þ 0,6III þ 0,1 colchicine. Anthers at early meiotic stages were were observed). Equational division of univalents 15th ICC: Abstracts 113 turned out a basic source of meiosis anomalies. the DNA mismatch repair protein MLH1 sug- As a result, gametes varied from 14 to 28 in the gested that while there might be inter-individual chromosome number in the F1 hybrids. Classes variation there was no consistent difference 17^18 (33,2%), 14 (27,2%) and 21 (24,8%) pre- between sexes. Thus the higher rate of recombi- vailed, unreduced gametes with 35 chromosomes nation in human oocytes is not a consequence of did not occur. Gametes with 21 chromosomes more closely spaced crossovers along the chro- (7R7A7B) balanced for genome composition were mosomes. We propose that SC length, and there- formed at partial meiotic unreduction with the fre- fore the size of the physical platform for quency of 0,3^2,2%. This process includes a nor- crossing-over, is the principal factor determining mal division of rye bivalents, predominantly the difference in rate of recombination in male and reduction division and uneven segregation of A-, female germ cells. One fundamental outstanding B- and R-univalents, or equational division and question is the background for the sex-specific meiotic elimination of R-univalents. Partially, architecture of meiotic pachytene chromosomes. unreduced gametes were the basis for producing This could be manifested, for example, by varia- secalotriticum according to the applied scheme tions in the number and sizes of the chromatin already in the second generation (F1BC1 was iden- loops associated with the axes of pachytene ti¢ed by C-banding). chromosomes. Our preliminary FISH observa- The developed secalotriticum is a genome- tions indicate that SC loops may be considerably balanced stable haxaploid form with high values smaller in oocytes than in spermatocytes. of agronomic traits.

PO:08:30 PO:08:29 Chromosome linear elements and Synatonemal complex length is the meiotic recombination hot spot principal factor determining the activation in S. pombe difference in rate of recombination R. McFarlane1, J. Loidl2, P. David1, in male and female germ cells 1 1 1 1 1 W. Jenny , S. Julia , D. Emma and M. Hulte´n and C. Tease A. Lorenz2 1 Department of Biological Sciences, University 1NWCRF Institute, School of Biological Sciences, of Warwick, Coventry CV47AL, UK University of Wales Bangor, Deiniol Road, Bangor, Gwynedd, LL57 2UW, UK; Crossing-over at meiosis can be analysed using 2Department of Cell Biology and Genetics, either genetic or cytogenetic methods (reviews in Institute of Botany, University of Vienna, Hulte´n and Tease 2003, Nature EHG 2, pp 882– Rennweg 14, A-1030, Vienna, Austria. 887 and 876–881). The cytogenetic approach provides an overview of patterns of meiotic The mechanisms regulating the function of recombination that cannot be obtained in any recombination during meiosis are heavily influ- other way. Spermatocytes have approximately 50 enced by the processes which control recombina- crossovers per cell in comparison to more than tion initiation, including homologue pairing and 70 in oocytes (corresponding to average genetic activation of specific hot spot chromatin. map lengths of 2500 and 3500 cM, respectively). We have identi¢ed Rec10 as a central reg- Meiotic prophase I bivalents (Synatonemal Com- ulator/component of the proteinacious structures plexes, SCs) are considerably longer in human known as linear elements (LEs), which form in oocytes than spermatocytes. We have examined meiotic prophase and are implicated in inter- the influence of this factor on meiotic recombina- homologue pairing. During meiotic prophase tion in male and female human germ cells. immunostaining using antibodies against Rec10 Analyses of inter-crossover distances (and pre- reveals that Rec10 is an integral part of LEs. Ana- sumptively crossover interference) along SCs by lysis of the amino acid sequence of Rec10 reveals 114 15th ICC: Abstracts a region of homology with the Red1 protein of there were some disturbances in the tapetal tis- S. cerevisiae which is essential for the formation sue. The analyses showed that microsporogenesis of structures analogous to LEs in this organism, was normal in male fertile, but in male sterile, it indicating a highly conserved mechanism for chro- was disturbed in the tetrad stage. At this time, a matinregulationinmeiosisI. degenerative process was observed. The tetrad We have identi¢ed a unique mutant allele of cell cytoplasm became wrinkled and with rec10, rec20-144. Analysis of recombination in vacuoles. The tetrad callose was thicker in male this mutant demonstrates that there are di¡er- sterile than in male-fertile plants. Section cuts ential context-dependent requirements for activa- showed that tapetal cells were slightly larger in tion of the meiotic recombination hot spot M26. male sterile than in male fertile. According to the These data implicate Rec10 in the regulation of observations, these disturbances did not involve the localised chromatin dynamics during hot spot structural chromosome changes, and chiasma activation. Previous work has also shown a simi- mean value differences in metaphase I, observed lar di¡erential requirement for RED1 in hot spot among all the plants were not significant. The activation in S. cerevisiae, providing further evi- degenerative process also took place in young dence of a highly conserved function for LE com- male buds; they became shrunken, full of aborted ponents. Given that the majority of meiotic mature tetrads and fell down. It seems that this recombination is likely to be initiated at hot process is under nuclear control since half of the spots, the study of the role of Rec10 and LEs in offspring inherited it. Male sterile may be a hot spot activation gives us an important insight useful tool in breeding programs. into the mechanisms regulating proper chromo- some segregation during meiosis. PO:08:32 Rec8 distribution throughout meiotic PO:08:31 prophase I in euploid and aneuploid Cytological analyses on a male sterile human oocytes ramie (Boehmeria nivea Gaud.) and in 1 1 1 1 some of its F1 offspring P. Robles , R. Garcia , I. Roig , J. Egozcue and M. Garcia1 1 1 1 N. Pierozzi , C. Pinto Maglio , R. Baroni 1 2 Unitat de Biologia, Departament de Biologia and R. Benatti Jr. Celular, Fisiologia i Immunologia, Facultat de 1CPD Recursos Gene´ticos Vegetais, Instituto Medicina, Universitat Autonoma de Barcelona, Agron Mico (IAC), Av. Baro de Itapura 1481, 08193 Bellaterra, Spain Campinas, SP, Brasil, CEP:13001-970; 2NPD Jardim Botanico, IAC, Brasil Recent publications have shown the role of cohe- sin Rec8 in sister chromatid cohesion during Boehmeria nivea (ramie) is a perennial, wind pol- mammalian meiosis. Most of the studies are linated, monoecious plant, native from Asia, with based on male meiosis since the meiotic prophase small diclinous flowers. It is known for its textile I occurs during the fetal life period in female, so fibers. Cytological analyses were carried out on a few samples are available. male sterile diploid ramie plant, obtained after a We have studied the distribution of Rec8 previous treatment with colchicine solution for throughout meiotic prophase I in human oocytes 72 hours in seed phase, and on a normal male applying inmuno£uorescence against Rec8 and fertile control plant and in some F1 descendents SCP1, which was used as control to elucidate the of this crossing. Young buds in different develop- pairing process since it appears at synapsed ing phases were collected, fixed in Carnoy and regions. stored. Cytological preparations were done by Rec8 appeared at preleptotene, the signal was squashing with acetic carmine. Some buds were dispersed whereas at leptotene and zygotene was selected for histological section cuts to know if organized in a ¢brillar pattern forming the axial 15th ICC: Abstracts 115 elements of the synaptonemal complex. In addi- During zygotene, pairing of the homologues chro- tion in this stage, zygotene, colocalization of mosomes occurs, then oocytes having three sig- SCP1 and Rec8 was observed in the synapsed nals (21.67%), two (36.67%) and one signal regions. At pachytene homologues pairing was (41.67%) were found. At pachytene, the vast completed and colocalization was observed along majority of the oocytes (91.92%) analysed pre- the whole length of the synapsed chromosomes. sented one single signal, suggesting that pairing of At diplotene, Rec8 staining was retained along the three homologues 18 was completed. Never- the ¢bers. theless some oocytes presented two separate sig- For the ¢rst time, we also studied Rec8 pattern nals, most likely corresponding to a bivalent plus in aneuploid fetuses: (47,XX þ 21), (47,XX þ 18) a univalent. Diplotene oocytes displayed gen- and (45,XO-47,XXX). Rec8 was always observed erally one signal (83.33%). This results suggest along the whole length of all the homologues. that chromosome 18 pairing is an e⁄cient process Alignment of the three homologues at pachytene which ensures formation of a bivalent before in trisomic oocytes 18, 21 and X was observed ori- pachytene in trisomic cases. In this sense, we com- ginating a bivalent plus an univalent partially monly observed at zygotene that trivalent forma- synapsed, a bivalent plus a completely unsy- tion, or alignment of the three chromosomes 18, napsed univalent or a complete synaptic adjust- was preceded by the existence of a bivalent plus ment originating a trivalent. an univalent. In conclusion, Rec8 staining dynamics Surprisingly, presence of an extra chromosome observed in euploid and aneuploid oocytes in this 18 had not detectable e¡ects on other chromo- study are in agreement with previous studies pub- somes pairing. Chromosome 13 pairing study in lished. Furthermore, existence of synaptic adjust- aneuploid and euploid cases gave non distinguish- ment may delay meiotic prophase progression in able results. aneuploid oocytes. These ¢ndings supports the existence of a pair- ing checkpoint at human female pachytene and PO:08:33 contradict the hypothesis of an interchromosomal e¡ect seen in other studies. Homologue chromosomes pairing process analysis in 47,XX þ 18 human oocytes PO:08:34 I. Roig1, P. Robles1, R. Garcia1, J. Egozcue1, Meiotic studies in OAT patients using H. Schertan2 and M. Garcia1 M-FISH 1Departament de Biologia Celular, Fisiologia i Z. Sarrate1, J. Blanco1, S. Egozcue1, F. Vidal1 Immunologia.Universitat Autonoma de and J. Egozcue1 Barcelona, 08193-Bellaterra, Spain; 2Inst.Radiation Biology, BW, Neuherbergstr.11, 1Unitat Biologia Celular, Universitat Autonoma 80937-Mu¨nchen, Germany Barcelona, Bellaterra 08193, Barcelona, Spain

Aneuploidy is one of the major causes of preg- A method combining the analysis of classical nancy loss in humans and it is mainly caused by meiotic preparations followed by M-FISH has chromosome non-disjunction during oogenesis. been developed to assess the frequency of chias- Little is known about mammalian female meiotic mata and to determine if anomalies affect specific first stages because these ocurr during fetal life bivalents in testicular material. period. We performed an analysis of the homo- Samples were obtained by unilateral biopsy logues chromosomes pairing process occurring at from 8 OAT patients and processed for meiotic meiotic prophase in a 47, XX þ 18 case and a studies. Prior to the application of M-FISH proto- euploid case by FISH. col, slides were stained with Leishman. MI and II At leptotene, most oocytes (71.43%) presented spermatocyte images were captured, coordinates three independent chromosome 18 signals. were noted and chiasma frequency was 116 15th ICC: Abstracts determined for every single bivalent. M-FISH pro- by transposable elements and viruses. Silencing cedures were subsequently applied (Spectra of unpaired segments during meiosis has also Vysion Assay, Vysis Inc) and analysis was per- been documented in Caenorhabditis elegans;in formed using a Cytovision 2.7 system (Applied this case silencing of the single X chromosome Imaging Corporation). during XO male meiosis is critical for imprinted Speci¢c results for every single bivalent (rang, X inactivation in XX offspring, and is abolished mean and standard deviation) in all cells studied when the single X chromosome is provided with were obtained through the analysis of 113 MI a pairing partner (XX, sex reversed males). We spermatocytes. Overall, a slight reduction in the now demonstrate that silencing of unpaired number of autosomal chiasma frequency was meiotic DNA also takes place in the mouse. In detected (47.8) compared to controls (51.3; mouse mutants harbouring errors in meiotic pair- Laurie and Hulte¤ n, 1985). Sex bivalent was ing (synapsis) such as XO females, males carry- observed in 83.3% of the MI analyzed. Synaptic ing the X-autosome translocation T(X;16)16H anomalies a¡ecting the sex chromosomes (struc- and Msh5 null males, unpaired chromosome seg- tural reorganization), 9 (break) and 22 (asy- ments are transcriptionally silenced, as revealed napsis) were observed in three di¡erent cells. by a recently developed Cot-1 RNA FISH tech- 43 MII cells were analyzed. Chromosome nique that identifies nascent nuclear transcripts. anomalies were detected for chromosome 2 This silencing mechanism is dependent upon the (2.6%; abnormal morphology), 3 (5.1%; break), 6 tumour suppressor gene Brca1, which has pre- (2.4%; break), 9 (7.3%; break) and gonosomes viously been implicated in two related process; (2.6%; numerical anomaly). Somatic X Chromosome Inactivation and Meio- Results con¢rm the high presence of synaptic tic Sex Chromosome Inactivation (MSCI). Silen- errors resulting in a reduction in the number of cing of unpaired DNA during mouse meiosis chiasmata in OAT patients. Non-disjunctional provides novel insights into the relationship errors or small reorganizations, overlooked in between asynapsis and sterility in mammals classic meiotic preparations, were identi¢ed. Pre- and a broader understanding of the biology of liminary results seem to indicate that gonosomes Brca1. and chromosome 9 are more a¡ected by synaptic anomalies than others.

Acknowledgments: Project-SAF2003-04312 (Spain) PO:08:36 Human arti¢cial chromosomes as tools for transgenesis and for analysing PO:08:35 mammalian meiosis. Silencing of unpaired meiotic DNA T. Voet1, J. Vermeesch1, E. Schoenmakers2, 3 4 1 in the mouse B. Liebe , S. Carpentier , C. Labaere , J. Verbeeck1, H. Scherthan3 and P. Marynen1 J. Turner1, S. Mahadevaiah1, C. Deng2 1 and P. Burgoyne1 Department of Human Genetics, University of Leuven, Belgium; 2Department of Medicine, 1MRC National Institute for Medical Research, University of Cambridge, UK; London, UK; 2National Institutes of Health, 3Max-Planck-Institute for Molecular Genetics, Bethesda, Maryland, USA Berlin, Germany; 4Laboratory of tropical plant sciences, University of Leuven, Belgium In Neurospora, DNA unpaired in meiosis is both silenced and induces silencing of all DNA homo- We have generated a circular chromosomal vec- logous to it, including genes that are themselves tor (CV) consisting of human alphoid 20 and paired. This mechanism has been called Meiotic 1p21-22 sequences, while detectable telomeres are Silencing by Unpaired DNA (MSUD) and is lacking. In addition to transgenic applications, thought to protect the host genome from invasion such artificial chromosomes represent a powerful 15th ICC: Abstracts 117 technology for studying meiotic chromosome A balanced view is that the study of chromo- behaviour and checkpoints. somes should form a small but important part of Proof-of-principle was provided for both: (1) population genetics. Chromosomal variants are the CV is mitotically stable in di¡erent mamma- easily scored characters that can, for instance, be lian cell lines, (2) the CV is transferrable between valuable as makers in phylogeography and for cell lines by microcell-mediated chromosome inferring population structure. They can also transfer, (3) plasmids and PACs can be inserted reveal population genetic principles such as the by the Cre/loxP system without disturbing its rele- selection pressures involved in meiotic drive and vant properties, (4) mouse ES-cells carrying the in the maintenance of hybrid zones. However, CV with or without insert can be used to generate chromosomal variants also have a role as evolu- trans-chromosomal mice, (5) genes present on the tionary agents in their own right. They may, for CV are expressed in a tissue-speci¢c manner at example, be involved in maintaining high genic levels compatible with the hemizygous presence variation and in speciation. This talk will run of the transgene and (6) male and female mice, through these various aspects of population cyto- either monosomic or disomic for the CV, show genetics as a prelude for the detailed case studies germline transmission of the CV. Detailed sper- that follow. matogenic analysis revealed a telomere-indepen- dent mechanism for homologous CV pairing and L128 illuminate escape routes to meiotic checkpoints. Recently, linearisation of the circular CV with On the role of chromosomal the CRE/loxP system was performed. Potential rearrangements in speciation linear CVs were detected by telomeric PNA-FISH K. Livingstone1 and L. Rieseberg2 and are currently being characterised in cell lines. Comparing the mouse meiotic data of the circular 1Department of Biology, Trinity University, San CV with this of a telomere-bearing version will Antonio, TX, USA; 2Department of Biology, further clarify the relevance of functional telo- Indiana University, Bloomington, IN, USA meres on chromosomes in mammalian bouquet formation, in bouquet-facilitated homologue pair- Recent data from a wide range of organisms and ing and in meiotic checkpoint bypass. Addition- new theoretical developments have again sug- ally, a linear CV will bene¢t from improved gested that chromosomal rearrangements may mitotic stability as it will eliminate CRE/loxP- have a causative role in the speciation process. and sister chromatid exchange (SCE)-mediated These developments will be reviewed along with duplications. new data from the Solanaceae in support of this hypothesis.

Population Cytogenetics Symposium L129 How much is temperature responsible L127 for latitudinal chromosomal clines in Population cytogenetics: the study of Drosophila? An experimental approach chromosome variation in natural A. Fontdevila1, M. Santos1, L. Serra2, 2 1 populations J. Balanya and W. Ce´spedes 1 J. Searle1 Departament de Genetica i Microbiologia, Universitat Autonoma de Barcelona; 1Department of Biology, University of York, 2Departament de Genetica, Universitat de PO Box 373, York YO10 5YW, UK Barcelona, Spain

Chromosomes have drifted in and out of favour Chromosomal polymorphism in Drosophila sub- as subject material for population geneticists. obscura shows concordant latitudinal clines 118 15th ICC: Abstracts between old Palaearctic original populations and In order to support conservation efforts and recent colonizing populations of the Americas. to identify long-term glacial refugia in Europe This parallel observation in two hemispheres has we are working with the fern genus Asplenium been taken as the most convincing evidence that as a model system to investigate patterns and natural selection is at work. Yet, the environ- processes in polyploid plant evolution in the mental causes of these chromosomal gradients Mediterranean Basin. Long-term glacial refu- are largely unknown, being temperature the gia are areas where plants (and animals) have most favoured selective agent. In order to dis- survived successive glaciation cycles, i.e. warm entangle temperature effects, a controlled and cold stages during the Pleistocene. In experiment using laboratory populations under order to find such areas we have explored the different thermal regimes, in which other puta- discontinuities in ploidy levels, distribution tive selective agents (namely, larval density) patterns, breeding systems and genetic diver- have been kept constant across temperatures, sity in several auto- and (segmental-) allopoly- has been carried out. Results show a quick and ploid complexes in the fern genus Asplenium, consistent shift in gene arrangement frequencies combining field work and extensive laboratory in response to thermal selection regime, qualify- studies (allozyme electrophoresis and cpDNA ing temperature as an effective selective agent. sequencing). Patterns observed in three autop- However, the analysis of arrangement frequency olyploid complexes, A. ceterach, A. petrarchae changes unveils a complex pattern of response and A. trichomanes, give us interesting insights to temperature often not consistent with the into the dynamic polyploid evolution in the observed trends in natural clines. For example, Pleistocene and allow us to infer the locations Est inversion is the most responsive in both of long-term Pleistocene refugia in the natural and experimental populations, yet the Mediterranean Basin. trend observed in the latter is the opposite of that predicted from latitudinal clines. Interest- ingly, a decrease of chromosomal diversity is L131 observed at the warmest (sub-optimal) tempera- ture, which is consistent with a similar diversity Episodic patterns of Robertsonian change in long-term studies related to climate fusion; causes and consequences warming in natural D. subobscura populations. 1 In sum, our results indicate that temperature is D. Rowell an important selective agent but does not 1School of Botany and Zoology, Australian explain by itself the observed trends in chromo- National University, Canberra, Australia somal latitudinal clines. Some groups of animals experience repeated Robertsonian rearrangements while their L130 karyotypically similar relatives show marked sta- Locating long-term glacial refugia in bility. Even within these, the pattern of rearran- gement varies dramatically, with some groups the Mediterranean Basin. showing rapid and repeated fusion across the Autopolyploid complexes in Asplenium whole karyotype (‘catastrophic pan-fusion’), as a model system while others are characterized by the sporadic 1 1 1 occurrence of individual fusions followed by fixa- J. Vogel , S. Russell , M. Grundmann , tion or extinction. Fissions are considerably rarer F. Rumsey1, H. Hunt1, H. Schneider2, 3 4 5 and do not appear to follow the same patterns. I. Pinter , M. Gibby and J. Barrett The mechanism(s) by which fusion occurs has 1The Natural History Museum, London, UK; not been elucidated, although a hereditary pre- 2Universitaet Goettingen, Germany; 3University disposition to fusion in one species points toward of Budapest, Hungary; 4Royal Botanic Garden, the involvement of a p-like transposable element. Edinburgh, UK; 5University of Cambridge, UK This presentation explores characteristics of 15th ICC: Abstracts 119 fusion events and their potential adaptive L133 significance. ECARUCA: an internet-based European database for collection of L132 rare chromosomal abnormalities Identi¢cation, variation and evolution I. Feenstra1, D. Koolen1, A. Siezen1, C. Evans2, of common fragile sites J. Fang2, R. Winter3, A. Schinzel4, M. Lees3, I. Greenbaum1 and P. Dahm2 B. De Vries1 and C. Van Ravenswaaij1 1Department of Biology, Texas A&M University, 1Dept Human Genetics, Nijmegen, The College Station, TX, 77843 USA; 2Department Netherlands; 2School of Computing Science, of Statistics, Texas A&M University, College Middlesex University, London, UK; 3Institute of Station, TX 77843 USA Child Health, London, UK; 4Institute of Medical Genetics, Zurich, Switzerland Although well studied in relation to human cancer, the genetics, variation and evolution of During recent years a considerable improvement the so-called common fragile sites (CFS) in diagnostic techniques has enabled cytogeneti- remain undetermined. As molecular analyses cists to find more and smaller chromosomal have failed to identify any associated signature aberrations. However, accurate clinical knowl- sequence, studies of CFS continue to depend edge about rare chromosome disorders is fre- on analyses of the distribution of chromoso- quently lacking, mostly due to a significant mal breaks/gaps. To investigate the basic decline in published cases. On the other hand, genetics and evolution of CFS, we have adop- there is an increasing demand from parents and ted the null hypothesis that the per-individual physicians for reliable information about the dis- presence or absence of any particular CFS order of their child or patient. reflects genetic variability among individuals Therefore, we established the European Cyto- and approximates a codominant allelic model. geneticists Association Register of Unbalanced As this approach is dependent on the accurate Chromosome Aberrations; http://www.ecaruca. per-individual identification of CFS, we (Ba¨hm net/. et al. Hum Genet 95:249, 1995) developed a This internet-based database, funded by the multinomial statistical model (FSM) for the European Union, collects cytogenetic and clinical identification of CFS as chromosomal bands data of (non-) published cases with a rare chromo- that express significantly nonrandom breakage some abnormality. This can be microscopic visual relative to the distribution of total breakage in aberrations,ormicro-deletionsand^duplications. data from single individuals. FSM analyses Genetic centres can submit data to the register were used to obtain initial estimates of pre- by using the downloadable forms from the web- sence/absence variation of aphidicolin-induced site, although online-submission will be possible CFS among deer mice (Peromyscus manicula- as from June 2004. At present, ECARUCA tus) and humans. In both cases, the analyses contains around 4000 cases. indicated that most CFS are polymorphic; the The availability of collected data is subdivided number of CFS per individual ranged from 7– in a two-level system: each internet user can view 20 with approximately 75% of the CFS occur- general information about speci¢c chromosome ring at frequencies less than 0.5 and only aberrations and the accompanying clinical about 5% being fixed. Consistent with the features. Whereas, account holders can receive hypothesis that CFS variation is allelic and speci¢c data per case, including additional polymorphic, cladistic analysis of preliminary research and clinical pictures. CFS characterizations for four species of Per- The collection and exchange of clinical and tech- omyscus yielded evolutionary relationships nical knowledge will allow for accurate informa- entirely concordant with the corroborated tion on clinical aspects of rare chromosome phylogeny of these species. disorders that can be used by professionals 120 15th ICC: Abstracts involved. Moreover, correlation study of chromo- in these segments are then summarized. Poly- some aberrations and their phenotypes will be morphisms in the constitutive heterochromatin invaluable for localizing genes involved with are delineated for 600 persons of Central Eur- mental retardation and congenital anomalies. opean origin and then compared to 2 groups of different ethnic origin. Then, from the own inves- L134 tigation group, structural rearrangements with breakpoints in eu- and heterochromatic bands Determination of risk factors in are analyzed as to their frequency and aberration carriers of peri- and paracentric type. One subgroup of precisely defined chromo- inversions some aberration after QFQ-staining, namely Robertsonian chromosomal translocation C. Pagenstecher1, B. Mub1 and G. Schwanitz1 (RobCT), were studied with the aim to estimate the frequency of the unbalanced progeny in case 1Institute of Human Genetics, University Hospital Bonn, Germany of inherited form of these RobCT. Genetic risk estimation has been performed according to the In 41,000 patients chromosome investigations method of Stengel-Rutkowski and Stene. Risk were performed because of suspicion of chromo- values for maternal and paternal carriers have somal aberrations. We found a frequency of been obtained. inversions of 2.2 per mill (91 patients). 90 patients had one inversion, one patient two. In 55 cases the inversion was pericentric, in 37 para- L136 centric. Familial cases were predominant: there Population-genetic consequences of the were 92% familial versus 8% de novo inversions. The three most frequent reasons for chromosome Chernobyl accident in different analysis were prenatal diagnosis because of mammalian species advanced maternal age (38.5%, accidental find- V. Glazko1 and T. Glazko1 ing), a child with malformations (17.5%) and 1 increased abortions (12.0%). Analyses of inver- Research Institute of Biology, Rostov State sions were done by conventional cytogenetics in University, Rostov-on-Don, 344090 Russia 87 and by FISH in 4 cases. There was aneusomy by unequal recombination which will be illustrated The increases of cytogenetic anomalies in the in detail. The three most frequent inversions were bone marrow cells of mice lines BALB/c, those in chromosome 2, 10 and Y. Our results C57BL/6j, CC57W/Mv, subjected to ionizing will be compared to findings in the literature. radiation (near 0.6 Sv) were revealed. However, only those types of cytogenetic anomalies were increased which were spontaneously highly vari- L135 able in an age- or season-dependent manner in the same mice lines not subjected to radiation. In Chromosome mutations in acrocentric other experiments, three species of voles (Micro- chromosomes: frequencies and genetic tus arvalis, Microtus oeconomus, Clethrionomys relevance glareolus), trapping in zone of alienation of 1 1 2 Chernobyl NPP with different levels of ionizing G. Schwanitz , L. Kalz , A. Jelska and < * * 2 2 irradiation ( 5; 200; 1000 Ci/km ) were A. Midro investigated. Among them, the evolutionary 1Institute of Human Genetics, University Hospital youngest species of common vole (Microtus arva- Bonn, Germany; 2Department of Clinical lis) characterized by comparative high karyotype Genetics, Medical University Bialystok, Poland instability in area was the most sensitive to ioniz- ing radiation. This interspecies comparison First, the different DNA structure of the short thus confirmed that an increase of ionizing radia- arm regions of the acrocentrics is given. Changes tion does not induce new genetic damages, but 15th ICC: Abstracts 121 destabilizes the preexisting genomic a˜hot spotsa¨ inheritance pattern, although coordination of the that are either species-specific (and more char- twochainsisessential.Thisisthe¢rststable,dou- acteristic to evolutionary young species) or geno- ble chain system found in any organism, and the type-specific (different laboratory lines of mice). coordination of segregation patterns between two In generations of cattle, born in experimental distinct translocation chains is unprecedented. farm (*200 Ci/km2) near Chernobyl NPP, dis- Analysis of this chromosomal race will permit the turbance of equiprobable transmission of alleles investigation of the origins of fusion hetero- in a number of molecular genetic markers, zygosity in D. cancerides, and may shed light on increase of heterozygosity and radio resistance basic principles of meiosis such as the mechan- (on decrease of cytogenetic anomaly frequencies isms behind chromosomal segregation, and sex in blood cells in generations) were observed. The chromosome evolution. result of selection for irradiation resistance in voles emerged through approximately 26 genera- tions after the beginning of the ionizing radiation exposure and it was dose-dependent. No increase PO:08:37 in the quantity of constitutive mutations in inves- Translocations of chromosome 13 in tigated genes, ISSR-PCR markers or chromoso- the course of family investigation mes in cattle and Rodentia species was observed. G. Bujdoso´1,P.So´tonyi1, O. Bellovits1, E. Csonka2 and G. Hadlaczky2 L137 1Hungarian Academy of Sciences, Semmelweis Co-segregation of two sex linked University, Institute of Forensic Medicine, Budapest, Hungary; 2Institute of Genetics, translocation chains in the Australian Biological Research Center, Hungarian Academy social huntsman spider Delena of Sciences, Szeged, Hungary cancerides. 1 1 During determination of origin studies certain H. Sharp and D. Rowell deviations and diseases surface in individuals 1Department of Botany and Zoology, The coming from the normal population. Australian National University Canberra, The ¢rst case is one from 30 years ago. The 0200, Australia putative father did not accept his paternity of the eight years old boy, despite the blood group and Delena cancerides incorporates five well char- anthropology results showing the possibility of acterised chromosomal races. The ancestral form his fatherhood. During the o⁄ce visit of father has a completely telocentric karyotype, while the and child for evaluation the intelligence of both other races are saturated for different combina- was above average and they showed peculiar psy- tions of Robertsonian fusions. Three of these chological behaviour. Based on these facts, this races form chains of chromosomes at male meio- was the ¢rst time ^ even internationally ^ that sis due to fixed fusion heterozygosity. One race chromosome studies were performed on all three has a chain of three chromosomes, another a parties (mother, child, defendant). chain of five, and the most spectacular, a chain Karyotyping results included a normal in the of nine chromosomes. These chains include a sex mother and 46XY t(2q ;13q þ ) both in the child chromosome, and segregate as a complex XY and the putative father. The paternity was system. This ensures that karyotypic integrity is proven. maintained over successive generations, and the Meanwhile the boy grew up and after thirty races remain stable. years he returned to our laboratory with his two We have recently found a new, distinct race of children, owing to some serious behavioural pro- D. cancerides. At male meiosis this race forms blems with them. According to our chromosome two chains of ¢ve chromosomes. The presence of investigation the daughter showed the same X chromosomes on both chains ensures a stable balanced translocation that her father and grand- 122 15th ICC: Abstracts father had. No translocation was found in the chromosomes from flower buds. Chromosome boy, but further genetic test results are pending. counting is performed on fluorochrome staining In our other case a girl born at term (with of leaf chromosomes. The 18S.26S ribosomal 1600 g, 45 cm) was searching for her genetic genes are mapped by fluorescence in situ hybridi- father. The chromosome studies resulted in the zation (FISH) on both mitotic and meiotic chro- child and probable father having translocation of mosomes using the wheat pTa71 ribosomal DNA chromosomes 13;14. as probe. Genomic DNA from all samples are At the time of the studies the girl was a dainty analysed for restriction fragment length poly- 21 months old, much smaller than her compa- morphisim (RFLP) using the same rDNA clone. nions, but well proportioned and healthy, both The results will be presented. No such work on physically and mentally. genetic diversity of this family in Thailand or Southeast Asia has been reported before.

PO:08:38 Genetic diversity of Fagaceae from PO:08:39 Chiangmai province, northern ‘Ages’ of chromosome translocations Thailand, based on 18S.26S ribosomal t(15;17), t(8;21), and inv(16) in human genes acute myeloid leukaemia (AML). P. Chokchaichamnankit1, W. Chulalaksanakul1 M. Colovic1, D. Vukomanovic2, M. Andolina3 and K. Anamthawat-Jonsson2 and G. Jankovic1 1Botany Department, Faculty of Sciences, 1Institute of Haematology, Belgrade, Serbia and Chulalongkorn University, Phayathai Road, Montenegro; 2Mathematical Institute, Belgrade, Bangkok 10330, Thailand; 2Biology Department, Serbia and Montenegro; 3Istituto per L’Infancia, University of Iceland, Askja-The Natural Science Trieste, Italy Building, Sturlugata 7, Reykjavik, IS-101, Iceland Patients with AML may(þ) or may not() carry The oak family (Fagaceae) in Thailand consists chromosome translocations (CT) associated with mainly of three genera: Castanopsis, Lithocarpus underlying oncogene fusions. Leukaemogenic and Quercus. They are most common in northern mutations do not accumulate with time but are Thailand, and the species diversity is impressive, purged in each generation. Nevertheless, propor- i.e. 40 o¨ 60 species have been estimated from this tions of their respective CT polymorphisms (þ/ ) region. Their habitats are relatively diverse, from may have changed, but not remained frozen, hill evergreen forests down to dry dipterocarp during evolution. Initial proportion [Ro] of the forests and mixed deciduous forests with open CT () variant of leukaemogenic allele fusions grassland. We have selected areas of study that ought to had been virtually 1.0 at some point include all three forest types in Chiangmai pro- back in time. Do present proportions of the () vince. At each site, trees were identified at reg- polymorphism in leukaemia mark time carrying ular intervals between them, regardless of species implications of a historical chronometer in dating identity (based on local names, Fagaceae flora of evolutionarilly neutral CT? If the propensity for Thailand is not yet complete), marked for further recurrent CT-generation in leukaemia patients visits. Various sample types were collected from follows the evolutionary dynamics of repetitive each plant, for herbarium and morphological elements underlying this propensity, then we assessment, for chromosome and DNA isolation. could age-date specific CT. Contemporary pro- About 100 trees are included in this study and portion [R] of the CT() variant of oncogene they are likely to represent more than 20 species. fusions characterizing three distinct subtypes of Total genomic DNA has been isolated from AML, and their incidence rates [substituted in young leaves, where as mitotic chromosomes place of a specific decay constant, (lambda)] were have been isolated from leaf buds and meiotic entered in a mathematical expression [t ¼ 11n 15th ICC: Abstracts 123

Ro/R] that relates exponential decay and the long survival. Comparative Genomic Hybridisa- time[t] elapsed since the CT first appeared. The tion (CGH) has allowed to delineate the cytoge- age estimates obtained are 230,170 t(15;17), netics anomaly. 182,958 t(8;21), and 163,248 (inv16) years. Pre- sent proportions of the () polymorphisms [R] may represent enduring palaeocytogenetic trace PO:08:41 of archaic tumour karyotypes. The age estimates of morbid CT concur with the emergence of first Human chromosomal modern humans 250,000 to 150,000 years ago. Q-heterochromatin regions in individuals of various age A. Ibraimov1 PO:08:40 1National Center of Cardiology and Internal A CGH and FISH detection of a de Medicine, Togolok Moldo str. 3, Bishkek, novo 22q13-qter trisomy and 2pter Kyrgyzstan, 720040, C.I.S. monosomy in a patient: clinical The quantitative content of chromosomal Q-het- presentation and review of the erochromatin regions (Q-HRs) was studied in literature individuals of different age groups taking into account their racial and ethnic affiliation. It was C. Hans1, Y. Sznajer2, E. Vamos2, B. Pichon1 1 shown that chromosomal Q-HRs are most and L. Duprez numerous in the genome of neonates, while they 1Laboratoire de Ctoge´ne´tique, Hopital Erasme, are the least numerous in the genome of elderly Universite´ Libre de Bruxelles, Belgique; 2Hopital subjects (aged 60 years and older) regardless of Universitaire des Enfants Reine Fabiola, the ethnic features of the individuals studied. Universite´ Libre de Bruxelles, Belgique Our data indicate that 1) in a population there is a clear-cut tendency towards a decrease in the The phenotype of distal 22q trisomy is char- number of chromosomal Q-HRs with age, acterised by intra-uterine growth retardation, regardless of racial and ethnic features on the hypotonia, mental retardation, microcephaly and individuals; 2) of all the age groups the genome feeding difficulties. Hypertelorism, narrow pal- of neonates contains the greatest number of Q- pebral fissures, small nose with anteverted nares, HRs; 3) decreases in the number of Q-HRs with a thin upper lip, cleft lip/palate, low set and dys- age are not due to the ‘loss’ of Q-hetero- plastic ears delineate the dysmorphic features. chromatin on individual loci or chromosomes, Hypogenitalism is an additional finding in males but occur simultaneously in all the seven Q- (cryptorchidism, small penis, hypospadias). polymorphic autosomes, in keeping with all our Most of the a¡ected patients reported to date previous observations. It should be noted that die shortly after birth or have a short lifespan. decreases in the number of chromosomal Q- The phenotypic picture of distal 2p monosomy HRs with age do not occur smoothly appar- associates post natal growth retardation, micro- ently. Thus, according to our data, in the Kyr- cephaly, bulging metopic suture, low set ears, £at ghyz and Russian populations 39.6% and 47.6% nasal bridge, micrognathia, high arched palate, of chromosomal Q-HRs ‘disappeared’ by the single palmar crease, foot deformities and devel- age of 60 years, respectively. Of them the por- opmental delay. We report clinical and cytoge- tion of new-born Kyrghyz and Russian infants netic ¢ndings in a 19-year-old boy with distal 22q amounted to 70.6% and 58.4%, respectively. It trisomy and distal 2p monosomy: the features dis- is supposed that the lesser amount of Q-HRs in played by our patient are rather related to 22q tris- the genome of elderly subjects is due to the omy than 2p monosomy. Clinical comparison is selective advantage in their survival to old age. established with the published cases. Our patient The possible selective value of chromosomal isthe¢rstreportofpartial22trisomywithsucha Q-HRs is discussed. 124 15th ICC: Abstracts

PO:08:42 1Laboratory of Human Genetics, National Center of Cardiology and Internal Medicine, Togolok Chromosomal Q-heterochromatin Moldo str. 3, Bishkek, Kyrgyzstan, 720040, C.I.S. variability in neonates deceased during ¢rst year of age. The quantitative variability of chromosomal Q- 1 heterochromatin regions (Q-HRs) was studied in A. Ibraimov alcoholics and drug addicts in various racial and 1Laboratory of Human Genetics, National ethnic groups. It was found that in the genome Center of Cardiology and Internal Medicine, of alcoholics the amount of chromosomal Q-HRs Togolok Moldo str. 3, Bishkek, Kyrgyzstan, is significantly lower than in control samples and 720040, C.I.S. drug addicts, whereas the latter have the greatest amount of chromosomal Q-HRs. There is analyzed the amount of chromosomal Alcoholics are characterized by the lowest Q-heterochromatin regions (Q-HRs) in a genome value of the mean number of Q-HRs per indivi- of neonates deceased during first years of life. dual in the population (¼ 1.45 0.09) and by the Chromosome preparations were made from narrowest range of variability of the number of umbilical-cord blood with the subsequent Q- chromosomal Q-HRs (from 0 up to 4) among all staining in 145 Kyrghyz and 37 Russian neo- the samples studied. Although subjects with drug nates. During the first year of life 17 Kyrghyz abuse typically had the highest value (4.1 0.11), and 5 Russian neonates have deceased from var- nevertheless it should be noted that the range of ious diseases in stationary conditions, the diag- variability of the number of Q-HRs was as nar- nosis of which is confirmed by pathological– row (from 2 to 6) in the as that in alcoholics. How- anatomical dissection. Mean numbers of Q-HRs ever prevailed individuals with great numbers of per individual in newborn population were Q-HRs in their genome. We just draw attention 3.16 0.13 in Kyrghyz and 3.59 0.23 in Rus- to the fact that in the vulnerability of man to alco- sian, whereas in neonates died ¼ 4.58 0.23 and holism and drug addiction is also of signi¢cance, ¼ 4.80 0.37 respectively. Neonates are char- in addiction to other things, the amount of chro- acterized by a high range of variability in the dis- mosomal Q-HRs in his genome, since the values tribution of Q-HRs (from 0 up to 7). But died of absolute and relative Q-HR frequencies on Q- neonates, besides high value, differ by extremely polymorphic autosomes in all samples are encoun- narrow range of variability of Q-HRs in popula- tered with an expected frequency on all the poten- tion: number of Q-HRs in a karyotype changes tially Q-polymorphic chromosomes, indicating from 4 up to 6. Q-HRs in all samples studied are that Q-heterochromatin is not locus-speci¢c mate- encountered with an expected frequency on all rial in the human genome. the potentially Q-polymorphic autosomes, i.e. in no group there was preferential Q-HR localiza- tion, and this is again suggesting that Q-hetero- PO:08:44 chromatin is not locus-specific material in the genome. It is supposed that individuals with the Variability of chromosomal greatest amount of Q-HRs in the newborn popu- Q-heterochromatin regions in patients lation have greater probability to die in the first years of life other conditions being equal. with alimentary obesity A. Ibraimov1 1 PO:08:43 Laboratory of Human Genetics, National Center of Cardiology and Internal Medicine, Togolok Variability of chromosomal Moldo str. 3, Bishkek, Kyrgyzstan, 720040, C.I.S. Q-heterochromatin regions in Variability of the amount of chromosomal alcoholics and drug addicts. Q-heterochromatin regions (Q-HRs) was studied A. Ibraimov1 in individuals with alimentary obesity and in 15th ICC: Abstracts 125 controls from two ethnic groups living in Bish- acute myeloblastic leukaemia (AML)] and acute kek, Kyrghyzstan. It was shown that obese indi- lymphoblastic leukaemias (ALL) leukaemias ‘age’ viduals differ from controls in the extremely low estimates of the Ph itself were obtained. The inci- amount of Q-HRs in their genome. Namely, dence rates were entered in place of decay con- females with obesity, regardless of their ethnic stant lambda) in a relation between exponential origin, are characterized by a consistently low decay of Ph-negativity and time [t] elapsed since value of the mean number (¼1.30 0.08) and by the Ph first appeared. The Ph-negativity char- narrow range of variability in the distribution of acterized all BCR/ABL fusions in the ancient the Q-HR numbers in the samples (from 0 up to state [Ro ¼ 1.0]. Proportions of the Ph-negative 3) as compared with controls (¼2.94 0.14 and variant of BCR/ABL chimerism [R] were from 0 up to 7, respectively). The values of abso- obtained as *10 percent of the proportion of Ph- lute and relative Q-HR frequencies on Q-poly- positive patients in analogy with CML where the morphic autosomes in obese individuals are Ph-negatives constitute *0.1 of all patients. encountered with an expected frequency on all Using the equation t ¼ 1/(lambda) ln Ro/R, the potentially Q-polymorphic autosomes, indi- identical values for t (age of the Ph) are obtained cating, as we suppose, nonlocusspecifity of Q-HR for otherwise biologically quite distinct but Ph- in the human genome. The question as to the positive leukaemias. The estimates of t (in years) possible role of the amount of Q-HRs in the gen- are 230,258 (CML), 230,170 (AML), 231,229 ome in the vulnerability of man to the develop- (adult ALL), and 230,472 (paediatric ALL). The ment of alimentary obesity is discussed. Ph age estimates (*230,000 years) concur with the 200,000 years estimated for the most recent common mitochondriarch. PO:08:45 The age of the Philadelphia PO:08:46 chromosome (Ph): dating the origin Kuwait Chromosomal Abnormality of tumour karyotype aberration. Registry (KCR) (1979^2003) G. Jankovic1, M. Colovic1, P. Wiernik2 D. Krishnamurthy1 and S. Al-Awadi1 3 and M. Andolina 1Cytogenetic Laboratory, Kuwait Medical 1Institute of Haematology, Belgrade, Serbia and Genetics, Center, Maternity Hospital, Kuwait Montenegro; 2Our Lady of Mercy Cancer Center, New York, USA; 3Istituto per L’Infancia, Trieste, The Kuwait Medical Genetics Center, was estab- Italy lished in the year 1979, to provide Cytogenetic diagnostic services in the referred population sus- Neither the chromosomes fossilize nor the recur- pected for chromosomal abnormalites. During ring chromosome translocations (CT) in human the period from 1979–2003 chromosomal analysis leukaemias accumulate over time (they are has been carried out in 16,850 cases among the purged in each generation), so it is not obvious 2.3 million heterogeneous population. Cytoge- how could their frequencies in patient popula- netic analysis were carried out as per the stan- tions be used to age-date modern tumour kar- dard procedures, and the karyotype was yotype. We assume that (i) CT have been interpreted according to the ISCN. The registry selectively neutral during human evolution and includes the common numerical and structural as that (ii) the evolutionary DNA dynamics (muta- well as some very rare cases. One of the note- tion) underlying the propensity for CT-forma- worthy observation in this population is recur- tion, in association with leukaemogenic allele rent regular trisomy 21, a very high incidence of fusion, was essentially random in time. Based on trisomy 21 (3.3 per 1000 births), clustering of proportions of the Ph polymorphism (þ/) asso- trisomy 18 and cri-du-chat syndrome cases. The ciated with the BCR/ABL oncogene fusion in data presented is one of the largest series in the myeloid [chronic myeloid leukaemia (CML), Middle East population. The significance of 126 15th ICC: Abstracts consanguinity, inbreeding and non-availability of 1Shahid Beheshti Medical University, Department prenatal diagnosis services (due to socio-cultural of Genetics, Evin Ave, Tehran, Iran.; 2Ghazvin reasons) in relation to increased chromosomal Medical University, Ghazvin, Iran abnormalities in this population will be discussed. We report the results of an analysis of congenital malformations and genetic diseases of 24513 chil- PO:08:47 dren from infancy to age 19 years, each with Population cytogenetics of Acrotylus varying clinical manifestation from normal to humbertianus (Sauss) (Fam: Acrididae; severe congenital anomalies including metabolic, heart, blood, infertility, neurology, deafness, Sub.Fam: Oedipodinae) from made over 5 years period in a major pediatric South India hospital and genetic division centre Ghazvin S. Mayya1, J. Hegde1 and S. Kanale2 state, and results of 76 cases drawn from this work, Taleghani hospital Tehran. In this survey, 1 Department of Biosciences, Mangalore 78 cases with abnormal chromosomal have been University, Mangalagangotri, Mangalore, India; 2 investigated employing conventional cytogenetic Department of Applied Zoology, Mangalore GTG- technic by chromosome banding. Down’s University, Mangalagangotri, Mangalore, India syndrome was the most common anomalies fol- lowed by Patau-Edward’s-Wolf Hirosh Horn Meiotic chromosomal analysis of Indian short syndromes among autosomes. Among sex chro- horned grasshopper Acrotylus humbertianus from mosome, Turner-KFS-super female-super male- three natural populations, Maravanthe, Some- Fragile X syndromes, was the most common. shwara and Calicut in the west coast of South This date gives an idea of the spectrum of cyto- India has been studied. As per the data on back- genetic disorders seen in Iran from this region, ground gamma radiations in the west coast more detail will be discussed. (Radhakrishna et al. 1993), it is found that the three study areas experience varied levels of radiations. i.e. Maravanthe, 3-5nGyh-1; Some- PO:08:50 shwara 215nGyh-1; Calicut 56nGyh-1. Hence it was proposed to carryout chromosomal studies FRAXA leading mutational pathways using testis material on commonly available among Basques grasshoppers. The study revealed different kinds O. Pen˜agarikano1,M.Te´lez1, B. Ortega1, of chromosomal anomalies which include sticki- L. Valverde1, P. Flores2, B. Criado3 and I. Arrieta1 ness and clumping (16.24%), bivalent segregation (10.84%), chromosome bridges (5.96%), laggards 1Dpto. Gene´tica, AntropologaFsica y Fisiologa (5.42%), polyploidy (1.48%), gaps and breaks Animal, Facultad de Ciencia y Tecnologa, (0.98%) etc. in different degrees in all the three Universidad del Pas Vasco, Bilbao, Spain; 2Dpto. populations. The frequencies of these anomalies FarmacologaClnica y Diete´tica, Escuela de in relation to background radiation have been Enfermera, Universidad del Pas Vasco, Bilbao, 3 discussed. Spain; Instituto Portugue´s de Oncologa, Porto, Portugal

PO:08:49 Fragile sites are heritable points on a chromo- some where gaps and breaks tend to occur. They Incidence of chromosomal are mainly designated as common or rare based abnormalities; A prospective study in on their frequency of expression. In addition, a referrd Iranian population. they are subdivided according to the specific cul- ture media needed for their expression. FRAXA, 1 2 1 A. Movafagh , Z. Pirzadeh , H. Moosavi , a fragile site located in Xq27.3, is a rare folate- 1 2 S. Ayenehchi , M. Ghasemi Barghi sensitive fragile site associated to fragile X syn- and H. Javadi2 drome (MIM#309550). Fragile X syndrome is 15th ICC: Abstracts 127 the most common form of inherited mental retar- approach to identify a susceptibility locus for dation. The publication in 1991 of both the mole- schizophrenia. Chromosomal abnormalities may cular basis of the fragile site and the syndrome, be helpful to find not only canditate regions for the massive expansion of a CGG repeat in the linkage studies, but also valuable materials for FMR1 X linked gene, focused much attention on positional cloning. We performed cytogenetic trinucleotide repeat expansion as a mutational examinations on peripheral lymphocytes of 39 mechanism. In addition, even the slight expan- (22 women, 17 men) schizophrenia patients. G- sion of this microsatellite sequence is associated banded metaphases were examined according to to other disorders such as premature ovarian the ISCN (1995). One male and one female failure (POF) and tremor/ataxia syndrome patients revealed only normal karyotypes. There (TAS). The most important factor that influences were not good quality metaphases to examine in on the trinucleotide repeat expansion is the two cases. In 23 cases, there were non-recurrent length of the repeat as well as its internal struc- chromosomal abnormalities along with normal ture (the presence/absence of anchoring interrup- karyotypes. In 12 cases, we observed recurrent tions). In order to ascertain the stability of the chromosome abnormalities which include FMR1 CGG repeat in the Basque Country we del(X)(q26q27), dup(6)(p22)?, add(22)(q12)?, have analyzed these factors in 158 healthy males X, þ mars, 15, 22 (in two cases), der(7)?, from the Spanish Basque population. Basques may 18, del(9)(q12), 21, der(21) (in two cases), represent one of the oldest human isolates. As a der(1), add(4)(q35?). result, they show distinctive cultural and genetic characteristics. The data show a lower prevalence This work was supported by the Research Fund of Istanbul of large CGG alleles among this population. University. Project Number: 1619/30042001 However, analysis of the AGG interspersion pat- tern suggests the existence of different mutational PO:08:52 pathways that could lead to longer CGG repeats. Fragile site expression in hypertensive patients treated with atenolol PO:08:51 M. Te´lez1,O.Pen˜agarikano1, B. Criado2, Cytogenetic ¢ndings in 39 C. Lostao1, P. Flores3, E. Ortiz4 schizophrenic patients and I. Arrieta1 Y. Tarkan-Argu¨den1, S. Yilmaz1, A. Egriboz1, 1Dpto. Gene´tica, Antropologia Fisica y Fisiologia B. Gunel2, A. Yilmaz2, A. Duran2 and Animal, Facultad de Ciencia y Tecnologia, S. Hacihanefioglu1 Universidad del Pais Vasco, Bilbao, Spain; 2Instituto Portugue´s de Oncologia, Porto, 1Dept. of Medical Biology, Istanbul University, Portugal; 3Dpto. Farmacologia Clinica y Cerrahpasa Medical School, 34300, Istanbul, Diete´tica, Escuela de Enfermeria, Universidad del Turkey; 2Dept. of Psychiatry, Istanbul University, Pais Vasco, Bilbao, Spain; 4Dpto. Especialidades Cerrahpasa Medical School, 34300, Istanbul, Me´dico-Quirrgicas, Universidad del Pais Vasco, Turkey Bilbao, Spain

Schizophrenia is a common and serious psychia- FS are specific points of chromosomes at which tric illness with peak age at onset in early adult- the chromosome is liable to break. The latest hood. Family twin and adoption studies have compendium comprises over 117, classified as demonstrated that schizophrenia is pre- common and rare according to their relative dominantly genetically determined and has high occurrence in the population. Both common and heritability. Although many linkage and associa- rare FS are further subdivided according to the tion studies have been performed, no gene has yet tissue culture conditions required for their been established as giving major susceptibility to expression. Their location has been correlated schizophrenia. The use of chromosomal examina- cytogenetically with locations of recurrent trans- tions could be an alternative or complementary location and deletion breakpoints in cancer cells 128 15th ICC: Abstracts as well as the integration sites of oncogene loci. they are not completely sterile, they can produce Previous studies have shown that genotoxic morphologically variable progeny after back- agents increase the expression of FS. crossing. However, these plants are not variable The analysis of the FS signi¢cantly expressed in in their chromosome numbers – they are diploid, our sample allowed the identi¢cation of possible triploid and tetraploid. No aneuploids have ever speci¢c FS on chromosomes of the hypertensive been found, suggesting genetic control over meio- patients. On one hand, the analysis of FS expres- sis or strict gametic selection in the hybrids. In sion de¢ned 28 di¡erent bands in patients and 21 this study we characterize the genomes in natural in controls considered to be FS in the sample; triploid hybrids using rDNA-FISH mapping FRA3B, FRA6C, FRA7C and FRA16D were the together with Southern genomic hybridization four most frequently expressed in both groups. and cpDNA-RFLP analysis. We also test our On the other hand, FRA1E, FRA1F, FRA2D, hypothesis that the selection against aneuploidy FRA5D, FRA7B, FRA7H, FRA11G, FRA11H, in the hybrids is during meiosis, by looking at FRAXB, FRAXC and the band 17q12^21 were chromosome behaviour and microsporogenesis. FS exclusively expressed in patients. The band Triploid hybrids seem to produce two sizes of 17q12^21, where known FS have not been repor- viable gametes (pollen), possibly corresponding ted, was in the ¢fth position in order of expres- to genome sizes as predicted. The significance of sion and was expressed in 63.64% of patients. interspecific hybridization is probably in main- Until now, linkage analysis has been pursued to taining genetic variability and gene flow between identify hypertension susceptibility genes but the two species, via introgression. Such mechan- results have been di⁄cult to reconcile. One nota- ism may be the major force behind the species ble exception is the study by Lathrop et al., which survivability in harsh environments like in provides reasonably compelling evidence for an Iceland. hypertension susceptibility locus on chromosome 17. Our results seem to support that ¢nding. Solid Tumours Symposium

PO:08:53 L138 Cytogenetic investigations of natural Cancer cytogenetics: potentialities and triploid birch hybrids in Iceland limitations of the current technology A. Thorsson1 and K. Anamthawat-Jonsson1 H. Tanke1 1University of Iceland, Department of Biology, 1Department of Molecular Cell Biology; Leiden Askja, Sturlugata 7, Reykjavik 101, Iceland Medical Centre; Leiden; Netherlands

Triploid hybrids (42 chromosomes) between tet- Conventional cytogenetic investigations of raploid downy birch (Betula pubescens) and Giemsa banded metaphase chromosomes gener- diploid dwarf birch (B. nana) are found in about ated from cultured solid tumours have been ham- 10% of birch plants in birch woodlands through- pered by both biological as well technical out Iceland. The two species co-exist in Iceland problems. Biological factors relate to tumour cell and in northern parts of Europe, and due to heterogeneity and cell culturing artefacts (c.q. short growing season their flowering periods selection), whereas major technical obstacles are overlap significantly and the species hybridize. encountered with respect to poor quality (or no!) Crossing experiments produce triploid inter- metaphase chromosomes, insufficient banding specific hybrids with high frequency, especially resolution and very complex rearranged karyo- when B. nana is the seed parent. Ribosomal gene grams. mapping by fluorescent in situ hybridization Techniques developed in the past two decades (rDNA-FISH) confirms the presence of parental such as 48 colour FISH karyotyping, comparative genomes in the hybrids. The hybrid plants have genomic hybridisation (chromosomal as well as intermediate leaf characters and growth form. As array CGH) and culturing techniques that 15th ICC: Abstracts 129 produce high quality metaphases by chemical micro-ampli¢cation of known and novel genes. induced PCC (premature condensation of chro- The repeatability and robustness of SAGE on lim- mosomes), justify a revisit of the ¢eld of cancer ited lung samples has been demonstrated. More cytogenetics. This paper outlines the state-of-art than 500 14mer tags of which 411 mapped to of the current technology illustrated by selected genes were found to be more than 50x over expres- samples from routine clinical practice (mainly sed between normal and cancer tissue. A small soft tissue tumours). subset of the possible screening markers have been evaluated on a set of cytology microarrays. The performance of a few of these markers L139 approach that required for a usable screening test. Genome wide approaches (high resolution BAC array CGH and SAGE) for analysis of lung cancer L141 C. Macaulay1, K. Lonergan1, C. Garnis1, Chemically induced chromosome B. Coe1, S. Zuyderduyn1, M. Marra1, condensation technique in tumor 2 3 1 1 A. Gazdar , M. Tsao , S. Lam and W. Lam cytogenetics 1 British Columbia Cancer Research Centre, K. Szuhai1 Vancouver, British Columbia, Canada; 2University of Texas Southwestern Medical 1Department of Molecular Cell Biology, Leiden Centre, Dallas, Texas, USA; 3Ontario Cancer University Medical Center, Leiden, Institute, Princess Margaret Hospital, Toronto, The Netherlands Ontario, Canada In the last decade several new molecular techni- Genetic alterations and expression changes are ques have been developed to identify genomic known to be associated with the neoplastic pro- alterations associated with tumor development cess in the lung. Recently developed tools enable and progression. However, there is no such an the genomic wide evaluation of genetic copy approach that allows genome-wide screening for number changes (at a resolution 10–50 times unknown chromosomal alterations with balanced greater than conventional array CGH) and rearrangements. These limitations necessitate the known gene and novel gene expression changes use of classical cytogenetic approaches to visua- (SAGE) from minute lung samples. lize chromosomal alterations. However, several The objectives of our study were (1) To exam- tumor types characterized with low mitotic rate ine the data from a CGH array designed to pro- or unable to adapt to the culture conditions, vide complete coverage of the human genome. (2) which results in the lack of tumor cell metaphase To identify genes important to disease progres- or in the metaphases from the contaminating sion (in 40 plus neoplastic lung specimens). (3) normal cells. An alternative technique that indu- Correlation of these with changes seen in 40 plus ces a rapid condensation of chromosomes inde- SAGE lung libraries. (4) Evaluate candidates on a pendent of the numbers of M-phase cells might recently developed cytology microarray platform. be a solution to these problems. Cell cycle inde- Arrays of 32,433 ampli¢ed BAC DNA clones pendent condensation of chromosomes can be spanning the whole genome has been constructed. achieved by the chemical premature chromosome Experimental conditions for array CGH for clin- condensation (cPCC) technique. This approach ical materials and the impact of tissue hetero- will particularly valuable to study tumor cells geneity and DNA quality/quantity in the with a slow transition from G2 to M phase. The generation of an accurate array CGH pro¢le have value of the cPCC technique was evaluated on a set been assessed. This has identi¢ed recurrent of 60 solid tumors, bone marrow and peripheral genetic alterations. In addition, the tiling resolu- blood samples of hematological malignancies. tion of the array facilitated the detection of The use of chemically induced chromosome con- 130 15th ICC: Abstracts densation was found to be the most informative detect new recurrent genomic imbalances that might in tumors with slow growth rate resulting in 4-10 lead to the discovery of genes involved in normal fold increase of the number of tumor metaphases neuroblast differentiation and NB development. compared to the classical Colcemid harvesting. In summary, our work and that of others illus- This technique can easily adapted to fit routine trates that, together with current e¡orts of gene practice, cPCC in combination with multicolor expression pro¢ling, the study of the genomic FISH is considered as an important tool in alterations occurring in tumours (in casu NB) tumor cytogenetics in addition to classical cyto- provides critical prognostic information and also genetic investigation. contributes to the identi¢cation of candidate NB genes. L142 Pro¢ling of chromosomal imbalances L144 as a tool for prognostic strati¢cation MAC (Mammalian Arti¢cial and gene discovery in cancer Chromosome) based elimination test in F. Speleman1, N. Van Roy1, B. Menten1, 1 2 the functional identi¢cation of tumor A. De Paepe , G. Laureys and growth antagonizing genes J. Vandesompele1 A. Szeles1, G. Hadlaczky2 and S. Imreh1 1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium; 2Dept of Pediatric 1Karolinska Institutet, MTC Karolinska Inst. Oncology, Ghent University Hospital, Ghent, MTC, Stockholm, Sweden; 2Institute of Genetics, Belgium BRC, Szeged, Hungary

Neuroblastoma (NB) is a paediatric neural crest We have developed a functional test system; the derived tumour, that shows a remarkably hetero- microcell hybrid (MCH) based ‘elimination test’ geneous clinical behaviour. Karyotyping typically (Et). The MCHs are tested for the elimination or showed 1p-deletions and MYCN amplification retention of specific human chromosome 3 but was in the majority of cases only applicable (chr.3) regions after passages in SCID mice. to high stage NB. Molecular studies, mainly We have defined a commonly eliminated region investigating loss of heterozygosity, lead to iden- (CER). We have covered these regions with tification of other chromosomal regions of recur- PACs (bacteriophage P1 based artificial chromo- rent deletions involving chromosome regions 3p, some) and identified several genes. 4p, 11q and 14q. Molecular cytogenetics intro- Our aim is to reintroduce the PACs and genes duced new possibilities for the genetic analysis of derived from the CER into cancer cell lines. For NB, revealing a high incidence of 17q gain due this purpose we are using mammalian arti¢cial to unbalanced chromosome 17 translocations in chromosome (MAC) vectors. Our MACs are NB cell lines and high stage tumours. Compara- satellite DNA based arti¢cial chromosomes tive genomic hybridisation (CGH) provided a (SATACs). Among the genes identi¢ed on CER genome wide view on the genomic imbalances lactoferrin (LF) is one of the candidates. We have associated with all clinical subtypes of NB. Sta- constructed human LF carrying mouse SATACs tistical analysis of CGH data from a large cohort (mSATAC-hLF) in a mouse (LMTK) and human of patients clearly demonstrated the presence of LF carrying human SATACs (hSATAC-hLF) in three major subgroups. Furthermore, chromo- a human ¢brosarcoma lines (HT1080). The mSA- some 17q gain and normal chromosome 17 TAC-hLF carrying mouse and hSATAC ^ hLF status were shown by multivariate analysis to be carrying human ¢brosarcoma lines were used for the strongest independent prognostic parameters studiesofthee¡ectofthehLFtoin vitro and in for poor outcome. More recently, using high- vivo tumour growth. Our results have showed that resolution copy number analysis by arrayCGH the exogenous hLF had no consistent e¡ect on in analysis and SNP chip analysis we were able to vitro growth. mSATAC-hLF was maintained in 15th ICC: Abstracts 131

78% and hSATAC-hLF in 92.5% of cells in vitro. orderliness at the transition from Phase I to Phase Next, we have tested the cells in SCID mice. We II/III is also evident by a drastic difference in have found that the majority of cells in SCID karyotypoic entropy. derived tumors lost the SATAC-hLF. It can be concluded, that elimination of the exogenous hLF carrying SATACs provides to the mouse and L146 human ¢brosarcoma cells with in vivo growth advantage. Sequence copy number changes in Barrett’s adenocarcinoma: Array-CGH and quantitative L145 Sequential Multi-Locus (SML-) FISH The statistical behavior of complex analysis cancer karyotypes B. Albrecht1,K.Ba¨wering2, H. Zitzelsberger3, 1 2 3 M. Ha¨glund , A. Frigyesi , T. Sall , T. Wiech1, M. Hausmann1,H.Ha¨fler2, 1 1 D. Giselsson and F. Mitelman M. Werner1 and A. Walch1 1 Department of Clinical Genetics, University 1University Hospital Freiburg, Institute of 2 Hospital, Lund, Sweden; Centre for Pathology; Freiburg i. Br., Germany; 2Technical Mathematical Sciences, Mathematical Statistics, University of Munich, Institute of Pathology; 3 Lund University, Sweden; Department of Munich, Germany; 3GSF-National Research Genetics, Lund University, Sweden Center for Environment and Health, Institute of Molecular Radiation Biology; Neuherberg, Epithelial tumors commonly show complex and Germany variable karyotypes obscuring the identification of general patterns of the karyotypic evolution. Array-CGH (aCGH) allowed the identification of To overcome some of these problems, we have DNA sequence copy number changes in for- previously analyzed systematically the accumu- malin-fixed, paraffin-embedded tissue samples of lated cytogenetic data for individual tumor Barrett’s adenocarcinomas (BCA). The micro- types using various statistical means. In the pre- arrays used contained 287 genomic targets cover- sent investigation we perform several meta-ana- ing oncogenes, tumor suppressor genes and DNA lyses of data obtained from a number of sequences localized within previously reported epithelial tumors, including head and neck, kid- changed chromosomal regions in BCA. DNA ney, bladder, breast, colorectal, ovarian, and sequence copy number gains and losses were lung cancer, as well as malignant melanoma, identified for a panel of approximately 50 genes, and Wilms tumor, with the specific aim to dis- mostly so far not described in BCA. For a first close common patterns of the karyotypic evolu- validation, the DNA sequence copy number tion. We show that the tumors frequently develop changes of selected clones (SNRPN, CMYC, through a hypo- or a hyperdiploid pathway and CErbB2, ZNF217) were confirmed by fluores- progress by an increasing number of alternative cence in situ hybridization (FISH). However, the imbalances through at least two karyotypic pha- results were obtained from DNA solutions repre- ses, Phase I and Phase II, and possibly a third, senting an average DNA over a lot of BCA cells Phase III. During Phase I the karyotypes exhibit neglecting individual cellular variability and com- a power law distribution of the number of chan- plexity. Therefore, aCGH was a starting point ges per tumor as well as a Zipf distribution of the for further systematic microscopic studies of the frequency by which bands are involved in breaks. numerical organization of gene loci and genome At the transition from Phase I to Phase II/III the architecture in BCA. Selected cases were used for observed power law and Zipf distributions systematic studies by 3D confocal laser scanning are lost indicating a transition from an ordered microscopy after sequential multi-locus FISH and highly structured process to a disordered (SML-FISH). Different states of aneuploidia of and chaotic pattern. The change in karyotypic the chromosomes 7, 8, 11, 17, 20 were analyzed 132 15th ICC: Abstracts in correlation to the copy number changes of the Mus) show evolutionary plasticity around the oncogenes CMET, CMYC, CyclinD1, CErbB2, mouse/human conservation breakpoint, that may and 20.q13.2. Due to SML-FISH it was possible explain the regional involvement in cancer related to detect simultaneously existing copy number deletions. changes of these genes and coincident intra-cel- Yang Y et al. PNAS 98, 1136–1141, 2001; Kost-Alimova M lular aneuploidia in individual cells. The correla- et al, PNAS 100:6622–6627, 2003 tion of the analyzed parameter indicated the complex changes in the genome and supported findings of intra-tumor heterogeneity being typi- PO:08:54 cal for Barrett’s adenocarcinoma. A clue for recessive inheritance pattern in familial colorectal cancer in Iranian L147 population Comparative genomics and F. Bishehsari1, M. Mahdavinia1, R. Ansari1 chromosome deletions in cancer. and R. Malekzadeh1 Lessons of the microcell hybrid based 1Digestive Disease Research Center, Shariati ‘elimination test’ Hospital, Kargar Shomali, 14114, Tehran, Iran S. Imreh1 The incidence of colorectal cancer in Iranian 1Karolinska Institutet, MTC, Stockholm, Sweden young adults is growing, the aim of this study was to investigate the familial aggregation of col- The elimination test developed by us is based on orectal cancer and also evaluate the hereditary the transfer of single human chromosomes into pattern of cancer in these families. tumor cells of human or murine origin, via In a pilot study, we conducted detailed inter- microcell fusion. The microcell hybrids are tested views with 327 Iranian colorectal cancer patients for the elimination vs. retention of specific chro- about their family histories of malignancies, age mosome regions after one or several passages in at diagnosis and consanguinity. SCID mice. On chromosome 3 the group gradu- Overall 118 patients (32%) had at least one ally narrowed down a common eliminated region ¢rst-degree relative with cancer. Twenty ¢ve on 3p21.3 designated CER1. A common retained patients (7.6%) had more than two ¢rst- or sec- region (CRR) preferentially retained after 4 con- ond-degree relatives with colorectal or other secutive SCID passages has been identified at HNPCC related cancers; suggestive of Lynch I or 3q26-qter. It contains among others the p53 II by the Amsterdam II criteria. Examining the induced WIG1 gene and the RNA component of family pedigrees, most of the family clustering of telomerase. In parallel we proved that in human/ disease was suggestive of autosomal dominant human MCHs based Et similar regions are elimi- pattern of inheritance, but two families showed nated as in the human/murine system and di¡erent patterns. One family with history of CER1-LOH characterizes 83% of human tumors. colon and gastric cancer in father’s grand father The transcriptional map of CER 1&2 has been and early-onset colon cancer in mother’s mother assembled, that contain 26 genes 6 of them novel, and endometrial cancer in two of mother’s aunts, cloned by the group. LIMD1 and LF are tumor but no history of any cancer in second generation, inhibitory gene candidates on functional basis but parents are ¢rst cousins while four of nine, in the presence of 10 CCRs, that arised by gene dupli- thirdgeneration(theirchildren)hadearly-onset cations, in CER1&2 is the most provocative. We colon cancer. The other family with colon and described the mouse synteny of 3p deletions and extra colonic cancers in both side grandparents found that contains both the borders of conserved relatives and no history of such disease in parents, segments and a hairpin forming (TATAGA)n in third generation two colon and one breast repeat. Invertebrates (Caenorhabditis elegans, cancer before age of forty, parents were not Drosophila) in contrast with vertebrates (Fugu, biologically related. 15th ICC: Abstracts 133

These results suggest that recessive contribu- PO:08:56 tions could be responsible in some family clusters of colorectal cancer, which needs further Cytogenetic and molecular cytogenetic molecular assessment of CRC related genes. analyses in brain tumors A. Cirakoglu1, Y. Tarkan-argu¨den1, S. Yilmaz1, D. Kuru1, M. Inan2, M. Uzan2, PO:08:55 E. Ozyurt2 and S. Hacihanefioglu1 Oesophageal carcinoma in South 1Dept. of Medical Biology, Cerrahpasa Africa: Possible involvement of FHIT Medical School, Istanbul University, Istanbul, Turkey; 2Dept. of Neurosurgery, Cerrahpasa J. Brown1, A. Stafne1, S. Engelbrecht1, Medical School, Istanbul University, Istanbul, R. Veale1 and P. Willem1 Turkey 1 University of the Witwatersrand, There are reports in literature on chromosome Johannesburg, SA abnormalities specific to different brain tumor subgroups. We set up tumor tissue cultures of 37 Oesophageal cancer (OC) is the third most cases who had been operated due to brain common malignancy in South Africa (SA), tumors. Chromosomes were obtained from 16 affecting 1 in 20 and 1 in 76 black males and cases, pathologically diagnosed as 3 astrcytomas, females respectively. The dominant type is 2 oligodendrogliomas, 1 oligoastrocytoma, 3 neu- squamous cell carcinoma (SSC), an aggressive roglial tumors, and 7 meningiomas. There was disease showing a poor prognosis largely due not a common numerical chromosome abnorm- to late diagnosis after the onset of symptoms. ality in astrocytoma cases. The breakpoints The precise mechanisms of development and involved in structural chromosome abnormalities progression of this disease are still unclear and of the astrocytoma cases were; 3q28 in one case, the identification of genetic changes associated 1p36, 1q31, 2q37, 3p21, 6q14, 7q36, 8q21q24, with these tumours may shed light on its 9p24,9q10, 11p15, 11q23,16q24 in one case, and aetiology in SA, as well as provide early diag- Xq27, 3p21, 7p22, 10p15, 22q13, 19p or q13 in nostic tools. The Fragile Histidine Triad gene another case. dmins and ring chromosomes were (FHIT) is a tumour suppressor gene encom- obtained in two cases. X was the only clonal passing the most common fragile site FRA3B, chromosome abnormality in one oligoden- located at 3p14.2. Loss of heterozygosity at the droglioma case. Y, 21, and 22 were present FHIT locus has been detected in a high per- in the oligoastrocytoma case. Aneuploidies in centage of primary tumours and pre-cancerous meningiomas were, 22 (in 5 cases), X, 10 lesions of the oesophagus elsewhere, suggesting (in 3 cases each), 3, 5, 6, 9, 12, 16, that it maybe an early event in oesophageal 17, 19, 20, 21 (in 2 cases each), and 2, carcinogenesis. This gene has not been investi- 4, 8, 11, 14 (in one case each), þ3, þ5, gated in SA oesophageal cancer. We looked at þ9, þ11, þ14 in one case and þ1, þ13, and the integrity and expression of FHIT in 5 þ16 in another case. The structural abnorma- oesophageal cancer cell lines established in SA. lities found in meningiomas were del(1)(p21) in Due to heterogeneous deletions within FHIT, one case, inv(2)(p15q11), add(7)(q36) in another we used fluorescence in situ hybridisation with case. Interphase FISH using alpha-satellite FHIT specific probes as a screening tool for probes specific to chromosomes X, Y, 7, and gene integrity. Significant rearrangements of the 10 was performed 16 cases and obtained FHIT gene were detected in 2 of the cell lines. results from 7 cases. We observed one case with RT-PCR detected aberrant transcripts in the þ7and 10, one case with þ7, and one case same 2 cell lines. These findings suggest that with 10. FHIT may have a role to play in oesophageal carcinogenesis in SA patients. Investigation of This work was supported by the Research Fund of the FHIT’s role in OC in SA patients is pertinent. University of Istanbul, project No. T-1056/19022001 134 15th ICC: Abstracts

PO:08:58 have the potential to signi¢cantly improve the detection of urothelial carcinoma. Comparison of £uorescence in situ hybridization (FISH) and melanoma PO:08:59 antigen gene (MAGE) Tests for the detection of primary bladder cancer Incidence and characteristics of Ph- negative in chronic meyloid lukemia S. Koo1, T. Jeong1, J. Kang1, K. Kwon1, 1 1 1 J. Park1,Y.Na2 and J. Lim2 A. Movafagh , F. Isfahani , H. Attarian , 1 1 1 1 A. Hajfathali , M. Ghadiani , S. Baharizadeh Department of Clinical Pathology, Chungnam and N. Varma2 National University Hospital, Taejon , South 2 Korea; Department of Urology, Chungnam 1Shahid Beheshti Medical University, Tehran, National University Hospital, Taejon, Iran; 2PGI, Chandigareh, India South Korea Philadelphia (Ph) chromosome negative chronic Our aim was to evaluate and compare the Uro- myeloid leukemia (CML) can be distinguished Vysion (Vysis, USA) fluorescence in situ hybridi- from clinically similar disorders on the basis zation (FISH) and Melanoma Antigen Gene of presence of rearrangement of the breakpoint (MAGE) tests for improved detection of bladder cluster region (bcr) of chromosome 22. The leu- cancer in urine specimens. kemic cells of most patients with CML carry a Forty ¢rst-morning urine specimens, 27 with a t(9;22) (q34;q11). Standard cytogenetic analysis history of urothelial carcinoma and 13 with was performed on bone marrow or peripheral benign prostatic hyperplasia (controls), were ana- blood samples cultured for 0–48 hours in the lyzed. A mixture of £uorescent labeled probes to absence of mitogen, using G-banding technic. Of the centromeres of chromosomes 3, 7 and 17, and the 150 CML cases analysed 81 were males and band 9p21 (P16/CDKN2A gene) was used for 69 females, the median age of these patients, at FISH to assess urinary cells for chromosomal the time of cytogenetic examination, was 31(14 to abnormalities indicative of malignancy. In addi- 73 years), 140 cases (93.3%) were Ph-positive and tion, reverse transcriptase^polymerase chain reac- the remaining 10 cases (6.6%) were Ph-negative. tion (RT-PCR) and the nested PCR with the All of the 10 Ph-negative cases, except 2 cases MAGE common primers (C1/C2, and C3/C4) with chromosomal abnormalities showed a nor- designed to detect MAGE 1-6 genes were con- mal karyotype. The incidence of Ph-negative ducted in the same samples and con¢rmed with cases, which was low (6.6%), was considerably the form of band on agarose gel electrophoresis. lower than usually reported incidence of about Twenty-¢ve of the 27 patients showed FISH- 15%, though it has been pointed out that the inci- positive analysis. The sensitivity of FISH for pTa, dence varies from 0% to more than 30% among pTis and pT1-pT4 tumors was 60%, 94% and different publications. Preliminary evidence sug- 90%, respectively, with an overall sensitivity of gests that these cases respond to chemotherapy 93%, compared to 55%, 88% and 87%, respec- and behave clinically as typical Ph-positive CML. tively, for MAGE, with an overall sensitivity of 85%. FISH was signi¢cantly more sensitive than PO:08:60 MAGE for all tumors (p ¼ 0.045). The speci¢city of FISH and MAGE among patients with benign Molecular cytogenetic abnormalities prostatic hyperplasia was 96% and 95%, respec- in Thai colorectal tumors by tively ( p ¼ 0.627). comparative genomic hybridization The sensitivity, but not speci¢city, of FISH for technique the detection of urothelial carcinomas is superior to that of MAGE. Even though further pro- S. Poeaim1, B. Rerkamnuaychoke2, spective studies are required, FISH and MAGE S. Jesdapatarakul3 and A. Campiranon4 15th ICC: Abstracts 135

1Department of Applied Biology, Faculty of 1Dept. of Genetics and Molecular Biology, Science, King Mongkut’s Institute of Technology Medical School, Isfahan University of Medical Ladkrabang. Bangkok 10520, Thailand; Sciences, Isfahan 81744-176, Iran 2Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, In order to define relationship between cytoge- Thailand; 3Department of Pathology, Vajira 4 netic abnormalities and prognosis and the sub- Hospital Medical College, Thailand; Department types of the non-Hodgkin’s lymphoma (NHL) of Genetics, Faculty of Science, Kasetsart patients, we employed the technique of Com- University, Thailand parative Genomic in situ Hybridization (CGH). This technique allows, in a single, rapid analysis, Little information is know about the genetic the detection of chromosomal gains and losses in changes of colorectal cancer (CRC) in Thailand. the complete tumor genome. It also requires only The aim of the present study was to identify a small amount of tumor material and allows the copy number aberration of potential oncogene detection of genetic abnormalities independent of and tumor suppressor gene locations in CRC. the presence of metaphase spreads. Comparative Genomic Hybridization (CGH) In this study, we investigated 33 lymph node technique was used to screen 40 cases of biopsy samples from patients diagnosed with B- CRC that were divided into 3 stages: Dukes’ A cell non-Hodgkin’s lymphoma using control (n ¼ 11); Dukes’ B (n ¼ 12) and Dukes’ C (n ¼ 17). DNA, FITCE labeled, and tumor DNA, Texas The most frequent gains in all CRC were on red labeled, were co-hybridize to a normal meta- chromosome arms 16p (15%: 6 of 40 tumors), phase spread. Images were acquired using an epi- 7p, 7q, 12q, 13q, 17q (17.5% each), 19p (20%), £uorescence microscope and the commercially 19q (22.5%), 8q (25%) and 20q (60%) and the available image analysis system ISIS from Meta- most frequent losses were on 4p (17.5%), 4q (20%) Systems. Ratio pro¢les of the £uorescence inten- and 18q (25%). The smallest region of copy num- sities of tumor DNA and control DNA were ber aberration was mapped to gain of 8q24 in calculated for each individual chromosome. five cases. The precised locations found in this Our results demonstrated that, using CGH, study are quite relevant to previous reports. In 24/33 (73%) of the patients exhibited chromoso- addition, these results highlight several chromo- mal imbalances. Some chromosomal regions somal regions that may harber important genes such as 3q22, 3q26, 8q24, Xq26, 2p23, 2p14 and for CRC. A correlation analysis between the 18q21were frequently ampli¢ed while frequent copy number aberration and the stage in our deletions were found at some other chromosomal total material showed a statistically significant (p regions such as 5q11-13, 8p2, 9p2, 13q21-32, 0.05). Increased copy number of chromosome and 17p. arms 7p, 12q and 16p (p ¼ 0.0389 each) were sig- In general it appeared that there might be a cor- nificantly associated with Dukes’ B and Dukes’ relation between the total number of events (gains C, while increased copy number of chromosome and losses) per tumor detected by CGH and arm 17q (p ¼ 0.0471) was associated more with aggressiveness of the tumor. Even though the Dukes’ A and Dukes’ C. Our data suggesting exact target of the observed ampli¢cations and that genes on those chromosomes may play key deletions by CGH may not be known, but the role in CRC progression and provide several total number of events detected by CGH might be starting points for the isolation of candidate considered as a prognostic marker. oncogenes and tumor suppressor genes.

PO:08:62 PO:08:61 A novel FISH assay for SS18-SSX Evaluation of CGH for the molecular fusion type in synovial sarcoma diagnosis of NHL subtypes C. Surace1, F. Mertens1, I. Panagopoulos1, M. Salehi1, R. Salehi1 and M. Behjati1 E. PŒlsson1, M. Rocchi2 and N. Mandahl1 136 15th ICC: Abstracts

1Department of Clinical Genetics, Lund Deletions in 4q, 8p, 13q, 16q and 17p were University Hospital, Lund, Sweden; 2DAPEG, frequently reported in hepatocellular carcinoma Section of Genetics, University of Bari, Bari, Italy (HCC) by comparative genomic hybridization and loss of heterozygosity studies; yet, detailed Synovial sarcoma is a morphologically, clinically chromosome structural aberrations are not known. and genetically distinct entity that accounts for We therefore characterize structural changes in 5 to 10% of all soft tissue sarcomas. The these arms and identify the minimal deleted majority of synovial sarcomas carries a t(X;18) regions and breakpoints. By multiplex-color (p11.2;q11.2) chromosome translocation, that is a FISH (M-FISH) with chromosome region-spe- specific cytogenetic rearrangement, being present cific probes (CRPs), chromosome structural aber- in more than 90% of the cases of synovial sar- rations of 4q, 8p, 13q, 16q and 17, in six HCC coma and not in any other tumours. It produces cell lines, BEC7402, QGY7703, PLC8024, three types of fusion gene formed in part by HepG2, H2M and H4M were studied. All CRPs, SS18 from chromosome 18 and by SSX1, SSX2, which were generated from microdissected DNA, or, rarely, SSX4 from the X chromosome. It has were specific and strong in intensity, and sensi- been shown that in synovial sarcoma a clear cor- tive enough to detect chromosome structural relation exists between type of fusion gene and aberrations including translocation, deletion and clinical outcome. SS18-SSX fusion transcript can amplification of target regions. Chromosome be detected by PCR amplification, requiring structural aberrations were matched with the access to good quality RNA. In order to obtain results of CGH, yet additionally several cryptic an alternative tool to diagnose and follow this breakpoints, especially those resulted from trans- malignancy, we developed a fluorescence in situ location. The findings by M-FISH using CRPs hybridization (FISH) assay that could distinguish effectively narrowed down the position of break- between the two most common fusion genes, i.e., points, making the further BAC screening more SS18-SSX1 and SS18-SSX2, which cannot be dis- efficient. Chromosome structural aberrations criminated using conventional chromosomal were matched with the results of CGH, yet addi- banding methods. The specificity of the selected tionally several cryptic breakpoints, especially BAC clones used in this FISH assay, as well as those resulted from translocation. The findings the sensitivity of the analysis in metaphase and by M-FISH using CRPs effectively narrowed interphase cells, was examined in a series of 22 down the position of breakpoints, making the synovial sarcoma samples without awareness of further BAC screening more efficient. The break- fusion gene status (previously determined by RT- points in 16q22 and 13q14 were finally mapped PCR). In all samples, the type of fusion gene was at 341K23 and between 240M20 and 456B18, correctly identified by FISH, comparing with the repectively. results obtained by PCR. Thus, the assay descri- bed here should be useful for identifying fusion gene status in samples from which insufficient PO:08:64 amounts of RNA could be extracted. A translocation t(3;11) in an ovarian carcinoma cell-line inactivates the FHIT gene. PO:08:63 1 Characterization of 4q, 8p, 13q, 16q P. Willem Belot and N. Reckviashvili 1 and 17 in six hepatocellular carcinoma Department of Haematology and Molecular Medicine, National Health Laboratory Services cell lines using region-speci¢c and the University of the Witwatersrand, multiplex-FISH probes Johannesburg, SA W. Tjia1, S. Sham1,L.Hu1 and X. Guan1 The molecular events driving the pathogenesis of 1Department of Clinical Oncology, University of ovarian cancer have not been clearly elucidated. Hong Kong Genetic instability and the involvement of 15th ICC: Abstracts 137 common fragile sites are being reported in an sentation will summarize data on trisomies 16, 18 increasing number of human cancer studies. In and sex chromosome trisomies as they relate to particular the putative tumor suppressor gene this model. FHIT (fragile histidine triad) which lies within Additionally, we will summarize cytological stu- the most common fragile site, FRA3B, has been dies designed to build chromosome-speci¢c shown to be involved in a variety of tumors includ- human genetic maps. These studies rely on ing a subset of ovarian carcinoma. In most tumors recently developed immuno£uorescence metho- the preferred mechanism of FHIT gene inactivation dology; speci¢cally, immunostaining of pachytene is through large intragenic deletions or promoter preparations using antibodies against MLH1 (a hypermethylation. Only four tumor associated mismatch repair protein thought to localize to translocations have been reported so far of which meiotic recombination nodules) and SCP3 (a com- one was described in a breast cancer cell line. ponent of the lateral element of the synaptonemal We describe a translocation t(3;11)(p14;p15) complex) makes it possible to visualize the sites of present in an early passage ovarian carcinoma cell meiotic exchanges. Our initial analyses of several line. Using £uorescence in-situ hybridisation thousand pachytene stage cells from 50 males (FISH) with selected YACs and BACs clones we make it clear that this approach does, indeed, ful- mapped the breakpoint on chromosome 3 within ¢ll criteria predicted for a molecule that ‘marks’ the FRA3B loci. reverse transcriptase polymerase the sites of exchanges. Thus, in subsequent studies chain reaction (RT-PCR) further re¢ned the we have used this approach to characterize the breakpoints and showed that the translocation number and location of exchanges in a series of resulted in the complete loss of FHIT expression. males, and to ask whether abnormalities in the This ¢nding supports a role for FHIT in some recombination pathway are evident in infertile ovarian carcinoma. males. The results of these analyses will be pre- sented at the symposium. Aneuploidy Symposium L149 A model system for increased L148 non-disjunction in older oocytes Recombination and aneuploidy S. Bickel1 in humans 1Dartmouth College, Hanover, NH 03755 USA T. Hassold1 1Department of Genetics and Center for Human As women age, the incidence of meiotic non- Genetics, Case Western Reserve University, disjunction rises dramatically. Although increased Cleveland, Ohio 44106-4955, USA maternal age has long been correlated with errors in chromosome segregation, the molecular defects Despite the clinical importance of human aneu- that give rise to age-dependent meiotic non- ploidy, we have been ignorant of the causes of disjunction in human oocytes are largely meiotic nondisjunction, the process that gives rise unknown. One major limitation has been lack of a to trisomic progeny. Over the past decade, how- suitable model system that recapitulates the ever, genetic mapping studies have led to the higher levels of meiotic nondisjunction observed in identification of the first molecular correlate of older human oocytes. We have developed an human nondisjunction; i.e., altered levels and experimental regimen using Drosophila melano- positioning of recombinational events. These gaster to study error prone segregation in older observations have led to the idea that human oocytes. Using a sensitized genetic background in meiotic nondisjunction may involve ‘two hits’: which sister chromatid cohesion is compromised, first, the establishment in prophase of a ‘vulner- we have shown that Drosophila oocytes exhibit a able’ bivalent and second, abnormal processing significant increase in meiotic nondisjunction of the bivalent at metaphase I or II. The pre- following aging. I will describe these initial 138 15th ICC: Abstracts experiments and our continued analysis of 1Department of Human Genetics, Emory segregation defects in older oocytes. University, Atlanta GA; 2Department of Biostatistics, University of Pittsburgh, USA L150 Advancing maternal age has long been identified The role of the meiosis-speci¢c protein as the primary risk factor for human chromo- SCP3 in chromosome segregation and some trisomy. In addition, altered patterns of aneuploidy meiotic recombination have now been associated with an increased risk of nondisjunction. For 1 C. Ho¨o¨g trisomy 21, these ‘susceptible’ patterns include 1Department of Cell and Molecular Biology, exchange near the centromere or single, telomeric Karolinska Institutet, Stockholm, Sweden exchanges. To date, however, no relationship has been found between these patterns on chromo- Aneuploidy (trisomy or monosomy) is a leading some 21 and maternal age. As part of an ongo- genetic cause of pregnancy loss in humans and ing study of maternal trisomy 21 (ts21), we results from errors in meiotic chromosome segre- examined the recombination status of 562 ts21 gation. We have developed a novel mouse model cases. The cases were genotyped at STR markers system where a single gene defect predisposes to spanning chromosome 21 and divided into aneuploidy (Yuan et al (2000) Molecular Cell 5, groups based upon maternal age at the time of < > 73–83; Yuan et al (2002) Science, 296, 1115– the trisomic birth ( 29, 29–34, 34 years of age) 1118). We show that absence of Synaptonemal and tetrad analysis used to infer the meiotic Complex Protein 3 (SCP3) promotes aneuploidy exchange patterns. Achiasmatic tetrads accoun- in murine oocytes by inducing defective meiotic ted for 39%, 21% and 28%, respectively, of each chromosome segregation. The abnormal oocyte age population. For tetrads with exchanges, the karyotype is inherited by embryos, which die location of the exchanges varied between the in utero at an early stage of development. In groups in an age-dependent manner. Among the addition, aneuploidy and embryo death in SCP3- youngest group, 62% of nondisjoining oocytes a~ deficient females increases with advancing mater- had susceptiblea¨ exchange patterns. These pat- nal age. We find that SCP3 is required for the terns were less frequent in the mid (44%) and organization of the cohesin core, for the older (33%) age groups, which instead contained structural integrity of meiotic chromosomes and increased proportions of stable, medially located for chiasmata formation, suggesting that altered exchange. These data suggest that oocytes of chromosomal structure triggers chromosomal younger women, with functional meiotic appara- non-disjunction. The SCP3-null mouse model tus and/or robust ovarian environment, are able system is used for studying the cause of aneu- to properly resolve all but the most susceptible ploidy in female germ cells, how such cells exchange patterns. As women age, however, are (inefficiently) eliminated and the con- meiotic mechanisms erode, making it difficult to sequences of aneuploidy for germ cell and resolve even stable exchange events. embryo development. L152 L151 Is there a relationship between trisomy risk and rate of ovarian aging? Examination of recombination 1 1 1 patterns by maternal age among W. Robinson , K. Bretherick , C. Brown , S. Watson2 and W. Lam2 nondisjoined chromosomes 21 of 1 maternal origin. Dept. of Medical Genetics, U. of British Columbia, Canada; 2Dept. of Pathology, S. Sherman1, E. Feingold2 and N. Lamb1 U. of British Columbia, Canada 15th ICC: Abstracts 139

Most trisomies arise as consequence of abnormal proximal 50% segment of chromosome 16 and chromosome segregation during oocyte matura- the distal 37% of the X chromosome. These cell tion and occur more frequently with maternal lines were uniformly unable to form typical age. Some data indicate that trisomy risk may be embryoid body failing in the formation of the more closely related to ovarian age than mater- outer endoderm layer. Cell aggregates they nal age. We thus hypothesize that genetic factors formed remained undifferentiated in suspension leading to premature loss of ovarian follicles culture for more than two weeks, while kar- should be increased in women experiencing a tri- yotypically normal ES cell lines gave rise to some somic pregnancy at a relatively young age. Large balloon-like cystic embryoid bodies by the 8th sized alleles (>34 repeats) at the FMR1 locus day of differentiation. These mutant cell lines and X-chromosome rearrangements are two fac- produced much fewer and smaller tumors with a tors that have been associated with premature longer latent period after subcutaneous injection ovarian failure (POF). FMR-1 allele size was into nude mice than chromosomally normal ES determined in 192 women who had a maternal cells. Unexpectedly, the degree of tissue differ- meiotic nondisjunction event leading to a preg- entiation in these tumors was comparable to nancy with trisomy or uniparental disomy. The those derived from karyotypically normal ES resulting distribution of allele sizes was similar to cells. A chromosome study showed, however, that found in 107 controls. Specifically, there was that the majority of tumor cells had lost the X16 no excess of alleles >34 repeats overall (4.7% of [der(X)] chromosome and gained an additional patient versus 5.1% of control alleles) or when copy of chromosome 16. The present observation subdivided by maternal age or type of abnorm- suggests that the monosomy for the proximal ality. We previously reported an increase in segment of chromosome 16 is mainly responsible skewed X-chromosome inactivation (XCI) among for the failure of endoderm differentiation. It women experiencing a trisomic pregnancy. The remains unknown, however, whether the gen- possibility of a microdeletion or microduplication erally low differentiation potential in these ES of the X as an explanation for skewing is cur- cell lines is attributed to the partial monosomy rently being investigated in a subset of these 16, or non-inactivation of X16 chromosome, or women using a high-resolution (*300kb) genomic both. array. While FMR1 status and X-chromosome abnormalities may contribute little to overall risk L154 of trisomy, it is still likely that there is some varia- bility in risk of trisomy at a particular maternal Array-CGH in the search for tetralogy age due to cumulative effects of common genetic of Fallot genes on chromosome 8 variants affecting ovarian and chromosomal aging. M. Poot1, M. Eleveld1, M. Van Dam1, R. Hochstenbach1 and J. Giltay1 L153 1Department of Medical Genetics, University Karyotype-speci¢c failure of mouse ES Medical Centre Utrecht, P.O. Box 85090, 3508 cell differentiation in vivo and in vitro AB Utrecht, The Netherlands 1 2 3 N. Takagi , M. Shoji and T. Tada After an uneventful pregnancy of 37 weeks a 1Hokusei Gakuen University, Sapporo; child with multiple dysmorphic features of head, 2Youdosha, Tokyo; 3Inst. Frontier Med. Sci., hand and feet, and a Tetralogy of Fallot (ToF) Kyoto University, Kyoto was born. The mother had previously given birth to three healthy children. The karyotype of the Differentiation potential was studied in three child was initially defined as 46,XY,i(8)(q10). The mouse ES cell lines carrying an unbalanced form mother proved to be carrier of a pericentric of Searle’s translocation, T(X;16)16H. The unba- inversion with 8p22 and 8q21.2 as the break- lanced karyotype designated as 40,XY, 16, points. DNA of the child was examined by þder(X),t(X;16)mat is doubly monosomic for the array-CGH with an array containing 3,343 140 15th ICC: Abstracts

FISH-localised BAC DNA probes, covering the Hypodiploid aneuploid cells have regularly been entire genome with an average spacing of reported in the Pacific oyster, Crassostrea gigas. approximately 1 Mb (Vissers et al. (2003) Am J A negative correlation between this phenomenon Hum Genet 73:1261–1270). This analysis revealed and growth as well as evidence for a genetic a 6.1 Mb deletion of the distal part of 8p basis have been shown. Furthermore, non-ran- (8p23.1pter) and a 30.0 Mb duplication of the dom chromosome loss was also demonstrated in distal part of 8q (8q21.3qter), as a result of G-banded aneuploid karyotypes of C. gigas. recombination within the inverted segment. Chromosome pairs 1, 5, 9, and 10 were char- In 55% of the patients with a del(8p23.1pter)- acterised by the loss of one chromosome. The dup(8q22.1qter); (OMIM 179613) conotruncal present study investigated the effects of the her- heart defects have been found. None of the 15 bicide atrazine on the level of aneuploidy in this genes with known function in the deleted region species. Firstly, C. gigas adults and juveniles of 8p of our patient have been linked to ToF. In were subjected to different atrazine doses (10 mg/l contrast, the duplicated region of chromosome 8q representing a peak value found in a polluted contained a zinc ¢nger protein gene (ZFMP2; environment and 100 mg/l). A positive relation- OMIM 603693; also known as FOG2). This gene ship between atrazine concentration and aneu- may be a candidate gene for ToF, because point ploidy was observed. Moreover, the aneuploidy mutations have been associated with ToF. The level of a sample of juveniles, previously exposed ToF candidate gene GATA4 gene is not con- to atrazine, which were subsequently transferred tained within the 8p deletion. We conclude that to non polluted conditions, remained significantly the improved resolution of array-CGH will help different between treatments. Furthermore, a to identify chromosomal regions and candidate progeny of oysters exhibited significantly higher genes involved in the pathogenesis of ToF. aneuploidy levels when their parents had pre- viously been exposed to atrazine. Thus, the aneu- ploidy phenomenon persists in time both within L155 and between generations. Additionally, restric- tion endonuclease banding was used on the pro- Etiology of recurrent trisomy geny to identify which chromosomes were D. Warbuton missing. The identity of chromosomes lost is not influenced by atrazine as the same chromosome Babies Hospital South, 3959 Broadway, pairs were affected by the loss of one chromo- Room 406, New York, NY 10032, USA some. Further investigation is required to enable a better understanding of aneuploidy in oysters, No abstract was submitted for this talk. especially as to why cells tolerate the loss of these chromosomes, and why some chromosomes are more easily lost than others. PO:08:65 Effects of the herbicide atrazine on PO:08:66 aneuploidy in Paci¢c oysters, The role of viruses (EBV, CMV, HPV Crassostrea gigas etc) in reproduction and aneuoploidy K. Bouilly1, A. Leit1, H. Mccombie1, V. Culic1 2 2 1 R. Chaves , H. Guedes-Pinto , P. Boudry 1 1 Department of Medical Genetics and Genetic and S. Lapague Counseling Unit, Clinical Hospital, Split, Croatia 1IFREMER, Laboratoire de Ge´ne´tique et Pathologie, 17390 La Tremblade, France; Each chromosome contains two centrioles which 2University of Tra´s-os-Montes and Alto Douro, adhere to each other through the cell cycle and Centre of Genetics and Biotechnology, 5001-911 normally separate only once during the G1-to-S Vila Real, Portugal cell cycle transition, resulting in centrosome 15th ICC: Abstracts 141 duplication. Centrosome duplication is regulated in 1,4 times higher comparatively to average by many intracellular events that are essential in index in 1985–1994 according to Lviv regional maintaining genomic stability. Abnormal centro- medical–genetic center data. The Down syn- some duplication is tightly linked to aneuploidy. drome is confirmed cytogenetic among 94,4% Spontaneous abortions appear with the incidence ones those diagnosed by the phenotype createria of 12–15% in population. In spontaneous abor- in the maternity hospitals. The cytogenetic cheks tions there are 25–60>% of chromosome have showed 95,2% Trisomy 21, 3,0% mosaicism abnormalities, and in most cases there are tri- for Trisomy 21, 1,8% Robertson’s translocation ploidies, tetraploidies and polyploidies. The genes of all cases. associated with cell cycle abnormalities are p53, Brca1, Brca2, Gadd45, human papillomavirus PO:08:68 type E6 and E7 also leads to mitotic defects. The theory of ‘two hits’ for one unstable cell cycle Phenotypic and molecular analysis in resulting with aneuploidy is still in bases of these 90 girls with Turner’s syndrome events. From the couples in the genetic counsel- 1 1 ling process with normal karyotype and aneu- R. Fernandez and E. Pasaro ploidy in aborted material analysed by flow 1Department of Psychobiology, University of A cytometry, we found CMV and EBV reactivation Coruna, Spain or new infection in both parents before and/or during pregnancy. We analysed those with aneu- We performed a phenotypic and molecular ana- ploidy in aborted material and significant ser- lysis of sex chromosome mosaicism in 90 Turner ologic findings in both parents. A retrospective syndrome patients. The investigation was carried analysis was done over 500 couples with one or out in three phases: 1) Cytogenetic (G-banding more spontaneous abortion and 296 paraffin and FISH); 2) Molecular (PCR and SRY gene embedded samples were found. 41 placentas was sequencing); 3) Comparative analysis of the analysed by flow: 27 (66%) diploid and 14 (34%) phenotype. aneuploiyd. From all number of 290 88 (30%) The application of classical alpha-satellite had IgM or/and high IgG for EBV and 12 (4%) probes (CEP-X and CEP-Y), painting probes for CMV. HPV positive diploid paraffin (WCP-X and WCP-Y), two subchromosomal embedded sample from one couple with three painting libraries (SCPL116, SCPL102) covering consecutive spontaneous abortion was found. the short and the long arm of the X chromosome, XIST and DXZ4 probes allowed us to ¢nd a sec- ond cell line (mosaicism) in 90% of the patients; PO:08:67 only 10% of the patients were de¢ned as 45,X non-mosaic. The most frequent mosaic was 45,X/ The Down syndrome frequency 46,XX; the presence of isochromosomes or dynamics in Lviv region (Ukraine) for pseudo-isochromosomes comprised 25%, frag- 1985^2001 period ments 5% and Y chromosome 4.4%. The patients who had been previously diagnosed as mosaics dis- Z. Fedoryshyn1, O. Hnateiko1 and 1 played a higher complexity in their karyotypes D. Zastavna due to the presence of new cell lines. Only one of 1Institute of Hereditary Pathology of Academy the patients showed mental retardation due to a of Medical Science of Ukraine small ring der(X). The Y chromosome and the SRY gene were pre- The Down syndrome average in Lviv region sent in blood and ovarian tissue in four patients. (1985–2001 period) composed 0,7 on 1000 new- The analysis of the SRY sequence in one of the borns or 1:1430 newborns. Analysis of received patients indicated the presence of two copies of results showed the increased frequency of Down the SRY gene, simulating a heterozygous state of syndrome during this period. The average index the gene. One of the copies presented a point of Down syndrome frequency in 1995–2001 was mutation Arg59Gly within the HMG-box. 142 15th ICC: Abstracts

PO:08:69 PO:08:70 Demonstration of trisomy 18 in an Cytogenetic analysis chromosomal autolytically altered fetus: a case status of subjects from the regions in report the vicinity of uranium contaminated U. Gamerdinger1, D. May1 and A. Seidel1 areas 1 1 1 1Institute of Pathology, University of Gieen, D. Jovicic , S. Milacic and R. Kovacevic Germany 1Institute of Occupational Health and Radiological Protection ‘Dr Dragomir Prolonged time interval between intrauterine fetal Karajovic’, Belgrade, Serbia and death (IUFD) and abortion commonly leads to Montenegro advanced autolytic alteration of the fetus. In such cases culture failure often impedes karyotype ana- The study was aimed at determining possible lysis. Here we present a case in which the combina- karyotype genotoxic effects in individuals from tion of fetal pathology and molecular-cytogenetic the regions close to the contaminated areas. Bio- methods was helpful in the evaluation of chro- logical dosimetry was performed using modified mosome abnormality associated with fetal death. Moorhead’s micromethod. Our studies included A fetus aborted after IUFD at 18th gestational the targeted group of 29 patients from the affec- week showed advanced autolysis at outer ted regions. The subjects were averagely aged inspection and also for the inner organs. External 39.5 þ 2.8 years. Average age of the control fetopathologic examination demonstrated devel- group (K), unexposed to the effects of the known opmental delay, hygroma colli, cranio-facial dys- genotoxic agents comprising 22 individuals was morphias, anal atresia and ambiguous genitalia. 28.3 þ 1.2 years. The presented data evidenced The placenta was hypotrophic with abnormal villi that increased incidence of the chromosomal and a single umbilical artery. The anomalies were aberrations was found in 6 subjects, accounting suggestive of chromosome aberration. In order to for 20.6%. Dicentric type changes were evi- prove suspected chromosomal aneuploidy mole- denced, as well ring chromosomes and acentric cular-cytogenetic studies were carried out. fragments, which are, at the same time the most Fetal DNA for CGH was extracted from tissue frequent aberrations. The changes are considered samples of kidney and lung. Despite DNA-degra- reparable aberrations accounting for 2–3% in dation CGH revealed a gain of the entire metaphases of the unexposed individuals. Statis- chromosome 18. To verify the result an inter- tical data processing evidenced significant differ- phase-FISH with a probe for 18cen was per- ence (p < 0,005) between structural chromosomal formed on cryo-sections of the kidney. Most aberrations in the studied and control groups, as nuclei exhibited three signals but also several well as in the number of chromatid aberrations showed only two signals. The latter were inter- (p < 0.05). Based on the obtained data it may be preted as artifact due to truncated nuclei or concluded that human karyotype changes were reduced hybridization e⁄cacy in the macerated present in the studied group, resulting from inter- tissue but a mosaic could not be excluded at least. action of ionizing irradiation and other genotoxic Postmortal diagnosis in fetal death is of impor- agents, with possibility of potent synergistic tance in counseling patients regarding recurrence effects. It is necessary to stress the importance risk. We could demonstrate that despite autolyic of further monitoring and control of the general alteration of a fetus which inhibits classical chro- population health, particularly due to mosome analysis molecular-cytogenetic methods possible late genetic effects that may effect future may be helpful in unraveling aneuploidy. generations. 15th ICC: Abstracts 143

PO:08:71 tool for identi¢cation of gene(s) responsible for recognition of chromosomal imbalance in Chromosome imbalance causes mammalian cells. elevated apoptosis in pre-mature neurons PO:08:72 Y. Kai1, Y. Kazuki2, S. Abe1, C. Okita1 1 Aneuploidy detection by FISH for and M. Oshimura postnatal, prenatal and 1 Department of Biomedical Science, Institute of preimplantation genetic diagnosis Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori (PGD) in India 2 University, Japan; Department of Molecular and P. Madon1, A. Athalye1, S. Dhumal1, Cell Genetics, Graduate School of Medical M. Kawle1, V. Bandkar1, A. Sopariwala1, Science, Tottori University, Japan N. Naik1, D. Naik1, S. Mohanty1 and 1 At least 8% of all human conceptions have major F. Parikh chromosome abnormalities and the frequency of 1Department of Assisted Reproduction and chromosomal syndromes in newborns is >0.5%. Genetics, Jaslok Hospital and Research Centre, Chromosome abnormality syndromes show non- Mumbai 400026, India specific phenotypic features. For example, most of the syndromes have a phenotype of mental Fluorescence in-situ hybridization (FISH) has retardation. Although these disorders make a become a rapid and reliable genetic diagnostic large contribution to human morbidity and mor- tool in medicine. Jaslok Hospital and Research tality, there are little data showing the molecular Centre is the first in India to routinely offer mechanisms. FISH on a wide range of tissues. We have stu- Previously, we showed that ES cells containing died over 1000 samples using the Vysis Aneuvy- an extra human chromosome 21 led to the ele- sion probe kit, Metasystems software and a Zeiss vated apoptosis in pre-mature neurons in vitro. Microscope. For PGD, we also use a combina- To test a hypothesis that the chromosomal imbal- tion of the PB probes for chromosomes 13, 16, ance itself might stimulate the apoptotic dis- 18, 21 and 22 together with CEP 18, X and Y in crimination, we made mouse ES cell lines with a 2-step procedure to detect aneuploidy of 7 chromosome abnormalities by two methods, and chromosomes on single blastomeres biopsied investigated the apoptosis during neurogenesis. from clevage-stage embryos obtained after ICSI. Firstly we used microcell-mediated chromosome FISH on blastomeres using the Aneuvysion kit is transfer (MMCT) to create ES cells containing a more cost-effective. FISH on arrested embryos human chromosome 6, 11 or 21, respectively. Sec- could detect chaotic embryos. ondly, we created ES cell lines with mouse chro- We have been able to detect X chromosome mosome aneuploidy by sub-cloning of normal ES mosaicism for the ¢rst time in cumulus cells from cells. We investigated the apoptosis during neuro- follicular £uid obtained at pick-up of oocytes dur- nal di¡erentiation (at day 3) in these chromoso- ing IVF in a patient with low-grade mosaicism mal aberrant ES cell lines using the SDIA detected on karyotyping and con¢rmed by FISH method. Accelerated apoptosis was observed in on buccal smears. Cumulus cells can thus serve as all ES cell lines with chromosomal imbalance com- an alternate source to detect mosaicism in women paredtoEScellswithnormalkaryotype.These undergoing IVF. data suggest that the increase in apoptosis at the FISH on sperm gives an insight into the percen- early neural stem cell stage might be responsible tage of hypo or hyperhaploid sperm. FISH on pro- for the reduction of brain neurons in chromoso- ducts of conception is routinely carried out to mal abnormality syndromes, which cause mental detect aneuploidies and triploidy. In Prenatal retardation. This in vitro di¡erentiation system Diagnosis, a rapid report on uncultured amnio- using chromosomal aberrant ES cells is a useful cytes by FISH helps in allaying the anxiety of 144 15th ICC: Abstracts parents when the Triple Test shows a high risk. In remaining 21 samples were revealed to be normal cases from rural areas we detected Down syn- by both methods. drome by LSI FISH alone to make the tests The results indicate that interphase FISH is a a¡ordable to the masses. useful and reliable tool in detection of mosaeic aneuploidies. However the technique has to be PO:08:73 used as a parallel to conventional cytogenetic methods, which allows detection of structural A comparative study on detection chromosome abnormalities. ef¢ciency of classical cytogenetic and interphase £uorescence in situ PO:08:74 hybridisation in diagnosis of mosaic Spectrum of chromosome anomalies form of X chromosome aneuploidies among the fetuses with an increased S. Mohaddes1, S. Tagavi1, J. Mohseni1, nuchal translucency Z. Moosavi1, N. Bageri1 and H. Farahman L. Samoylova1, A. Latypov1, O. Shipacheva1 1 1Medical Genetic Unit, Faculty of Medicine, and I. Boobis Tabriz university of Medical Sciences, Tabriz, 1Genetic Division of Tatarstan Republic Hospital, Iran Kazan, Russia

Standard cytogenetics technique is of value in The aim of the study was to investigate the chro- detection of numerical and structural chromo- mosome abnormality spectrum among the fetuses some abnormalities. However the detection of with an increased nuchal translucency in Tatar- chromosomal mosaicism is often difficult due to stan (Russia). time constrains and limited number of available A total of 97 fetuses with an increased nuchal metaphase cells for analysis. Interphase fluores- translucency at 11 to 14 weeks’ gestation were cence in situ hybridisation (FISH) can be utilized evaluated cytogenetic prenatal diagnosis on to study the number of copies of a specific chro- chorion villi samples (CVS). mosome in interphase cells. The technique has A chromosome anomalies was obtained in 46 fetu- valuable application in detection of chromosomal ses (47.4%). There are trisomy 21 ^ 11 cases (23,4%), mosaicism since, a minimum of 100 cells can be monosomyX^8(17,4%),trisomy18^7(15,2%),tris- analysed in a limited time. omy 13 ^ 8(17,4%). Another cases was marker chro- We employed the GTG-banding assay followed mosome ^ 8, þ C ^ 4 cases, trisomy 22 ^ 1 case and 1 by interphase FISH using DXZ1 (Q-Biogene) as casewithstructuralabnormalityat18chromosome. probe to enumerate the X chromosome on periph- Measurement of nuchal translucency thickness eral blood samples of 88 women demonstrating improve the detection of any kind of chromosome the clinical features, compatible with X aneu- trisomy in the ¢rst trimester of pregnancy. ploidies. Thirty-two samples were detected as Although numerically limited, our experience con- 45,X and 3 samples as 47,XXX by GTG-Banding ¢rms that increased nuchal translucency often method and con¢rmed by Interphase FISH. Nine observed in chromosomally abnormal fetuses. Samples were detected as suspicious 45,X/46,XX and 2 samples as 45,X/46,XX/47,XXX by conven- PO:08:75 tional cytogenetic studies and were subsequently con¢rmed by Interphase FISH. Five samples were Two de novo partial trisomies 9p diagnosed as normal by conventional cytogenetic (p13-p24) characterized by CGH in studies, while 4 samples were shown to be mosaeic 45,X/46,XX and the ¢fth sample 45,XX/46,XX/ two unrelated children 47,XXX by interphase FISH analysis. Six samples M. Santos1, C. Hernando1, A. Escalona1, showed structural aberrations of chromosome X, M. Carrera2, G. Rodriguez-Criado3 and which were undetectable by Interphase FISH. The C. Fuster1 15th ICC: Abstracts 145

1Unidad de Biologia, Facultad de Medicina, dysmorphic facial features. In recent years an Universidad Autonoma de Barcelona, E-08193 increasing number of patients with microscopic as Bellaterra, Barcelona, Spain; 2CPC Centro de well as cryptic terminal deletion involving band Patologia Celular, Londres 6, Barcelona, Spain; 22q13 has been described and their phenotype 3 Servicio de Gene´tica Clinica, Instituto Hispalense shows clinical features overlapping with patients de Pediatria, Sevilla, Spain with ring chromosome 22. Loss of DNA in the 22q13.3 region may lead to a clinically recognizable We report the use of comparative genomic hybri- syndrome named ‘‘22q13.3 deletion syndrome’’. didization (CGH) to define the extra chromo- We report on a patient with a ring chromosome some material detected in the karyotype of two 22 who has hypotonia, profound mental retarda- unrelated children. Both patients showed some tion, language impairment, dysmorphic features typical clinical features from partial 9p trisomy and behavioral disorders. syndrome. Cytogenetic analyses of peripheral To check if the critical region responsible of blood lymphocytes after Wright’s G-banding 22q13.3 deletion syndrome was absent in this þ þ revealed the presence of 13p and 22p chro- ring, a FISH analysis using a probe correspond- mosomes respectively. Parental karyotypes were ing to the ARSA locus has been performed; normal in both. Partial 9p trisomy was disclosed ARSA resulted deleted. A more detailed analysis by FISH analysis using multi-probe chromo- of the deletion extent then was performed using a somes analysis in one case and by 24-Multicolor- panel of £uorescent probes located within 22q13. FISH in the other. CGH studies revealed gains These experiments allowed the identi¢cation of in 9p13.2-p24 and 9p13-p24 chromosome regions the breakpoint between CTA-299D3 and RP5- and at the same time discarted other genomic 925J7 probe, located in 22q13.32. Deletion extent imbalances. These findings suggested that the dif- could be estimated to be about 2.5 Mb, and this ferences between both phenotypes could be due larger deletion may explain the severity of clinical to concomitant involvement of other chromo- features observed in our patient. somes (chromosome 13 or 22) and maybe to We would suggest considering this deletion in mild differences in the size of trisomic segments all children presenting with developmental delay/ involved. Our results, as previously reported mental retardation, absent or severely delayed cases which used methodologies other than the speech, autistic-like behavioral disorders, and CGH (YAC DNA probes), support that 9p22 minor dysmorphic features. High-resolution chro- may be the critical region in some aspects of the mosome analysis should be performed together trisomy 9p syndrome phenotype. with FISH or molecular studies to exclude this Acknowledgments: Financial support from SAF (2003-03894) deletion syndrome. and CIRIT (2001, SGR-00201).

PO:08:76 PO:08:77 A new patient with ring chromosome 22 A case with partial trisomy 7p and A. Valetto1, V. Bertini1, R. Battini2, monosomy 9p 2 2 1 A. Battaglia , G. Cioni , E. Rapalini , 1 1 2 1 S. Yilmaz , Y. Tarkan-Argu¨den , F. Tinelli and P. Simi A. Cirakoglu1, A. Deviren1, D. Kuru1, 1Cytogenetic and Molecular Genetic Laboratory, 1 1 1 2 G. Guven , E. Yosunkaya-Fenerci , H. Kurt , S.Chiara Hospital, Pisa, Italy; Stella Maris A. Yuksel1 and S. Hacihanefioglu1 Clinical Research Institute for Child and Adolescent Neuropsychiatry, Calambrone (Pisa), 1Dept. of Medical Biology, Cerrahpasa Medical Italy. School, Istanbul University, 34300, Istanbul, Turkey The clinical phenotype of patients with ring chro- mosome 22 includes mental retardation with This eleven months old boy, born to healthy, severe language impairment, hypotonia, and non-consanguinous parents referred to us for 146 15th ICC: Abstracts developmental delay and multiple congenital 1Division of Microscopic Anatomy and anomalies. The family history revealed that the Bio-imaging, Nigata University Graduate School parents had four first trimestr spontaneous abor- of Medical and Dental Sciences, Nigata, Japan tuses, three girls who died during neonatal period from unknown etiology and two healthy daugh- The present study aimed to introduce a method ters still alive. for observing immunostained chromosomes by a Our case was born at term in normal centiles. combination technique of atomic force micro- In the prenatal ultrasonographic examinations, scopy (AFM) and scanning near field optical left kidney showed multiple cysts and in the post- microscopy (SNOM). For this purpose, we natal period nephrectomy was done for renal com- designed an AFM combined with SNOM (i.e. plications. The physical examination revealed SNOM/AFM), which can collect both topo- developmental delay, trigonocephaly, scarce scalp graphical and optical images of the same portion hair, hypotrichosis, narrowing of the bitemporal of the samples simultaneously. We showed diameter, low-set dysmorphic ears, upslanting pal- SNOM/AFM images of human chromosomes, pebral ¢ssures, epikanthal folds, depressed nasal which were immunostained with an anti-BrdU root, high arched palate, micrognati, small mouth, antibody and Alexa Fluor 488 after incorpora- downturned corners of the mouth, congenital car- tion of BrdU into DNA. SNOM/AFM enabled diac anomalies, genital anomalies, ¢rst and third the collection of fluorescence images of differen- toes overlapping the second on the right foot. tially immunostained chromatid simultaneously Cytogenetic analysis done with GTL banding with topographical images of the same portion. to peripheral blood culture revealed add(9)(p24). The fluorescent images clearly showed the por- The cytogenetic analysis of the parents showed tion of sister chromatid exchanges (SCEs), and that the father’s karyotype was 46,XY and the the corresponding topographical images provided mother’s was 46,XX,der(9)t(7;9)(p21;p24). The precise information on the fine structure of those brother and two sisters were also examined and in portions. We then observed by SNOM/AFM the one of the sisters and the brother normal kar- human metaphase chromosomes immunostained yotypes were found while the elder sister with an anti-topoisomerase II antibody. By this appeared as balanced translocation carrier like technique, topoisomerase II immunoreactivity in her mother. Therefore, our proband’s karyotype the core of the chromosome arms and cen- was 46,XY,der(9)t(7;9)(p21;p24)mat. FISH analy- tromere was analyzable precisely in correlation of sis using whole chromosome painting probes for fluorescent images with the corresponding topo- chromosomes 7 and 9 was performed to identify graphical images. Since the spatial resolution of balanced and unbalanced translocations. the obtained fluorescent images was obviously Literature examination showed that the physi- higher than those by conventional fluorescence cal ¢ndings of the present case shows extensive microscopy, SNOM/AFM is expected to serve as overlap with both the ¢ndings of partial 7p a tool for future studies of the structure of chro- trisomy syndrome and partial 9p monosomy mosomes in relation to the localization of their syndrome. components.

New Methodologies Symposium L157 L156 Combined analysis of morphology and Application of scanning near ¢eld FISH for increased accuracy of cancer optical/ atomic force microscopy to the diagnosis observation of human metaphase M. Daniely1, L. Trakhtenbrot2, R. Rona3, chromosomes. G. Rechavi2, S. Law3, A. Guber4, N. Amariglio2, I. Leibovitch5, T. Kaplan1 E. Kimura1, O. Hoshi1 and T. Ushiki1 and M. Reichart1 15th ICC: Abstracts 147

1BioView Ltd, Rehovot, Israel; 2Dept. of L158 Pediatric Hemato-Oncology and Inst of Hematology, The Chaim Sheba Medical The ACE System: engineering Center, Tel-Hashomer, and Sacler School of arti¢cial chromosomes to rapidly Medicine, Tel-Aviv University, Tel-Aviv, generate high-expressing cell lines for Israel; 3Cytology Unit, Sapir Medical Center, Kfar-Saba, Israel; 4Dept. of Pulmonary, Sapir manufacture of recombinant proteins 5 Medical Center, Kfar-Saba, Israel; Dept. of M. Lindenbaum1, E. Perkins1, E. Csonka2, Urology, Sapir Medical Center, Kfar-Saba, 1 1 2 Israel A. Greene , E. Fleming , G. Hadlaczky , N. Macdonald1, A. Maxwell1, C. Perez1 and 1 Fluorescence in situ hybridization (FISH) is a H. Ledebur, jr. valuable tool in clinical practice of cancer. How- 1Chromos Molecular Systems, Inc., Burnaby, BC ever, high false positive and false negative rates Canada V5A 1W9; 2Institute of Genetics, complicate the interpretation of results. Com- Biological Research Center, Hungarian bined analysis of morphology and FISH may Academy of Science, Szeged, enhance the accuracy of diagnosis. The Duet Hungary scanning system (BioView Ltd, Rehovot, Israel) enables combined analysis of several parameters Mammalian artificial chromosomes are ideal for in each cell by performing multiple scans of the introducing large genetic payloads into cells in same slide under various stains. This approach an autonomously replicating, non-integrating was applied to blood and bone marrow samples format. We have developed a unique satellite for the detection of various hematological malig- DNA-based Artificial Chromosome Expression nancies as well as to urine and sputum samples (ACE) system. A platform architecture with for the detection of bladder and lung cancer, multiple targeting sites allows transfer of single respectively. 500 blood/bone marrow samples, 35 or multiple copies of genes into cells for use in urine samples and 8 sputum samples were ana- recombinant protein expression, generation of lyzed. The preparation of samples included transgenic animals and gene therapy. ACEs can Giemsa/IHC staining followed by stain removal be reproducibly generated de novo in different and FISH for chromosomal aberrations detec- cell lines, readily purified from the host cellsaˆ tion. chromosomes by flow cytometry and re-intro- In the hematological samples, the combined duced into a variety of recipient cells where analysis allowed (a) More accurate follow-up of they are stably maintained for extended periods MRD and bone marrow/stem cells transplanta- without selection. Platform ACEs form part of tion by determination of lineage, clonality and a biological engineering system including expres- maturity of cells carrying chromosomal rearrange- sion-optimized shuttle vectors to specifically ment (b) Enhancing FISH accuracy when using transfer genes, and a plasmid-encoded proprie- probes with high false positive rate. tary integrase to catalyze specific incorporation In urine samples the combined analysis detec- of shuttle payload onto the ACE. Since genes of ted as much as 100% of bladder cancer cases interest are introduced into a consistent genomic compared to only 60 and 80% detection rate environment that supports high-level expression, reported by cytology and FISH, respectively. In relatively few clones need be screened to identify sputum samples, a correlation was found high expressers, leading to rapid, reproducible between atypical morphology and chromosomal and efficient cell line generation. Cell lines changes. expressing industry-relevant levels of recombi- Overall, the combined analysis enabled nant protein (> 30 pg/cell/day) can be gener- increased speci¢city and sensitivity of cancer cells ated in less than 12 weeks; amplification is detection. These preliminary results indicate the unnecessary. Multiple rounds of loading can be advantage of using such approach in diagnosis of carried out onto previously loaded ACEs, which diverse cancer diseases. allows incorporation of supplementary genes to 148 15th ICC: Abstracts improve protein quality or cell growth char- well with those of individuals with a partial acteristics. By transferring ACEs to different cell monosomy of this 13q region described in the lines the optimum producer for a particular literature. product can be identified. The ACE System DNA array CGH analysis appears to be an advantages of speed, efficiency and versatility invaluable tool for diagnostic purposes, which, in provide an attractive alternative to conventional combination with traditional cytogenetic and methods of cell line generation. FISH analyses, will help us to unravel and iden- tify subtle imbalances that otherwise would remain undetected. L159 Identi¢cation of a complex L160 chromosome rearrangement by A new approach to investigating cytogenetic, FISH, and genome-wide transcription on lampbrush DNA array CGH analysis chromosomes N. De Leeuw1, E. Bongers1, H. Mieloo1, A. Saifitdinova1, T. Kulikova1 and J. Willemen1, J. Veltman1 and E. Gaginskaya1 C. Van Ravenswaaij1 1Biological Research Institute of Saint-Petersburg 1Department of Human Genetics, University University, 198504, Russia Medical Centre, Nijmegen, The Netherlands It is really interesting to investigate sequences of A 3-year old girl was referred to our centre for RNA-transcripts from oocyte nuclei because DNA array-based Comparative Genomic Hybri- they include sequences that are never transcribed disation (array CGH) analysis because of pre- in somatic cells. We have developed a new and postnatal growth retardation, microcephaly, approach to investigation transcription on lamp- cleft palate, an atrium septum defect, ataxia, brush chromosomes. Nuclear RNAs were iso- hypotonia, psychomotor retardation and expres- lated immediately from the manual dissected sive aphasia. An apparently balanced, de novo bird oocyte nuclei and treated with RNase free translocation t(6;9)(p21.3;q22.3) had been identi- DNase I. RNA quality was tested using FISH of fied elsewhere. labeled cDNA samples with lampbrush chromo- Routine cytogenetic analysis was repeated, and somes preparations. The results of FISH were in addition to the t(6;9), we found an interstitial the same as results of reverse transcription RNA deletion of the long arm of chromosome 13 labeling in situ. Reverse transcription PCR-ana- (del(13)(q?21q31)). Subsequent Fluorescence in lysis with specific primers used for RNA sam- situ Hybridisation (FISH) analysis with whole ples investigation. Reactions without Revertase chromosome paints of chromosomes 6, 9, and 13, were DNA presence controls. Transcription of respectively, showed that chromosome 13 mate- chicken Z-macrosatellite and Ssp1 sequences and rial was inserted into the derivative chromosome chaffinch centromeric sequence FCP was shown, 6 at the translocation breakpoint. The question whereas chicken WEcoRI and WXhoI repetitive remained whether the chromosome rearrange- DNAs are not transcribed. The results were ment was balanced or not. Therefore, array CGH confirmed by reverse transcription RNA labeling analysis was performed using a genome wide in situ with specific primers on lampbrush chro- array of 3,500 BAC clones, evenly spaced over mosome preparations. Quantitative analysis of the human genome with an average resolution of known RNA transcripts could be produced by 1 Mb. Array CGH analysis revealed a signi¢cant, real time reverse transcription PCR-analysis net imbalance due to a deletion of approximately with specific primers. This approach may be 7 Mb (9 clones) between 13q21.33 and 13q22.3. useful for making a cDNA library of oocyte The clinical features of our patient correspond nuclei transcripts. 15th ICC: Abstracts 149

This work was supported by Russian Foundation for Basic some of the oligonucleotides have additional Research (02-04-49116), Russian Ministry of Education binding sites somewhere else in the genome. (PD02-1,4-291) and Science (MK-2655.2003.04). Due to the diffraction limited resolution of a microscope, the fluorescence signals of the joined oligo probe set merges into a typical, L161 nearly homogeneous FISH spot. Typical sets COMBO-FISH: Speci¢c labeling of are introduced for tumor correlated genome genes and breakpoint regions by loci. Experiments were performed as a very first proof of principle of COMBO-FISH. The tech- computer selected oligo-probe nique was applied to human peripheral blood combinations lymphocytes, formalin-fixed and paraffin embed- M. Hausmann1, J. Finsterle2, G. Hildenbrand2, ded tissue sections, and routine bone marrow E. Schmitt3, C. Großmann2, A. Rapp4, smears. S. Stein2, M. Werner1 and C. Cremer2 1Institute of Pathology, University Hospital, L162 Albertstr. 19, D-79104 Freiburg, Germany; 2Kirchhoff-Institute of Physics, University of Analysis of small tissue samples by Heidelberg, Im Neuenheimer Feld 227, D-69120 CGH on Arrays Heidelberg, Germany; 3Dept. Biocomputing, Institute of Molecular Biotechnology e.V., P.O. P. Lichter Box 100 813, D-07708 Jena, Germany; 4 Deutsches Krebsforschungszentrum, Im Dept. Single Cell and Single Molecule Neuenheimer Feld 280, 69120 Heidelberg, Techniques, Institute of Molecular Biotechnology Germany e.V., P.O. Box 100 813, D-07708 Jena, Germany No abstract was submitted for this talk. The principle of fluorescence in-situ hybridiza- tion (FISH) with COMBinatorial Oligo (COMBO) probes is presented as a new L163 approach that permits specific labeling of any Tissue microarrays for given genomic sites. COMBO-FISH takes high-throughput early target advantage of homopurine/homopyrimidine oligo-nucleotides that form double strands as validation well as triple helices with intact genomic DNA. G. Sauter The latter can be performed without the need for prior thermal denaturation of the target Institute of Pathology, University of Basel, Schoenbeinstrasse 40, CH-4031 Basel, sequence. An analysis of the human genome Switzerland. data bases has shown that homopurine/homo- pyrimidine sequences longer than 14 DNA No abstract was submitted for this talk. bases are nearly homogeneously distributed over the genome and that they represent about 1–2% of the entire genome. Considering that the minimum observation volume in a microscope L164 equipped with a high numerical aperture lens Bioinformatics corresponds on average to a *250 kb chroma- tin domain, a set of distinct, uniformly labeled A. Ladurner oligo-nucleotide hybridization probes can be EMBL Heidelberg, Meyerhofstrasse 1, D-69117 configurated from human genome data base. Heidelberg, Germany This set is expected to exclusively co-localize within a 250 kb chromatin domain, although No abstract was submitted for this talk. 150 15th ICC: Abstracts

L165 parts of the chromosome. All of the transgenic eucalypt hybrids were hemizygous genotype. ChIP on Chip Roland Eils PO:08:80 No abstract was submitted for this talk. Physical nature of the chromatin dynamics: Higher order genome PO:08:78 structure is £exible independently Expression of a yeast enhancing of chromatin proteins in HAL2 gene for salt stress Schizosaccharomyces pombe tolerance in eucalypt and the T. Kobori1, S. Sugiyama2, K. Takeyasu3 2 localization was detected using and T. Ohtani FISH 1National Food Research Institute & Kyoto 2 1 1 University; National Food Research Institute; S. Apisitwanich , N. Thanananta , 3Kyoto University, Japan S. Siripatanadilok2, S. Peyachoknagul1 and 1 S. Suputtitada One of the structural bases for nuclear functions 1Department of Genetics, Faculty of Science, is a higher-order structure of chromatin that is Kasetsart University, Bangkok, 10900; expected to be flexible. Here we show one aspect 2Department of Forest Biology, Faculty of of such flexibilities that depends exclusively on Forestry, Kasetsart University, Bangkok, the physical nature of chromatin fibers by 10900 employing a nano-scale imaging technique, atomic force microscopy (AFM). The halotolerant gene, HAL2 of Saccharomyces AFM revealed that Schizosaccharomyces pombe cerevisiae Montache was cloned and constructed had a chromatin hierarchy as observed in other as the pBHATKU plasmid. This plasmid was eukaryotes, including the particles with 45 nm in transformed into the axillary buds of a eucalypt diameter that form a 350 nm ¢ber. An exposure hybrids (Eucalyptus camaldulensis x E. ter- of the chromatin to the high-salt solution (e.g., 1^ eticornis) using Agrobacteriu m mediated transfor- 2 M NaCl) extracts a complete set of chromatin mation. Eighteen percent of the tested explants proteins including histones. In S. pombe,how- using PCR gave a positive HAL2 fragment. ever, biochemical analyses showed that a brief Thirty one percent of those transformants (11 out exposure (*10 min) of chromatin to high-salt of 35) could grow on MS6 medium supplemented solution did not realize any release of the chroma- with 300 mM NaCl but the control parents and tin proteins. Under this condition, AFM demon- their hybrid could not. The expression of the strated that a stepwise structural transition took HAL2 gene in transgenic eucalypt hybrids in high place during a brief exposure to 400 mM NaCl; salt condition (300 mM NaCl) was confirmed by i.e., the 45 nm particles no longer existed, and, RT-PCR. The location of the inserted HAL2 gene instead, a newly formed structure, 110 nm beads, on chromosome was determined by fluorescence was stably maintained regardless of a further in situ hybridization (FISH). Some were localized increment of the salt concentrations. This process near centromere and some were at terminal of the was reversible, and the 45 nm particles were re- chromosome. Ten clones showed single positive formed again by removing the salt from the signal consistently at the same position in all cells 110 nm beads. This structural reversibility with- except one clone having single signal at the differ- out any changes in the protein composition sug- ent position in different cell indicating that this gests the importance of the physical properties of clone was mosaic. These results indicated that chromatin-environment interactions. A chroma- Agrobacterium-mediated gene, HAL2 could inte- tin-transition model can be proposed that grate into many sites and distribute in different predicts an ionsolvation in the transition. Careful 15th ICC: Abstracts 151 observation of the time-dependent unfolding path- ture against FISH treatments. The disposition way could give an insight into a way to establish of the FISH signals on the re-fixed chromo- chromatin architecture. somes and the potential of this method toward much precious gene mapping and analysis of higher order chromosome structure will be dis- cussed. PO:08:81 Disordered three-dimensional PO:08:82 structure of FISH treated Usefulness of calyculin A for the chromosomes: visual analyses by FISH diagnosis of trisomy 18 in AF culture and atomic force microscopy M. Srebniak1, A. Wawrzkiewicz1, 1 2 1 M. Shichiri , D. Fukushi , S. Sugiyama , W. Kazmierczak1, A. Wiczkowski2 and 1 1 T. Yoshino and T. Ohtani A. Olejek1 1National Food Research Institute, Japan; 1 2 Silesian Medical Academy, Department and Niigata University, Japan Clinic of Perinatology and Gynaecology, Zabrze, Poland; 2Silesian Medical Academy, In molecular genetic studies, the fluorescence in Department of General Medical Biology, Zabrze, situ hybridization (FISH) technique has been Poland widely used for DNA mapping. Although the FISH treatments should cause structural chan- Calyculin A, an inhibitor of protein phosphatases ges and/or damage of the chromosomes, the type 1 and type 2A-serine/threonine, has proved influences of FISH treatment on the chromo- to be useful in human karyotype examination. some morphology are not well understood. We The addition of Calyculin A to in vitro cultures investigated the morphologic changes of the leads to premature chromosome condensation barley chromosomes during standard FISH pro- (PCC). All phases of the cell cycle (G1, S, G2, cesses using an atomic force microscope (AFM) M) can be observed on one slide (Gotoh et al. that generates high-resolution topographic ima- 1995). We have previously reported that Calycu- ges of the samples. The chromosomes were lin A treatment does not influence chromosome observed in the selected four steps in which the banding patterns (Srebniak et al. 2003) and ban- samples were able to dry. The chromosomes ded chromosomes can be analysed from a lym- collapsed by each step of the process and finally phocyte culture. their heights decreased to one fourth of original The aim of our work was to investigate whether height. To further analyze the chromosome it is possible to detect chromosome aberrations in damages, we performed multicolor FISH using cultures treated with Calyculin A. human chromosomes and two or three probes Amniotic £uid culture previously diagnosed by targeting the genes only short distance apart a conventional karyotyping (Colcemid treatment) (0.7–1.5 Mb). The observed dispositions of the was used for that study. Additional cultures were FISH signals were not always same but rather subcultivated and treated with 100 nM Calyculin changeable among the samples. These results A for 45 min. indicate that the chromosomes are severely The karyotype was 47,XX, þ18anditcouldbe damaged and lose the exact nature of their diagnosed from the culture treated with Colce- three-dimensional structure after standard FISH mid. The GTG banding resolution occurred to be treatments. To avoid the destruction of the not satisfactory on preparations from the culture chromosome structures, we examined the effect treated with Calyculin A, but trisomy was clearly of the re-fixation of the chromosome with alde- seen (47 chromosomes). The aneuploidy in this hyde fixatives before FISH treatment. The pre- sample could be easily detected, chromosomes liminary experiment showed that the re-fixed were easy to count, but the GTG banding didnaat^ chromosomes had an ability to keep its struc- allow identifying particular chromosomes. 152 15th ICC: Abstracts

PO:08:83 HaematologicalMalignancySymposium Development and evaluation of a computer-based tutorial to teach L166 karyotyping in undergraduate Clinical relevance of genomic practical classes and to assist in the aberrations in lymphomas screening for hypoproli¢cacy in boars M. Bentz J. Stephenson1, N. Gibbons1, W. Morris1, A. Mileham2 and D. Griffin1 Medizinische Universitaetsklinik, Abt. Fuer Innere Medizin III, Robert-Koch Str. 8, 89081 1Cell and Chromosome Biology Group, Brunel Ulm, Germany University, UK; 2Sygen International, Berkeley, CA, USA No abstract was submitted for this talk.

The ability to karyotype human G-banded chro- L167 mosomes is an essential skill for chromosome biologists and forms an integral part of the curri- Chromosomal translocations involving culum in many biological degrees. Karyotyping the immunoglobulin genes in B-cell usually is taught by providing students with a malignancies photograph of G-banded chromosomes and scis- 1 sors. This has the disadvantage that excessive M. Dyer time is taken cutting and pasting and compara- 1Cancer Studies and Molecular Medicine, tively little in learning pattern recognition. Here Department of Pathology, University of Leicester, we report the development and evaluation of a Leicester LE1 7RH, UK computer-based student practical class ‘Kar- yoLab’. Opinion analysis suggests that students No abstract was submitted for ths talk. greatly prefer the computer-based approach, we have evidence that marks are not compromised by using it and that exercises can be done in sig- L168 nificantly shorter amounts of time. Peculiarities of nonrandom aneuploidy * It is thought that 10% of boars are subject to patterns in acute lymphoblastic reduced fertility and cytogenetic studies suggest that approximately 50% of these have a recogni- leukemia (ALL) sable chromosomal translocation. It is thus essen- O. Haas1 tial to ensure all boars are free of translocations 1 before embarking upon an insemination pro- CCRI, Vienna, Austria gramme as this could potentially save the pig breeding industry many millions of pounds in lost The simplest classification of childhood B-cell revenues from unproductive animals. G-band ana- precursor ALL is according to chromosome lysis is often thought to require highly skilled indi- number and ploidy level. The specific features of viduals however, in this study, we have adapted the aneuploid categories are the nonrandom human ‘KaryoLab’ to instruct users in the skill of numerical changes and the paucity of structural porcine karyotyping. Initial tests suggest that the rearrangements. Near-haploid near-triploid and tutorial that can be learned in the users’ own time near-tetraploid cases are rare. Near-haploid and improves the karyotyping skills dramatically. clones usually coexist with hyperdiploid ones. Indeed, users spotted the presence of an abnorm- They contain at least a haploid chromosome set ality in over 80% of cases. We propose that this with two copies of the sex chromosomes as well programme could be used for the screening of AI as of 10, 14, 18 and 21. The most common and boars prior to arti¢cial insemination. prognostically favorable hyperdiploid subgroup is characterized by nonrandom trisomies of 4, 6, 15th ICC: Abstracts 153

10, 14, 17, 18, 20, X and tetrasomy 21. Four pos- tional subtypes, including clinically-relevant sible mechanisms could account for the forma- subgroups within the class of AML with normal tion of such karyotypes: the doubling of a near- karyotype. Using a randomly-selected training set haploid set, an initial tetraploidization with sub- of 59 patients, we applied a novel supervised sequent losses or the sequential or simultaneous learning algorithm to build a gene expression- gain of chromosomes. The currently available based clinical outcome predictor, which was then data indicate that the last option is the default tested using an independent validation group pathway, whereas the duplication of a near-hap- comprising the 57 remaining patients. An opti- loid karyotype is another option. Also the acqui- mal 133-gene expression signature accurately pre- sition order of chromosome is nonrandom: 21 dicted overall survival for patients in the and X are present in virtually all cases, followed independent validation group (P ¼ 0.006), even by gains of 14, 6, 4, 18, 17, 10, 8, 5, 12 and 11. for the subset of AML cases with normal kar- Nevertheless, almost 70% of the over-expressed yotype alone (P ¼ 0.046). In multivariate analysis, genes in hyperdiploid ALL are located on chro- the gene-expression predictor was a strong [odds mosomes X and 21. Of particular interest is also ratio ¼ 8.8 (2.6 to 29); P < 0.001], independent the unique in vitro behavior of hyperdiploid leu- prognostic factor. Our findings support the uti- kemia. Since they rapidly undergo apoptosis, it is lity of gene expression profiling for improved virtually impossible to propagate them in culture. molecular classification and disease management It therefore also comes of no surprise that only a in adult AML. single cell line has been established so far from such a hyperdiploid ALL. L170 L169 Comparative genomic hybridisation on Gene expression pro¢ling identi¢es to array slides reveals speci¢c DNA prognostically relevant subgroups in copy number changes in acute adult acute myeloid leukemia lymphoblastic leukaemia 1 2 1 L. Bullinger ,K.Da¨hner , E. Bair , 1 2 3 2 2 1 J. Strefford , M. Griffiths , F. Ross and S. Fra¨hling , R. Schlenk , R. Tibshirani , C. Harrison1 H. Da¨hner2 and J. Pollack1 1Leukaemia Research Fund Cytogenetics Group, 1Stanford University, Stanford, California, USA; 2 Cancer Sciences Division, University of University of Ulm, Ulm, Germany Southampton, UK; 2Regional Genetics Laboratory, Birmingham Women’s Hospital, UK; In acute myeloid leukemia (AML), recurrent cyto- 3Wessex Regional Genetics Laboratory, Salisbury genetic aberrations are used to classify patients General Hospital, UK for appropriate therapy. However, the current classification system does not fully reflect the het- An important factor in the diagnosis of acute erogeneity of the disease, and treatment stratifica- lymphoblastic leukaemia (ALL) is that karyotype tion is difficult, especially for intermediate-risk is an independent prognostic indicator, with an cases with normal karyotype. To further char- impact on the choice of treatment. Although acterize adult AML at the molecular level, we pro- patient outcome can be related to changes in filed gene expression in 116 samples (including 45 chromosome number and structure, there remain with normal karyotype) using cDNA microarrays instances where no established chromosome representing 26,260 genes. Unsupervised and abnormality is available to aid prognostication. supervised analysis readily identified gene-expres- The aim of this study is to investigate the DNA sion signatures characterizing known cytogenetic from patients without informative cytogenetic subgroups, and suggested numerous genes with data using microarray-based comparative geno- potential pathogenic relevance. Further, unsu- mic hybridization (aCGH), to allow the identifi- pervised hierarchical clustering identified addi- cation of novel chromosome regions involved in 154 15th ICC: Abstracts disease development, progression and patient chromosomes on leukaemogenesis are known. To outcome. We have used 1Mb aCGH analysis to address the question of whether the characteristic accurately define DNA copy number changes trisomies in high hyperdiploid ALL are asso- throughout the genome in DNA from 50 ALL ciated with increased gene expression, we used com- patients and compared it with previous cytoge- parative expressed sequence hybridisation (CESH), netic and molecular cytogenetic information. a technique analogous to CGH that identifies chro- Although this project is ongoing, aCGH analysis mosomal regions with differential gene expres- has already allowed the accurate mapping of several sion. Relative expression of ALL blast cells previously described cytogenetic abnormalities versus peripheral blood mononuclear cells was and highlighted several novel regions. For exam- analysed in 18 high hyperdiploid ALL patients. ple, the size of 9p deletions varied from a single CESH profiles showed an increase in expression DNA clone (including the p16 gene locus) to for the trisomic chromosomes, with distinct peaks large deletions involving loss of 30-40 DNA clones. observed at: Xp22.1–22.2, 4q28, 6q14–15, 6q24, Small novel changes involving two or three DNA 10p13, 14q23–24, 17q21, 18q12, 21q21 in 28–89% clones were also identified on other chromosomes of cases. Importantly, increased expression with- and may harbour important tumour suppressor out underlying trisomy occurred at 7q11.2 in and oncogenes. In general, aCGH is valuable in 90% of cases. This was confirmed by quantitative the characterisation of complex karyotypes and PCR. When expression was reanalysed versus a the data compares well with conventional G-ban- range of purified normal cell fractions as refer- ded, FISH and MFISH analysis. In the future, ence, a reduction in the number of relatively this type of genetic information may be important over-expressed regions was observed with CD34þ in the development of effective treatment strate- 19þ cells, the putative normal counterpart for gies and defining therapeutic targets. the ALL blast cell. In particular, the expression peak at 7q11.2 disappeared, indicating the invol- L171 vement of genes within this region in the early ontology of normal B-cell development. Overall, The characteristic trisomies of this study has shown that the characteristic triso- childhood high hyperdiploid ALL are mies in hyperdiploid ALL lead to increase in associated with an increase in expression of associated sequences, and has high- lighted critical regions for further investigation. expression of associated sequences The importance of choosing the biologically L. Kearney1, S. Horsley1, A. Martinez relevant reference cell type for meaningful data Ramirez1, C. Harrison2, H. Kempski3, interpretation has also been demonstrated. A. Moorman2, F. Ross4, M. Griffiths5, M. Greaves1 and A. Gruszka-Westwood1 L172 1Section of Haematological Oncology, Institute of Genetic mapping and expression Cancer Research, London, UK; 2LRF Cytogenetics analyses of MYC-containing double Group, Cancer Sciences Division, Southampton General Hospital, Southampton UK; 3LRF Centre minutes in myeloid malignancies: forChildhoodLeukaemia,InstituteofChildHealth, detection of a commonly ampli¢ed London, UK; 4Wessex Regional Genetics 4.3 Mbregionwithoverexpressionofthe Laboratory, Salisbury District Hospital, Salisbury UK; 5Regional Genetics Laboratory, Birmingham C8FWgenebutnotoftheMYCgene Women’s Hospital, Birmingham, UK C. Storlazzi1, T. Fioretos2, K. Paulsson2, B. Strombeck2, C. Lassen2, T. Ahlgren3, High hyperdiploidy, defined as 51–67 chromo- G. Juliusson4, F. Mitelman2, M. Rocchi1 somes per malignant cell, occurs in 30% of child- and B. Johansson2 hood acute lymphoblastic leukaemia (ALL) cases. However, neither the causative mechanism, 1DAPEG, Section of Genetics, University of Bari, nor the relevant molecular consequences of extra Italy; 2Department of Clinical Genetics, Lund 15th ICC: Abstracts 155

University Hospital, Sweden; 3Department might very well be that in this group cryptic spe- of Hematology, Malmo¨ University Hospital, cific chromosome abnormalities are present that Sweden; 4Department of Hematology, Linko¨ping are not detectable using conventional karyotyping. University Hospital, Sweden; We used spectral karyotyping (SKY) and comparative genomic hybridisation (CGH) to Double minutes (dmin), the cytogenetic hallmark identify new specific chromosome rearrangements. of genomic amplification, are found in approxi- In 30 AML patients, frequent structural involve- mately 1% of karyotypically abnormal acute mye- ment of chromosomes 3, 5, 11 and 12 was observed loid leukemias (AML) and myelodysplastic with karyotyping only. Additionally, SKY showed syndromes (MDS). The MYC gene at 8q24 has frequent rearrangements of chromosomes 5 and been reported to be amplified in the majority of 17. In 14 ALL patients, chromosomes 9 and 22 the cases, and it has been generally assumed that were frequently involved based on karyotyping MYC is the target gene. However, only a few stu- only. SKY showed frequent involvement of chro- dies have focused on the extent of the amplicon mosomes 12 and 21 as well. In 16 MDS cases we or on the expression patterns of the amplified found many abnormalities of chromosomes 1, 2, 3, genes. We have studied 6 AML and MDS cases 5, 7 and 15. Using SKY, new structural aberra- with MYC-containing dmin. Detailed fluores- tions were found involving chromosomes 17 and cence in situ hybridization analyses identified a 22. In 3 cases we observed rearrangements of common 4.3 Mb amplicon, with clustered prox- apparently normal chromosomes: a der(1)t(1;22) imal and distal breakpoints, harboring 8 known and der(21)t(5;21) in ALL, a der(10)t(10;20) in genes (C8FW, NSE2, POU5FLC20, MYC, MDS and a der(2)t(2;3) in AML. In patients hav- PVT1, AK093424, MGC27434, and MLZE). The ing rearrangements of the same chromosomes, as corresponding region was deleted in one of the observed using SKY, CGH was started to study chromosome 8 homologues in 5 of the 6 cases, the chromosomal regions involved. Regions of 5q, suggesting that the dmin originated through extra 7q and 17q are frequently lost, suggesting that replication (or loop-formation)-excision-amplifi- genes present in a minimally lost region might be cation. Northern blot analysis revealed that MYC important for leukemogenesis. Therefore, study- was not overexpressed. Instead, only the C8FW ing these regions and the newly observed transloca- gene, encoding a phosphoprotein regulated by tions in more detail might lead to the identi¢cation mitogenic pathways, displayed increased expres- of new speci¢c chromosome aberrations and sion. These results exclude MYC as the target gene genes that might play a role in leukemogenesis. and strongly indicate that deregulation of the C8FW gene may be the functionally important consequence of 8q24 amplicons in AML and MDS. L174 Development and application of two L173 dual colour split signal FISH assays Detection of new speci¢c chromosome for detection of the t(5;14) involving aberrations in acute leukaemia using HOX11L2 or CSX in T-cell acute molecular cytogenetic techniques lymphoblastic leukaemia L. Van Zutven1, E. Van Drunen1, L. Van Zutven1, S. Velthuizen1, S. Velthuizen1 and B. Beverloo2 I. Wolvers-Tettero2, J. Van Dongen2, 3 4 5 1 T. Poulsen , R. Macleod , B. Beverloo and Department of Genetics, Erasmus MC, Rotterdam, 2 The Netherlands; 2Department of Clinical Genetics, A. Langerak Erasmus MC, Rotterdam, The Netherlands 1Department of Genetics, Erasmus MC, Rotterdam, The Netherlands; 2Department of Specific chromosome aberrations are observed in Immunology, Erasmus MC, Rotterdam, The 50% of acute leukaemia patients. In the other Netherlands; 3Department of Probe Application, 50% no or non-specific aberrations are found. It DakoCytomation, Glostrup, Denmark; 156 15th ICC: Abstracts

4Department of Human and Animal Cell Culture, Bari, Italy; 3Sezione di Genetica, DAPEG, DSMZ, Braunsweig, Germany; 5Department University of Bari, Bari, Italy of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands The Philadelphia chromosome (Ph), due to t(9;22)(q34;q11), is observed in more than 95% of The t(5;14)(q35;q32) is a novel cryptic transloca- chronic myeloid leukemia cases. Large deletions tion recently described in paediatric T-cell acute adjacent to the translocation junction on the lymphoblastic leukaemia (T-ALL). This transloca- derivative 9 chromosome have recently been tion involves either HOX11L2 or CSX on 5q35, identified in patients with CML. We report the whereas the 14q32 breakpoints are very hetero- presence of similar deletions on the third deriva- geneous. Because the t(5;14)(q35;q32) is cryptic tive other than chromosomes 9 and 22 in 4 CML and thus hard to detect using conventional kar- cases with variant translocations using fluores- yotyping, it is easily missed in routine diagnostics. cence in situ hybridization (FISH). Here we describe the development, validation and We studied 11 cases a¡ected by CML with variant application of split signal FISH assays for both translocations at diagnosis. Of the 11 cases, 7 (64%) HOX11L2 and CSX, for easy metaphase and bore large deletions on der(9). Among them, we interphase detection of t(5;14) possibly present in found 4 cases (57%) with microdeletions also on T-ALL patients. We also used a split signal probe the third derivative chromosome: t(6;9;22) combination for the possible involvement of IGH (p12;q34;q11), t(9;13;22)(q34;q14;q11), t(4;9;22) on 14q32. We identified 5 new cases with a t(5;14) (p15;q34;q11), t(9;11;22)(q34;q13;q11) in cases #1, involving HOX11L2 out of 32 new T-ALL cases; #2,#3,and#4,respectively.Thesecaseswerechar- in each case the 14q breakpoint was found to be acterized in detail by FISH analysis with appropriate centromeric of the IGH region. All 5 positive P1 Arti¢cial Chromosome (PAC) or Bacterial Arti¢- cases showed HOX11L2 expression. Additionally, cial Chromosome (BAC) clones. This study showed HOX11L2 expression was observed in 1 case with- an association between deletions on der(9) and the out the t(5;14)(q35;q32), showing that HOX11L2 loss of genomic sequences of other chromosomes expression may occur independently of t(5;14) involved in variant t(9;22). The deletions were detec- (q35;q32). We did not identify patients with a ted in each analyzed metaphase cell, suggesting that, t(5;14)(q35;q32) involving CSX, indicating that very likely,therearrangementand thedeletionoccur- the incidence of this alternative 5q translocation is red at the same time. We precisely de¢ned the exten- probably low. We also found that cases with the sion of genomic material losses. The size of the t(5;14)(q35;q32) involving HOX11L2 did not deletions at the third chromosome appears to be rela- show TAL1 abnormalities, whereas 5 HOX11L2 tively large, ranging from 2.7 to 20.4 Mb. Moreover, negative cases did. These results suggest that TAL1 the deleted sequences on the third derivative chromo- expression and HOX11L2 expression, or TAL1 somes included tumor suppressor genes and/or aberrations and the t(5;14)(q35;q32) trans location othergenesinvolvedinsignaltransductionorinmod- involving HOX11L2 are mutually exclusive. ulation of cell proliferation. These ¢ndings may yield furtherinformationaboutthepathogenesisofCML.

PO:08:84 Genomic deletions on other PO:08:85 chromosomes involved in variant Systematic screening for MLL status t(9;22) chronic myeloid leukemia cases at diagnosis in 209 unselected patients L.Anelli1,A.Zagaria1,F.Albano2,C.Storlazzi3, with acute myeloid leukemia 2 2 1 A. Pannunzio , C. Buquicchio , M. Roberti , B. Arnaud1, N. Douet-guilbert1, F. Morel1, 2 1 3 V. Liso , G. Specchia and M. Rocchi M. Lebris1, P. Morice2, P. Bourquard2, 2 1 1 1Hematology, University of Foggia, Foggia, Italy; S. Banzakour , C. Berthou , J. Abgrall and 2Department of Hematology, University of Bari, M. De Braekeleer1 15th ICC: Abstracts 157

1Faculte´ de Me´decine & CHU Morvan, Treatment for hematologic disease often includes Brest, France; 2CH Yves Le Foll, St Brieuc, allogeneic bone marrow transplantation after suc- France cessful induction chemotherapy and consolida- tion. Post-transplant leukemic relapse occurs A large number of abnormalities involving the when residual, therapy resistant host cells pro- MLL gene, located in the 11q23 chromosomal liferate leading to recurrence of the original or band, has been associated with hematological further progressed disease. However, successful malignancies, including acute lymphoblastic and eradication of recipient tumor cells and sub- myeloblastic leukemias and myelodysplastic syn- sequent transformation of donor cells is uncom- dromes. In this study, we investigated the occur- mon. Here we report two cases of AML in donor rence of MLL abnormalities in 209 unselected cells after related sex mis-matched allogeneic consecutive patients with acute myeloblastic leu- bone marrow transplantation. The first case was kemia (AML) using fluorescent in situ hybridiza- a 55-year old female with AML-M2 carrying a 8;21 tion (FISH) analyses with a commercial MLL (ETO;AML1) translocation. She responded well dual colour probe (LSI¨ MLL, Vysis). Twenty-six to treatment and transplant. However, after 2 patients (12.4%) showed MLL abnormalities. years she relapsed and developed cytopenia with a Nine showed a split signal ascertaining the invol- karyotype showing a (11;19)(q23;p13.3) translo- vement of the MLL gene in translocations (7 cation and trisomy 21 in all the male donor cells. cases) and insertions (2 cases). FISH analysis was No female cells were detected by XY FISH stu- necessary to identify the partner chromosome or dies. The second case was a 37-year-old female the exact mechanism of the disruption for 4 of diagnosed with paroxysomal nocturnal hemoglo- the patients. Seventeen patients showed over- binuria and had a normal karyotype. Her course representation of the MLL gene without evidence was complicated by neutropenic fevers and, graft of rearrangement. Trisomy and tetrasomy 11 versus host disease. She relapsed and developed were observed in 6 and 1 cases respectively. leukopenia. This time her karyotype showed tris- Other various abnormalities, such as double min- omy 11 and t(11;21)(q23.3;q22) in all the male utes and i(11) (q10), were the source of the MLL donor cells. FISH analyses on both cases sug- amplification. MLL rearrangements have been gested aberrations involving the MLL gene. much studied for a long time and shown to be Expression of chimeric MLL fusion products or associated with a worse prognosis. However, additional copies of MLL gene have been asso- MLL amplification has only been considered as a ciated with transformation. These two cases illus- new entity in AML quite recently, playing a trate the unusual occurrence of transformation putative role in leukemogenesis. Given the cryp- of donor cells by MLL-partner fusion proteins. tic nature of some rearrangements and the diffi- culty of identifying chromosome 11 segments in some abnormalities, we think that FISH analysis PO:08:87 with the MLL probe should be performed in all Identi¢cation of chromosomal AML cases at diagnosis. aberrations in chronic lymphatic leukemia (CLL) by interphase FISH PO:08:86 and multiplex ligation-dependent probe MLL-partner fusion and ampli¢cation (MLPA). transformation activity in donor cells A. Buijs1 and P. Krijtenburg1 after allogeneic transplant in two 1Department of Medical Genetics, University patients Medical Centre Utrecht, The Netherlands S. Brodie1 and N. Rao1 Chronic lymphatic leukemia (CLL) is the most 1UCLA School of Medicine, Los Angeles, CA common leukemia in adults. It has a highly vari- 90095, USA able clinical course. Attempts to correlate this 158 15th ICC: Abstracts apparent clinical heterogeneity to genomic het- human tumors. Chronic lymphocytic leukemia is erogeneity have been hampered by poor cytoge- unique among other malignancies, for survivin netics of CLL samples. To identify new markers mRNA and protein is undetectable in leukemic B we analyzed 41 CLL cases using multiplex liga- lymphocytes. tion-dependent probe amplification (MLPA) for We present a case of B-CLL patient that expres- 41 tumor-associated targets. To validate the sed survivin when analyzed by RNase Protection MLPA analysis, MLPA data of CLL samples Assay. were compared with results of interphase FISH Utilizing FISH technique we also found chro- analysis using ATM (11q22–23), centromere 12 mosome 12 trisomy, deletion in p53 locus and 13q and Rb-1 (13q14) specific probes. deletion. We found that 3/29 cases had a deletion of the Diagnosed with stadium III according to Rai ATM locus by FISH that could be con¢rmed by classi¢cation, this patient showed slow disease MLPA. In 6/8 cases with a trisomy chr.12 seen progression and poor chemotherapy response. by FISH, the result was con¢rmed by MLPA ana- lysis using target sequences speci¢c for CCND2 and BCL7A, located on chromosome 12. In two PO:08:89 cases, the percentages of interphase nuclei with þ12 were 20 and 40%, respectively, which may Trisomy 12 in B-cell chronic explain the false-negative result for MLPA. In lymphocytic leukemia detected by one case, MLPA con¢rmed trisomy chr.12, but £uorescence in situ hybridization also suggested a trisomy chr.19. This was con- 1 2 ¢rmed by M-FISH. Of 10 cases with a Rb-1 dele- D. Koczkodaj , E. Wasik-Szczepanek , 1 2 tion by FISH analysis out of 41 cases, 8 could be A. Filip , A. Dmoszynska and con¢rmed by MLPA. In two cases the deletion of J. Wojcierowski1 Rb-1 was found only in 15% and 30% of the inter- 1Department of Human Genetics with Molecular phase nuclei that may explain the false negative and Cytogenetic Diagnostics, Lublin, Poland; results by MLPA analysis. 2Department of Haemato-oncology and Bone We conclude that MLPA can be used to iden- Marrow Transplantation, Lublin, Poland tify new chromosomal aberrations that serve as markers for the identi¢cation of independent Among 75 untreated patients with typical predictors of disease and survival in CLL. (CD19 þ , CD5/CD19 þ , CD23/CD19 þ ) B-cell chronic lymphocytic leukemia (B-CLL) cytoge- netic aberrations of peripheral blood cells were PO:08:88 evaluated, using fluorescence in situ hybridization techniques. Two cytogenetic aberrations were Does elevated survivin level in£uence found: trisomy 12 and TP53 deletion. The clon- the progress of B-cell lymphocytic ality was determined when >9% of the cells had leukemia? trisomy 12 or TP53 gene deletion. Trisomy 12 1 1 1 1 was detected in 7 patients, while trisomy 12 and A. Filip , D. Koczkodaj , I. Nesina , B. Cisel , TP53 deletion were present simultaneously in 6 J. Korzeniowska1, E. Wasik-Szczepanek2, 2 1 patients. If the first group is linked to the second A. Dmoszynska and J. Wojcierowski one then 13 patients among 75 (17%) have tris- 1Department of Human Genetics with Molecular omy 12. In a group of patients with trisomy 12 and Cytogenetic Diagnostics, Lublin, Poland; and TP53 deletion, the percentage of cells with 2Department of Haematooncology and Bone trisomy 12 was almost two times more compared Marrow Transplantation, Lublin, Poland to patients with trisomy 12 as a single aberra- tion. It is possible that TP53 deletion facilitates Survivin is a member of IAP (inhibitor of apop- proliferation of clones with others genomic aber- tosis) gene family. Absent in almost all tissues rations. In two patients with trisomy 12, control except foetal, survivin is often overexpressed in cytogenetic study was performed. An increase in 15th ICC: Abstracts 159 the percentage of cells with trisomy 12 for 8% success or relapse. Patients with CML and AML- and 30%, respectively was detected. However, M3 were managed by repeatedly monitoring the proliferation of cells with TP53 deletion was also success of treatment by FISH. observed. Clinical course of B-CLL in a group of In ALL, some of the cytogenetic abnormalities patient with trisomy 12, trisomy 12 and TP53 detected were t(1;19)(q23;p13), a jumping translo- deletion simultaneously is more aggressive com- cation t(5;12), i(6p) and trisomy 8. A complex pared to the course of the disease in patients translocation t(2;21;14)(p12;p11;q32) was seen in with no cytogenetic aberrations. Frequency of acaseofCLL.ApatientwithATLLhad IGHV gene mutation occurrence was not ana- t(1;3)(p32;p21) with trisomy 4, trisomy 12 and lyzed in both groups of patients. But trisomy 12 inversion 6. Cytogenetic studies thus help in the together with unmutated IGHV gene is found by diagnosis, prognosis and management of patients some authors. The absence IGHV gene mutation with leukemia. is independent unfavourable prognostic factor.

PO:08:91 PO:08:90 Chromosome 1 abnormalities in Karyotyping and FISH in multiple myeloma: a multicolor FISH haematological malignancies in India study. P. Madon1, F. Parikh1, A. Athalye1, Y. Marzin1, D. Jamet1, F. Morel1, M. Lebris1, V. Bandkar1, S. Dhumal1, A. Sopariwala1, P. Morice2, P. Bourquard2, S. Banzakour2, M. Kawle1, S. Advani1, S. Parekh2 and C. Berthou1, J. Abgrall1 and M. Agarwal2 M. De Braekeleer1 1Jaslok Hospital and Research Centre, Mumbai, 1Faculte´ de Me´decine & CHU Morvan, Brest, India; 2Bombay Hospital and Medical Research France; 2CH Yves Le Foll, St Brieuc, France Centre, Mumbai, India Multiple myeloma (MM) is a B cell neoplasia Cytogenetic analysis was performed on over 1000 characterized by the proliferation of a malignant samples of bone marrow or peripheral blood at plasma cell population in the bone marrow. the Genetics laboratory of a private hospital in Patients with MM commonly present bone pain, Mumbai. FISH was carried out using Vysis anemia, renal failure and paraproteinemia. Given probes, the signals were visualized on a Zeiss the karyotypic complexity observed in most Axioskop microscope and the Metasystems soft- patients with advanced disease, conventional ware was used for image analysis. The use of both cytogenetic techniques usually fail to properly karyotyping and FISH gave additional informa- identify all the chromosome abnormalities. This tion especially when complex translocations were is why biomolecular techniques, such as multi- present in CML, leading to masked Philadelphia color fluorescent in situ hybridization (M-FISH), chromosomes. FISH on metaphases helped to are used to improve the interpretation of the kar- detect a case where the BCR/ABL fusion signal yotypes. Amongst the 56 patients studied since was present on chromosome 9 instead of 22. 2000, 37 presented chromosome 1 abnormalities. Karyotyping a patient in the blast phase showed Most of these abnormalities were unbalanced dup(1)(q22q24) with hyperdiploidy and two Ph translocations resulting in amplifications of the chromosomes. Clonal evolution was detected long arm (22 patients) or deletions of the short with trisomy 4, del(5)(q22-q33), del(7)(q22-q36), arm (12 patients). The minimum regions of over- der(17) or i(17q) in different cases of CML. The lap were 1q31q4 and 1p11p21 for the amplifica- BCR/ABL ES probe (Vysis) was used to detect tions (20 patients) and deletions (9 patients) deletions in the ASS region. In cases of sex-mis- respectively. The 31 breakpoints (18 patients) on matched bone marrow transplantation, FISH the chromosome partners of the unbalanced using probes for the sex chromosomes determined translocations were randomly distributed on the 160 15th ICC: Abstracts whole karyotype, but for 6 cases located on rent reciprocal translocation. The review of the lit- chromosome 16, including 5 in band 16q11. erature suggests the de¢nition of a new subgroup Almost half of the breakpoints occurred in the of Ph- CMPD which includes t(5;12)(q33;p13), pericentromeric regions. Balanced translocations eosinophilia, monocytosis and splenomegaly. Our involving chromosome 1 were also distributed in case is one of the few not associated with eosino- the whole karyotype and half of the breakpoints philia, which suggest that eosinophilia is a promi- occurred in pericentromeric regions. Never- nent but not invariable feature. theless, such translocations were rare (6 of the 37 The TEL-PDGFR fusion gene results in pro- patients), interesting the long or short arm with- duction of a constitutively active tyrosine kinase out predominance. As chromosome 1 abnormal- fusion protein that deregulates hemopoiesis. With ities are commonly considered of bad prognosis the advent of targeted treatment, patients with in MM, it is important to identify these rearran- t(5;12) are candidates for treatment with Imatinib gements, which can be achieved by M-FISH. Mesylate, a tyrosine kinase inhibitor. The treat- ment induces durable responses in patients in the PO:08:92 literature and in our case. A translocation t(5;12)(q33;p13.1) in a chronic myeloproliferative disease PO:08:93 without eosinophilia Variations of CCND1/IGH signal N. Nadal1, P. Flandrin1, J. Cornillon2, pattern in Mantle Cell Lymphoma E. Deladesse3, L. Mauvieux4, D. Olaru1, (MCL) by double-colour £uorescence S. Morel1 and L. Campos1 in situ hybridization (FISH) 1Laboratoire d’He´matologie, CHU Hopital Nord, R. Woroniecka1, B. Pienkowska–Grela1,A. 2 42055 Saint-Etienne, France; Service Pastwinska1, J. Rygier1 and A. Witkowska1 d’He´matologie, CHU Hopital Nord, 42055 Saint-Etienne, France; 3Laboratoire Central 1Centre of Oncology, M. Sklodowska-Curie d’He´matologie, CHU Necker-Enfants-Malades, Memorial Institute, Cytogenetic Department, 75015 Paris, France; 4Laboratoire d’He´matologie, Warsaw, Poland CHU de Hautepierre, 67000 Strasbourg, France Mantle cell lymphoma is characterised by A t(5;12)(q33;p13) has been described in a small t(11;14)(q13;q32). This translocation juxtaposes number of cases of BCR-ABL negative (Ph-) IGH sequences with the CCND1 (BCL-1). Clas- Chronic MyeloProliferative Disease (CMPD) sic cytogenetic analysis of MCL is frequently with eosinophilia. The translocation t(5;12) fuses impossible, due to low mitotic index and poor the Platelet-Derived Growth Factor Receptor morphology of metaphases. FISH detects the (PDGFR) gene at 5q31-33 to the ETS-Variant aberrations on interphases as well as metaphases. gene 6 (TEL/ETV6) at 12p13. We investigated this method in the new diag- We report a case of Ph- CMPD without eosino- nosed MCL patients. philia associated with an isolated t(5;12)(q33;p13) FISH analysis was carried out on lymphoid observed in 25 metaphase cells. Molecular analy- cells obtained from lymph node, bone marrow or sis detected the TEL-PDGFR fusion gene and peripheral blood. All known breakpoints of showed the absence of a cryptic BCR-ABL rear- t(11;14) were spanned by the use of two dual col- rangement. The diagnosis was atypical Ph- our probes: LSI IGH/CCND1 (Vysis) and CMPD. A good haematological and cytogenetic BCL1/IGH (Qbiogene). response was obtained after treatment with Imati- FISH study was carried out on 14 MCL cases. nib Mesylate. The patient was in good health The typical 1R1G2Y of signals was observed in after one year of treatment. 11 cases. Simultaneously, in 3 of them a sub- Ph- CMPD is a heterogeneous group of disease. population of cells with di¡erent signal pattern A minority of cases are associated with a recur- was observed. There were detected 2R2G3Y, 15th ICC: Abstracts 161

2R2G4Y, that indicated tetraploidy or 1R1G3Y, completely encompassing the gene; RUNX1 gene 1R1G4Y, that suggested ampli¢cation of fusion was detected by RP5-1107L6 probe. In the case gene. In one of the remaining 3 cases the signal with ins(8;21), the use of chromosome 21 specific pattern was 1R1G1Y. The analysis of metaphases clones revealed that a region of 4 Mb was inser- showed, that the missing yellow fusion signal on ted on the der(8) chromosome. In the remaining thederivativechromosome11wasprobably cases a functional fusion gene was detected on deleted. In the next case the signal pattern was the der(21) instead of the der(8) chromosome as a 2R2G and 2R3G. The detailed analysis of meta- consequence of ins(21;8). The insertion size was phases indicated a variant of translocation of established in all cases and turned out to be very CCND1 gene onto chromosome 2. In the last case heterogeneous, ranging from a minimum of 2.4 Mb hybridization demonstrated the lack of fusion to a maximum of 44 Mb. Sequences encompassing signals and 2R2G pattern. the breakpoints on chromosome 8 were compared FISH is enough not only for detection of the each other and with the region including RUNX1 CCND1/IGH fusion, but also for detection of gene on chromosome 21, using the Genalyzer deletions or ampli¢cations associated with the software. No significant similarity was observed. CCND1/IGH fusion. Insertions seem not to be associated with a sub- set of patients with common features in terms of PO:08:94 prognosis and are not linked to the presence of additional cytogenetic rearrangements. Our ¢nd- Insertions generating the ings suggested that the 5RUNX1/3CBFA2T1 5/RUNX1/3/CBFA2T1 gene in acute fusion gene plays a crucial role in leukemo- myeloid leukemia cases show variable genesis independently from breakpoint location and insertion size. breakpoints A. Zagaria1, L. Anelli1, F. Albano2, Post-genomic Era Symposium M. Mancini3, G. Saglio4, A. Liso1, L. Sebastio, R. La Starza, G. Specchia1 and M. Rocchi5 L175 1Hematology, University of Foggia, Foggia, Italy; 2Department of Hematology, University of Bari, Chromosomes in disarray: DNA 3 Bari, Italy; Dipartimento di Biotecnologie microarrays for the study of Cellulari ed Ematologia, University La Sapienza, Rome, Italy; 4Division of Hematology and chromosome rearrangements Internal Medicine, Department of Clinical and N. Carter1 Biological Sciences of the University of Turin, Turin, Italy; 5Sezione di Genetica, DAPEG, 1The Wellcome Trust Sanger Institute, Hinxton, University of Bari, Bari, Italy Cambridge, UK

Translocation t(8;21)(q22;q22) is detected in With the publication of the complete human gen- about 15% of acute myeloid leukemia cases. The ome sequence, a complete set of mapped and rearrangement involved RUNX1 and CBFA2T1 sequenced DNA clones are now available which genes generating a 5RUNX1/3CBFA2T1 fusion can be utilised as a valuable resource for mole- gene. We described 6 among 82 (7.3%) AML cular cytogenetics. The use of this resource in the cases showing insertion events responsible for the form of DNA microarrays to represent and RUNX1/CBFA2T1 rearrangement. All cases replace metaphase chromosomes provides us with were studied by RT-PCR and conventional cyto- new possibilities for the analysis of chromosomes genetic analysis. A detailed molecular cytogenetic and their aberrations. We have been using BAC characterization by FISH experiments revealed 1 and PAC clone DNA microarrays not only to case with ins(8;21) and 5 with ins(21;8). In detail, study gains and losses in tumours and to identify the CBFA2T1 locus was identified using a microdeletion and microduplications in patients mixture of RP11-118O8 and RP11-777J24 clones, with learning disability using comparative 162 15th ICC: Abstracts genomic hybridisation (CGH) but also to map out knowledge of their phenotypes. These results chromosome translocation breakpoints. sub-divided the patients into three groups. (1) The For breakpoint mapping, the derivative chro- translocation breakpoints of four cases appeared mosomes are ¢rst £ow sorted, ampli¢ed using simple and balanced at the resolution used. (2) DOP-PCR, di¡erentially labelled and hybridised Three cases were found to have complex rearran- to the array. The pattern of hybridisation identi- gements some including deletions, inversions and ¢es the chromosomes involved in the rearrange- insertions at or near one or both breakpoints. The ment and the position of the breakpoint. This new two deletions, del(10q) and del(11q) were both de approach, which we have termed Array Painting, novo. (3) Three cases in which the translocations dramatically reduces the time taken to map break- appeared balanced were found by microarray points by FISH. We have used these methods to to have previously unrecognised imbalances, identify additional complexity or genome imbal- dup(3p), del(6q) and del(18q). The dup(3p) was ance in 60% of patients with apparently balanced also present in the phenotypically normal father, rearrangement. These results, if generally con- the del(6q) was de novo and the status of the ¢rmed in the study of further patients, will have a del(18q) was unresolved. We are also conducting signi¢cant impact on current diagnostic investiga- a similar series of experiments to examine the tions of this type and provide an argument for the breakpoints in ten phenotypically normal indivi- more widespread adoption of microarray analysis duals carrying apparently balanced familial or other high resolution genome wide screens for translocations so that the level of complexity chromosome imbalance and rearrangement. involving the breakpoints and other genomic regions can be compared between the two groups. Our results on the de novo phenotypi- L176 cally abnormal patients will have a significant Array painting, array CGH and FISH impact on current diagnostic investigations of patients of this type and provide an argument for reveal complex chromosomal the more widespread adoption of microarray analy- abnormalities in a proportion of sis or other high resolution genome-wide screens patients with apparently balanced de for chromosome imbalance and rearrangement. novo translocations and abnormal phenotypes L177 J. Crolla1, N. Carter2, S. Gribble2, DNA array based analysis of E. Prigmore2, D. Burford2, S. Clegg2, chromosomes in leukaemia 3 1 1 N. Dennis , P. Jacobs , S. Thomas B. Young1 and R. Sandstrom4 1Molecular Oncology Group, Medical Oncology 1 Wessex Regional Genetics Laboratory, Salisbury Unit, Cancer Research UK Clinical Centre, UK District Hospital, Salisbury, SP2 8BJ. U.K.; 2The Wellcome Trust Sanger Institute, Wellcome Trust No abstract was submitted for this talk. Genome Campus, Hinxton, Cambridge, CB10 1SA. U.K.; 3Wessex Clinical Genetics Service, Princess Anne Hospital, Coxford Road, L178 Southampton, SO16 5YA. U.K.; 4Lower Columbia Pathologists, 121714thAve,Longview,WA,U.S.A. Human meiotic recombination: a high-resolution view We have used array-CGH and array painting to C. May1, K. Holloway1, L. Kauppi1, analyse constitutional de novo apparently T. Slingsby1, R. Neumann1, A. Webb1 balanced translocations in ten patients presenting 1 with abnormal phenotypes. With this approach and A. Jeffreys we identified additional complexity or genome 1Department of Genetics, University of Leicester, imbalance in six of the ten patients analysed with- Leicester, LE1 7RH, UK. 15th ICC: Abstracts 163

A detailed understanding of how meiotic recombi- The Human Genome Project’s recent completion nation shapes human DNA diversity will be key in of a high-quality sequence of the human genome developing successful strategies for mapping com- represents a landmark scientific accomplishment mon disease genes, a challenge that has been of historic significance. It also signifies a critical enthusiastically embraced by the research commu- transition for the field of genomics, as the focus nity. Global patterns of recombination can be shifts from elucidating the human genome gleaned from comparisons of physical and genetic sequence to determining the functional informa- maps, but higher resolution analysis relies on the tion it encodes. The comparison of sequences fine-scale characterization of many recombinants generated from different extant species has over intervals of just a few kilobases. To this end, emerged as a powerful strategy for identifying we have exploited the abundant SNP class of functionally important genomic elements. DNA marker and have developed PCR-based As a complement to ongoing e¡orts to sequence strategies to detect de novo recombinant molecules entire genomes, the NISC Comparative Sequencing directly from batches of sperm DNA. Data from Program is pursuing multi-species genome explora- different regions of the genome indicate that cross- tions by sequencing and analyzing the same ortholo- overs tend to cluster into very narrow hotspots gous regions in many di¡erent vertebrates. To date, and that these hotspots can profoundly influence we have generated over 450 Mb of comparative patterns of haplotype diversity within a popula- sequence data from more than 30 di¡erent species. tion. Gene conversion events co-localise with the Comparative analyses of these data are revealing peak of crossover activity but can be more fre- important insights about the patterns of sequence quent and are even more tightly distributed affect- conservation among species. In particular, we are ing haplotypes at an extremely localised level. developing approaches for utilizing multi-species sequence comparisons to identify highly conserved References non-coding sequences, which likely correspond to Jeffreys et al (2000) Human Molecular Genetics 9: 725–733. regulatory and other functionally important ele- Jeffreys et al (2001) Nature Genetics 29: 217–222. ments. In addition, we are examining the relative May et al (2002) Nature Genetics 31: 272–275. ‘informativeness’ of di¡erent speciesa^ sequences for Kauppi et al (2003) Human Molecular Genetics 12: 33–40. Jeffreys & May (2004) Nature Genetics 36: 151–156. identifying such highly conserved regions. In conjunction with ongoing and future whole- genome sequencing e¡orts, our studies should L179 advance the utility of comparative sequence ana- High-resolution genomic microarrays: lyses, and make important contributions towards applications for the study of unraveling the functional and evolutionary complexities of the human genome. chromosome biology D. Vetrie1 Prize Nominee Special Symposium 1Sanger Institute, Hinxton, Cambridge, UK L181 No abstract was submitted for this talk. FISHing with Locked Nucleic Acids L180 (LNA) Deciphering human genome function A. Silahtaroglu1, H. Pfundheller2, A. Koskin2, by multi-species sequence comparisons N. Tommerup1 and S. Kauppinen3 E. Green1 and Nisc comparative sequencing 1Wilhelm Johannsen Centre for Functional program1 Genome Research, Dept. of Medical Genetics, Inst. of Medical Biochemistry and Genetics, The 1National Human Genome Research Institute, Panum Institute, University of Copenhagen, National Institutes of Health, Bethesda, Denmark; 2Department of Chemistry, Exiqon, Maryland, USA Bygstubben 9, DK-2950 Vedbaek, Denmark; 164 15th ICC: Abstracts

3Department of Functional Genomics, Exiqon, 1Division of Microscopic Anatomy and Bygstubben 9, DK-2950 Vedbaek, Denmark Bio-imaging, Department of Cellular Function, Niigata University Graduate School of Medical 2 Locked Nucleic Acids (LNA) constitute a novel and Dental Science, Niigata, Japan; H.H.Wills class of RNA analogues having an exceptionally Physics Laboratory, University of Bristol, high affinity towards complementary DNA and Bristol, UK RNA. The ribofuranose ring in LNA is structu- rally constrained by a methylene bridge between The high-order structure of human metaphase its 20-oxygen and the 40-carbon atoms resulting in chromosomes is still controversial, due to the a locked 30-endo conformation, thereby reducing technical difficulty of its high resolution analysis its conformational flexibility and increasing the in a natural condition. Atomic force microscopy local organization of the phosphate backbone. (AFM), which has been recently applied to the LNA obeys the Watson-Crick base-pairing biological field, has the advantage in obtaining rules and LNA bases are furthermore linked by a sample images in various (vacuum, air and phosphodiester backbone, thus allowing synthesis liquid) environments at high spatial resolution. of chimeric LNA/DNA and LNA/RNA Thus, the aim of the present study is to clarify oligonucleotide probes using standard phosphor- the native conformation of the human meta- amidite chemistry. Finally, an important prac- phase chromosomes by observing them in a tical advantage is that LNA is fully soluble in liquid environment with an AFM. Metaphase water, which makes its handling and the experi- chromosomes were harvested from human lym- ments simple. phocytes, suspended in a methanol-acetic acid The development of molecular probes and image solution and spread on a clean glass slide. The analysis has made £uorescence in situ hybridization chromosomes 1 and 2 were observed using an (FISH) a powerful investigative tool. Although intermittent contact mode in a phosphate-buf- FISH has proved to be a useful technique in many fered saline solution. Symmetrical alternating areas, it is still a fairly time-consuming procedure ridges and grooves were present on the surface with limitations in sensitivity and resolution. Thus, of the sister chromatids in individual chromo- probes with higher a⁄nity would potentially somes. The number and arrangement of the rid- improvethesensitivityofthetechnique. ges and grooves were specific to the type of the Here we describe the development of LNA- chromosome. We also observed the change in substituted oligonucleotides as probes for £uo- structure of chromosome caused by the quina- rescence in situ hybridization on metaphase crine mustard treatment (Q-banding) using chromosomes and interphase nuclei. We have AFM in liquid. A comparison of images of the used several di¡erent human-speci¢c repeats such same chromosome before and after the staining as the classical satellite-2 repeats, telomere treatment showed that the basic structure of the repeats and alpha satellite repeats as targets and chromosome was not changed, except that the demonstrated that LNA oligos are excellent height difference between the ridges and grooves probes for FISH, combining high binding a⁄nity was increased. These results show that the AFM with short hybridization time and even with the observation in liquid is useful for analysing the ability to hybridize without prior thermal dena- chromosome structure and its change in relation turation of the template. to the effect of various staining treatments.

L182 Imaging of the human metaphase L183 chormosomes by atomic force Strand-speci¢c £uorescence in situ microscopy in liquid hybridization: CO-FISH O. Hoshi1, R. Owen2, M. Miles2 and S. Bailey1, M. Cornforth2 and T. Ushiki1 E. Goodwin3 15th ICC: Abstracts 165

1Colorado State University, Fort Collins, CO, 1Institute of Biophysics, Czech Academy of USA; 2University of Texas Medical Sciences, Brno, Czech Republic; Branch, Galveston, TX, USA; 3Los Alamos 2School of Biological Sciences, Queen Mary National Laboratory, Los Alamos, University of London, UK; 3Department of NM, USA Functional Genomics and Proteomics, Masaryk University Brno, Czech Republic; 4 The ability to prepare single-stranded chromoso- Jordell Laboratory, Royal Botanic mal target DNA allows innovative uses of FISH Gardens, Kew, Richmond, UK technology for studies of chromosome organiza- tion. Standard FISH methodologies require func- Analysis of telomeres within the plant order tionally single-stranded DNAs in order to Asparagales has shown a remarkable polymorph- facilitate hybridization between the probe and the ism of telomere DNA sequences. In association complementary chromosomal target sequence. with a phylogenetic tree of Asparagales, these This usually involves denaturation of double- results show original ‘typical’ plant telomeric stranded probe and target DNAs to induce tem- repeats [TTTAGGG]n have been partially or porary separation of the DNA strands. The fully replaced by the human-type telomeric strand-specific CO-FISH (Chromosome Orienta- sequence [TTAGGG]n in a distinct clade within tion-FISH) method involves selective removal of Asparagales. Correspondingly, telomerases of newly replicated strands from the DNA helixes these species synthesise human repeats with a within metaphase chromosomes. The result is sin- high error rate in vitro. Further, even more pro- gle-stranded target DNA. Single-stranded probes found change has occurred within this clade in can then be hybridized to the single-stranded chro- Alliaceae resulting in the absence of any known mosomal target DNA, resulting in strand-specific telomeric minisatellites and corresponding telo- hybridization – revealing a wealth of information merase activity. Telomerase mutation(s) respon- previously unattainable at the cytogenetic level. sible for the switch in telomeric DNA sequence Mammalian telomeric DNA consists of tandem could be expected to have an impact on interac- repeats of the (TTAGGG) sequence, oriented 50- tions of telomere-binding proteins involved in tel- to-30 towards the end of the chromosome. Based omere structure and function. However, our on this conserved structural organization, CO- results show that nuclear protein extracts from FISH with a telomere probe can be used to deter- plants with human-like telomeres form specific mine the orientation of DNA sequences along complexes with both typical plant and human chromosomes. Applications of the CO-FISH telomeric DNA sequence. approach will be presented and include detection This work has been supported by the Leverhulme Trust, of pericentric inversions and isochromosomes, dis- GACR (204/02/0027) and the Institutional support crimination between leading vs. lagging strand (MSM143100008 and AV0Z5004920). telomeres, identi¢cation of dysfunctional telo- mere-double strand break fusions, a combined L185 SKY-CO-FISH method, as well as revelation of The dynamic centromere: chromosome replication timing of mammalian telomeres via an approach we term ReD-FISH. evolution in marsupials R. O’Neill1 1 L184 Dept. Molecular and Cell Biology, University of Connecticut, U-2131, Storrs CT 06269, USA Species divergence in Asparagales is associated with the evolution of novel Studies of chromosome evolution have focused types of telomeres heavily on the evolution of conserved syntenic, gene-rich domains. It is obvious, however, that 1 2 3 E. Sykorova , K. Lim , Z. Kunicka , the centromere plays an equally important role in 3 4 4 G. Rotkova , M. Chase , M. Bennett , chromosome evolution, through its involvement A. Leitch2 and J. Fajkus1 in fissions, centric fusions, translocations, inver- 166 15th ICC: Abstracts sions and centric shifts. It is unclear how the cen- dimensional (1-D) SDS-PAGE and isoelectric tromere, either as a functioning unit of the chro- focusing and radical free and highly reduced two- mosome or as a DNA sequence motif, has been dimensional electrophoreses were identi¢ed to involved in these processes. Macropodine marsu- draw up a list of constituent proteins with quanti- pials (kangaroos and wallabies) offer unique tative information. Image analysis of 1-D SDS- insights into current theories expositing cen- PAGE pattern indicated most abundant proteins tromere emergence during karyotypic diversifica- were histones, of which population exceeds 60% tion and speciation. Tracing the phylogentic of total amount of chromosomal proteins. A total distribution of centromeric sequences, including of 158 di¡erent proteins were identi¢ed from chro- tandem repeats and retroelements, within this mosomes isolated with polyamine procedure fol- group of mammals indicates these sequences lowed by sucrose density gradient centrifugation. have played an important role in chromosome Classi¢cation according to known localization evolution through amplifications, segmental indicated that 32% (51 proteins) of the number of duplications, fissions and fusions. Hybrids the identi¢ed proteins belong to nuclear ones, of between different kangaroo species provide evi- which 25 proteins are reported to localize to mito- dence that the centromere is unstable within this tic chromosomes. The isolated chromosomes group of mammals and is involved in a large included substantial number of nucleolar pro- number of chromosome aberrations, including teins, indicating that these chromosomes contain the appearance of knobs. A better understanding not only chromosomal proteins. In addition to of the genetic and epigenetic factors that define chromatin binding proteins related to transcrip- centromeres and how centromeres may mediate tion, topoisomerase II alpha and subunits of con- changes in chromosome architecture are critical densin complex were identi¢ed, although the not only to our understanding of basic cellular amounts of these chromosome associated pro- functioning but also to our understanding of the teins are rather low. Finally, the identi¢ed pro- process of speciation. teins were classi¢ed into four groups based on the behavior during the puri¢cation and known data during mitosis. Present catalogue would provide L186 essential data for understanding metaphase chro- Proteomic analysis of human mosomes based on the constituent proteins. metaphase chromosomes S. Uchiyama1, S. Kobayashi1, H. Takata1, L187 T. Ishihara1, T. Sone2, T. Nirasawa3, The fate of primary aneuploid cells 4 1 1 T. Takao , S. Matsunaga and K. Fukui during mouse embryo development 1 Department of Biotechnology, Graduate School D. Lightfoot1 and C. Ho¨o¨g1 of Engineering, Osaka University; 2Institute for Microbiological Diseases, Osaka University; 1Dept. of Cell and Molecular Biology, Karolinska 3Bruker Daltonics, K. K.; 4Institute for Protein Institute, Sweden Research, Osaka University, Japan The absence of synaptonemal complex protein 3 Formation of mitotic chromosome is needed to (SYCP3) incites chromosomal nondisjunctions ensure the equal partition of the semi-con- during the first meiotic division in murine female servatively replicated genomic DNA into daugh- germ cells, resulting in the formation of primary ter cells during mitosis. Despite more than a aneuploid haploid oocytes. This model system is century of research on chromosome structure, it unique in that it can reliably produce large num- still remains poorly understood. bers of primary aneuploid oocytes. We have stu- We report the comprehensive list of proteins died the fate of fertilized mouse embryos involved in isolated human metaphase chromo- resulting from these abnormal cells and find that somes for the elucidation of chromosome higher primary aneuploid embryos can implant into the order structures. Proteins separated by one- female uterine tissue and initiate gastrulation 15th ICC: Abstracts 167

(E6.5). At E7.0, however, the embryonic cells axis and meiotic recombination is taking place. begin to display abnormal morphological signs. The existence of immunolabelling techniques for Spherical cellular blebbings originating from the SC proteins (SCP1, SCP2 and SCP3) and for inner wall of the developing embryo are seen DNA mismatch repair proteins present in late aggregating into the center of the proamniotic recombination nodules (MLH1) allow analyses of cavity, as they become dissociated from the ecto- both synapsis and meiotic recombination in the dermic cellular layer. The inner cellular layers of gametocyte I. The creation of recombination the embryo then degrade and the embryo forms maps for each chromosome and the analysis of an indiscernible mass by E8.5. The embryo each bivalent synapsis is possible by in situ hybri- degradation process follows a singular pattern, disation methods because chromatin is preserved initially seen within the embryonic ectoderm, after cell fixation for immunoanalysis. In this along the lateral median of the embryo, and pro- work we apply the seven-fluorochrome sub- gressing outward to the distal poles, and latter telomere-specific FISH assay (Fauth et al., 2001, the surrounding deciduea. Concurrent with the Hum Genet. 109:576) to human male meiotic morphological changes in the dying embryos, cells previously labelled by immunofluorescence nuclear abnormalities are observed showing that (SCP1, SCP3, MLH1, CENP) to assess its utility loss of embryo viability is caused by the activa- for human SC karyotyping. This FISH method tion of a spatially and temporally controlled consists of microdissected subtelomeric probes apoptotic mechanism. This apoptotic response to labelled combinatorially with seven different systemic primary aneuploidy is p53-independent fluorochromes. Results prove the usefulness of and does not result from a failure to progress this FISH method (stM-FISH) for the identifica- through mitosis. We find that primary aneuploid tion of all human SC. Furthermore, by labelling embryos display a different developmental fate subtelomeric regions this one-single-step method than what has been observed for secondary enables the characterisation of interstitial and (Robertsonian) or tertiary (partial, segmental) terminal SC fragments and SC delineation even if aneuploid conceptus. superposition is present in pachytene spreads.

L188 L189 Characterisation of all human male B chromosomes alter the metaphase synaptonemal complexes with checkpoint in grasshopper meiocytes subtelomere multiplex-FISH B. Herron1, S. Martin-Iluesma2, P. Arana3 M. Codina-Pascual1, J. Kraus2, M. Speicher2, and D. Wise1 1 1 3 M. Oliver-bonet , V. Murcia , J. Sarquella , 1 1 1 1 Department of Biological Sciences, Mississippi J. Egozcue , J. Navarro and J. Benet State University, Mississippi State, MS 39762, 2 1 USA; Max Planck Institut fur Biochemie; Unitat Biologia, Facultat Medicina, Dept. 3 Biologia Celular, Fisiologia i Immunologia, UAB, Universidad Complutense, Madrid, Spain Bellaterra, Spain; 2Institut fu¨r Humangenetik, Technische Universita¨tMu¨nchen and GSF The B chromosome polymorphism in Spanish Neuherberg, Germany; 3Unitat de Reproduccion populations of the grasshopper, Eyprepocnemis Humana i Diagnostic Gentic, Clinica Girona, plorans (Charpentier) is ancient and widespread. Girona, Spain Meiocytes containing B chromosomes were ana- lyzed in our laboratory using the 3F3/2 mono- During meiotic prophase I, homologous chromo- clonal antibody, which binds to a kinetochore somes synapse and recombine in pachytene stage. phosphoepitope whose degree of phosphorylation Both events are of vital importance for the suc- is sensitive to tension applied to the kinetochore. cess of meiosis. When homologous chromosomes Further, the tension created by the spindle at synapse a proteinacious structure called synapto- metaphase controls a checkpoint (the metaphase nemal complex (SC) appears along the pairing checkpoint) that allows the cell to begin ana- 168 15th ICC: Abstracts phase when all chromosomes are aligned at the chromosome formation and stable segregation metaphase plate. Fluorescence patterns of the without integration. We conclude that transient 3F3/2 phosphoepitope in cells containing B expression of an EGFP marker is not suitable to chromosomes were determined using confocal identify transfected cells, which will subsequently laser scanning microscopy. The phosphorylation form viable clones. One explanation could be pattern of kinetochores in these cells was shown that the high copy number required to con- to be different from that of cells without Bs. This sistently detect transient EGFP-marker expres- suggests that the metaphase checkpoint has been sion [1] impairs viability and clone formation. modified in some way. We propose that B chro- mosomes in these grasshopper populations may [1] Schindelhauer D., Laner, A. (2002) Visible transient have survived during evolution due to an altera- expression of EGFP requires intranuclear injection of large copy numbers. Gene Ther. 9: 727–30. tion of the metaphase checkpoint, making it more permissive to the presence of misaligned chromosomes. L191 Fundamental structural unit of the L190 chromosome identi¢ed by atomic force Human arti¢cial chromosome microscopy expression marker K. Takeyasu1, J. Kim1, M. Yokokawa1 A. Laner3, S. Christan2, T. Schwarz1 and S. Yoshimura1 2 and D. Schindelhauer 1Kyoto University Graduate School of Biostudies, 1Medizinische Genetik, Kinderpoliklinik LMU, Japan Munich, Germany; 2Livestock Biotechnology, Life Sciences Center Weihenstephan, TU-Munich, The proper function of the genome largely Freising, Germany; 3Medizinisch Genetisches depends on the higher-order architecture of the Zentrum, Munich, Germany chromosome. Nano-technology is essential to address questions regarding the structural basis Human artificial chromosomes are generated for such macromolecular dynamics [1–3]. Because using transfer of telomerized PAC constructs of its applicability to biological specimen (non- containing alpha satellite DNA of various human conductors) under physiological conditions, chromosomes. To monitor which cells obtained atomic force microscopy (AFM) was employed constructs and subsequently form stable clones for the structural analyses. After HeLa cells on a under blasticidin S selection, a CMV/EGFP cover glass were successively treated with a deter- expression marker was inserted into a chr 5 gent and a high-salt solution to remove the cyto- alpha satellite construct (142 kb). Lipofection plasmic and nucleoplasmic materials, the into HT1080 cells resulted in a subportion of interphase chromosome was found to be com- bright green cells under the microscope at day 1. posed of a granular unit of *80 nm in diameter, Areas containing such early green cells were forming a fiber of *80 nm width. When this on- marked and plates monitored over 2 weeks. In substrate procedure was applied to Escherichia only one out of 41 marked areas (from 2 experi- coli, the nucleoid was found to consist of a fun- ments) a viable clone developed. In the remain- damental fibrous structure with a diameter of ing 40 areas, the green cells ceased growth at the *80 nm. The nucleoid dynamically changed its 1–8 cell stage (similar results were observed in structure during the cell growth; In addition to sections taped to exclude light damage). In con- this ‘80-nm fiber’, a thinner ‘40-nm fiber’ and a trast, outside of the marked areas, 16 stable higher-order structure of ‘loop’ were identified in clones have formed, which became uniformly the log phase nucleoid. However, in the later green not before day 4–6. FISH analysis of iso- growth phases, the nucleoid turned into a ‘coral lated clonal lines demonstrated human artificial reef structure’ that also possessed the 80-nm fiber 15th ICC: Abstracts 169 units, and, finally, into a ‘tightly compacted chromosome packing is common in both prokar- nucleoid’ that was stable under a mild lysis buf- yotes and eukaryotes. fer. Mutant analysis demonstrated that these tight compactions of the nucleoid required a pro- tein, Dps. From these, we propose a structural 1. Curr. Bio., 12: 508–512; 2. Proc. Nat’l Acad. Sci. USA, 97: model that assumes a fundamental mechanism of 7266–7271; 3. Biochemistry, 39: 9139–9145.

Chromosome Research 12 (Suppl 1): 171–181, 2004. 171 # 2004 Kluwer Academic Publishers. Printed in the Netherlands

Author Index

L denotes Lecture abstract; PO denotes Poster abstract.

Abdulrazzak, H. L112, L118, PO:08:17, PO:08:19 Balanya, J. L129 Abe, S. L125, PO:08:71 Balmus, G. PO:06:04 Abeydeera, L. L11 Balu, S. PO:08:10 Abgrall, J.-F. PO:08:85, PO:08:91 Bandkar, V. PO:08:72, PO:08:90 Abramowicz, M. L46 Bang, J. PO:06:80, PO:06:82 Acevedo, M. PO:06:65 Banzakour, S. PO:08:85, PO:08:91 Adega, F. PO:06:01 Baroni, R. M. PO:08:31 Advani, S. PO:08:90 Barrett, J. L130 Affara, N. L101 Bartos, J. L79, PO:06:83, PO:06:92 Agarwal, M. PO:08:90 Ba´rtova´,E.PO:06:70 Agata, K. L65, PO:06:52 Battaglia, A. PO:08:76 Aguilera, M. PO:06:54 Battini, R. PO:08:76 Ahlgren, T. L172 Battle-Morera, L. L99 Ahn, N. PO:06:06 Baulcombe, D. L75 Al-Awadi, S. A. PO:08:46 Behjati, M. PO:08:61 Albano, F. PO:08:84, PO:08:94 Belil, I. PO:06:34 Albiez, H. PO:06:66, PO:06:69 Bellovits, O. PO:08:37 Albrecht, B. L146 Belmont, A. L34, L37 Ali, H. L88 Belyayev, A. L80, L85, PO:06:72 Alkhimova, O. L81, PO:06:56 Benatti Jr., R. PO:08:31 Allain, D. L10 Benet, J. L54, L188, PO:06:34 Alonso-Gonzalez, L. PO:06:22 Bennett, M. L184, PO:06:45, PO:06:46 Altinkut, A. PO:06:72 Bentz, M. L166 Amariglio, N. L157 Bermu´dez, M. L54, PO:06:34 Anamthawat-Jonsson, K. PO:06:73, PO:08:38, PO:08:53 Bernardino-Sgherri, J. PO:08:25 Ando, J. L65, PO:06:53 Bertaud, M. L10 Andolina, M. PO:08:39, PO:08:45 Berthou, C. PO:08:85, PO:08:91 Anelli, L. PO:08:84, PO:08:94 Bertini, V. PO:08:76 Ansari, R. PO:08:54 Betti, M. PO:06:61 Anton, E. PO:08:23 Beverloo, B. L173, L174 Apisitwanich, S. PO:08:78 Bhardwaj, R. PO:06:79 Arana, P. L189 Bheemineni, S. PO:06:15, PO:06:33 Bickel, S. L149 Aranyavalai, V. PO:06:02 Bickmore, W. L31 Arbour, L. L126 Bidau, C. L63 Arnaud, B. PO:08:85 Bird, A. L71 Arneric, M. L68 Bishehsari, F. PO:08:54 Arrieta, I. PO:08:50, PO:08:52 Bisoni, L. L99 Assenberg, M. PO:08:20 Blanco, J. PO:08:23, PO:08:34 Astakhova, N. PO:06:64 Bleuyard, J. PO:08:24 Athalye, A. PO:08:72, PO:08:90 Bonduelle, M. L49 Attarian, H. PO:08:59 Bongers, E. L159 Aulard, S. PO:06:50 Bonifacio, S. PO:06:35 Aveline-Wolf, L. PO:06:06 Bonne, G. L30 Ayabe, F. PO:08:18 Boobis, I. PO:08:74 Ayenehchi, S. PO:08:49 Boudry, P. PO:06:13, PO:08:65 Azuma, T. PO:06:08 Bouilly, K. PO:08:65 Bourquard, P. PO:08:85, PO:08:91 Babcock, J. L117 Bourrouillou, G. L46 Bacher, C. P. PO:06:67 Bo¨wering, K. L146 Bageri, N. PO:08:73 Boyle, S. L31 Baharizadeh, S. PO:08:59 Bozzini, G. PO:06:04 Bailey, S. L183 Brack-Werner, R. PO:06:69 Bair, E. L169 Bradbury, M. PO:06:63 172 15th ICC: Author Index

Branco, M. L39 Chuzhanova, N. L60 Brenton, J. L74 Ciampi, R. PO:06:61 Bretherick, K. L152 Cianchetti, C. PO:06:60 Bridger, J. L11, L30, L111 Cihalikova, J. L79, PO:06:83, PO:06:92 Britton-Davidian, J. L104 Cioni, G. PO:08:76 Brodie, S. PO:08:86 Cirakoglu, A. PO:08:56, PO:08:77 Brown, C. L97, L152 Cisel, B. PO:08:88 Brown, J. PO:08:55 Clegg, S. L176 Brown, L. L100 Clemente, E. L101 Brown, W. L116 Clouston, H. L51 Bruyere, H. L126 Cloutier, S. PO:06:26 Buijs, A. PO:08:87 Codina-Pascual, M. L188 Buiting, K. PO:06:71 Coe, B. L139 Bujdoso´,G.PO:08:37 Cohen, P. L123 Bullinger, L. L169 Collignon, P. L102 Buquicchio, C. PO:08:84 Colls, P. L54 Burford, D. L176 Colovic, M. PO:08:39, PO:08:45 Burgoyne, L. PO:06:03 Conn, C. L47 Burgoyne, P. L101, PO:08:35 Constantinou, M. PO:06:16, PO:08:06 Burn, J. L01 Cook, P. PO:06:38 Burt, D. L09, L56 Cooper, D. L60 Bystricky, K. L35 Cooper, J. L67 Coppola, G. PO:06:04, PO:06:04 Cabuy, E. L70 Cornforth, M. L183 Camats, N. PO:06:57 Cornillon, J. PO:08:92 Campiranon, A. PO:08:60 Corredor, E. PO:08:26, PO:08:27 Campos, L. PO:08:92 Cowan, R. S . PO:06:38 Canham, P. L26 Cox, H. L111 Cao, A. PO:06:60 Craig, J. L26, L27 Carniani, S. PO:06:35 Cremer, C. L161, PO:06:32 Carpenter, A. PO:06:22 Cremer, M. PO:06:32, PO:06:69 Carpentier, S. PO:08:36 Cremer, T. L33, L42, L76, PO:06:66, PO:06:69, PO:06:71 Carrera, M. PO:08:75 Criado, B. PO:08:50, PO:08:52 Carrillo, J. A. PO:06:27 Crisponi, G. PO:06:60 Carter, N. L31, L175, L176 Crolla, J. L176 Carvalho, A. L18 Crooijmans, R. L09 Carvalho, B. PO:08:15, PO:08:16 Csonka, E. L158, PO:08:37 Ceccarelli, M. PO:06:23 Culic, V. PO:08:66 Cesari-Weimar, F. L74 Ce´spedes, W. L129 Dafhnis-Calas, F. L116 Chadwick, B. L117 Dahm, P. L132 Chaminade, N. PO:06:50 Dalakiouridou, M. L50 Chantry-Darmon, C. L10 Dame, R. L93, PO:08:01 Chase, M. L184, PO:06:62 Daniely, M. L157 Chaumeil, J. PO:06:68 David, P. PO:08:30 Chaves, R. PO:06:01, PO:06:13, PO:08:65 De Braekeleer, M. PO:08:85, PO:08:91 Cheeseman, I. L19 De Carli, L. L36 Chetan, K. G. PO:08:10 De Jong, H. L88 Cheung, W. L112, L118, PO:08:19 De la Herra´n, R. PO:08:08, PO:08:09 Chiaroni, J. L102 de Leeuw, N. L159 Chicheportiche, A. PO:08:25 de Paepe, A. L142 Chokchaichamnankit, P. PO:08:38 de Rochambeau, H. L10 Choo, A. K. L07, L26, L27 de Vries, B. L133 Chow, J. L97 Dechyeva, D. PO:06:75 Christan, S. L190 Dedovic-Tanic, N. L15 Christopikou, D. L50 Defeu Soufo, H. L95 Chudzinska, E. PO:06:74 Dekker, C. L93 Chulalaksananukul, W. PO:06:02, PO:08:38 Deladesse, E. PO:08:92 Chung, W. PO:08:17 De-Lange, T. L66 15th ICC: Author Index 173

Delhanty, J. L47 Farahman, H. PO:08:73 Demidov, D. L87 Faridi, N. PO:06:76 Deng, C. PO:08:35 Farr, C. L06, PO:06:22, PO:08:22 Dennis, N. L176 Fay, M. PO:06:38 Derjusheva, S. L13, PO:06:37, PO:08:07 Fedoryshyn, Z. PO:08:67 Desai, A. L19 Feenstra, I. L133 Deviren, A. PO:08:77 Feingold, E. L151 Devroey, P. L49 Ferguson-Smith, M. L09, L41, L55, L61, L106 Dhumal, S. PO:08:72, PO:08:90 Fernandez, R. PO:08:68 Di Berardino, D. PO:06:04 Ferreira, I. L17, PO:06:07 Di Meo, G. PO:06:04 Fiegler, H. L31 Diaz, A. PO:06:12 Field, J. K. PO:06:25 Dickson, G. L113 Figgitt, M. L30 Dimitrijevic, B. L15 Filatov, D. L103 Ditchfield, C. L22 Filip, A. PO:08:88, PO:08:89 Dmoszynska, A. PO:08:88, PO:08:89 Filipe, S. L92 Do¨hner, H. L169 Fillon, V. L09 Do¨hner, K. L169 Finsterle, J. L161 Dolezel, J. L79, PO:06:56, PO:06:83, PO:06:92 Fioretos, T. L172 Dombrowski, C. PO:06:75 Flandrin, P. PO:08:92 Donev, R. L43 Fleming, E. L158 Donnison, I. PO:06:78 Flores, P. PO:08:50, PO:08:52 Dordari, S. PO:06:05 Fogel, M. PO:08:02 Douet-Guilbert, N. PO:08:85 Fontdevila, A. L129 Draper, J. PO:06:77, PO:06:78 Foresti, F. L17, PO:06:07 Duprez, L. L46, PO:08:40 Fo¨rstemann, K. L68 Duran, A. PO:08:51 Foster, H. L11, L30 Dutrillaux, B. PO:08:25 Fragouli, E. L47 Dyban, A. PO:06:20 Fransz, P. L88 Dyer, M. S. L167 Frederic, V. L104 Frigyesi, A. L145 Earle, E. L26 Fro¨hling, S. L169 Earnshaw, W. L18, PO:06:22 Fuchs, J. L45, L64, PO:06:28 Egozcue, J. L188, PO:06:34, PO:06:57, PO:08:23, Fujiwara, A. PO:06:53 PO:08:32, PO:08:33, PO:08:34 Fukagawa, T. L23, PO:06:22 Egozcue, S. PO:08:34 Fukova, I. L105, PO:08:04, PO:08:13 Egriboz, A. PO:08:51 Fukui, K. L14, L82, L83, L186, PO:06:08, PO:06:10 Eijpe, M. L122 Fukushi, D. PO:08:81 Eils, R. L76, L164, PO:06:67 Furlong, R. L101 Eleveld, M. L154 Fuster, C. PO:08:75 Ellis, J. A. L111 Ellis, P. L101 Gaginskaya, E. L13, L160, PO:06:37, PO:08:07 El-Mogharbel, N. L106 Gair, J. L126 Emma, D. PO:08:30 Galetti Jr., P. M. L17 Emrick, M. PO:06:06 Galkina, S. PO:06:31 Endo, T. PO:06:92 Gallardo, M. L63 Engelbrecht, S. PO:08:03, PO:08:55 Gallego, M. PO:06:58, PO:08:24 Enne, G. PO:06:04 Gamerdinger, U. PO:08:69 Enukashvily, N. L43 Gao, J. L76 Erhardt, S. L74 Garagna, S. L44 Errington, J. L94 Garcia, M. PO:06:51, PO:06:54, PO:06:57, PO:08:32, Escalona, A. PO:08:75 PO:08:33 Evans, C. L133 Garcia, R. PO:08:32, PO:08:33 Garcı´a, F. PO:06:54, PO:06:57 Fadaei, S. L77 Gardunia, B. R. PO:06:81 Faed, M. L47 Garnis, C. L139 Fairclough, H. L61 Garrido-Ramos, M. A. PO:08:08 PO:08:09 Fajkus, J. L184, PO:06:62 Gasser, S. L35 Fang, J. L133 Gassmann, R. L18 174 15th ICC: Author Index

Gazdar, A. L139 Hameister, H. L60, L98 Gelati, M. T. PO:06:23 Hanaoka, K. L125 Gerats, T. L88 Hans, C. PO:08:40 Ghadiani, M. PO:08:59 Hanson, L. PO:06:38 Ghasemi Barghi, M. PO:08:49 Harrison, C. L170, L171 Gibbons, N. PO:08:83 Harvard, C. PO:06:30 Gibby, M. L130 Hasezawa, S. L83 Gifford-Garner, J. PO:08:19 Hass, I. PO:06:40 Gilbert, N. L31 Hassold, T. L148 Giltay, J. L154 Hasterok, R. PO:06:77, PO:06:78 Girimaji, R. S. PO:08:10 Hausmann, M. L146, L161, PO:06:32 Giselsson, D. L145 Hayes, H. L10 Glazko, T. L40, L136 Heard, E. PO:06:67, PO:06:68 Glazko, V. L136 Hegde, J. PO:08:47, PO:08:48 Glazkov, M. PO:06:24 Helszer, Z. PO:08:06 Glover, D. PO:08:11 Hemleben, V. PO:06:47 Goidts, V. L60 Henzing, A. L18 Goodwin, E. L183 Hernando, C. PO:08:75 Goosen, N. L93, PO:08:01 Herr, A. L75 Gordei, I. PO:08:28 Herron, B. L189 Goswami, H. PO:06:39 Heslop-Harrison, J. S. L86, PO:06:01 Graumann, P. L95 Heun, P. L35 Graves, J. L61 Heyting, C. L122 Greaves, M. L171 Higashi, T. PO:06:08 Green, E. L180 Higgs, D. L72 Greenbaum, I. L132 Hildenbrand, G. L161 Greene, A. L158 Hnateiko, O. PO:08:67 Greensmith, J. PO:06:38 Hobza, R. PO:08:05 Gregor, T. PO:08:11 Hochstenbach, R. L154 Greilhuber, J. PO:06:88 Hodges, C. L122 Gribble, S. L176 Ho¨fler, H. L146 Griffin, D. L09, L11, L30, L50, PO:06:53, PO:08:83 Ho¨glund, M. L145 Griffiths, M. L170, L171 Holecova, M. PO:06:11 Grimes, B. L117 Holloway, K. L178 Groenen, M. L09 Homa, S. L50 Großmann, C. L161 Ho¨o¨g, C. L150, L187 Grundmann, M. L130 Horsley, S. L171 Gruszka-Westwood, A. L171 Horsthemke, B. PO:06:71 Grutzner, F. L61 Hoshi, O. L156, L182 Gru¨tzner, F. L106 Hoshiya, H. PO:08:18 Guan, X. Y. PO:08:63 Houben, A. L87 Guber, A. L157 Hu, L. PO:08:63 Guedes-Pinto, H. PO:06:01, PO:06:13, PO:08:65 Hudakova, S. PO:06:18 Guichaoua, M. L102 Hughes-Davies, L. L73 Guillaume, M. PO:06:58 Hulte´n, M. L48, PO:08:29 Gunel, B. PO:08:51 Hunt, H. L130 Guskov, E. PO:06:94 Hunt, P. L120, L122 Gustavino, B. PO:06:55 Hurtado, N. S. PO:06:09 Gutie´rrez-Mateo, C. L54, PO:06:34 Huxley, C. L112, L118, PO:08:17, PO:08:19, PO:08:21 Guven, G. PO:08:77 Iannuzzi, L. PO:06:04 Haas, O. A. L168 Ibraimov, A. PO:06:41, PO:06:42, PO:06:43, PO:08:41, Habermann, F. PO:06:37 PO:08:42, PO:08:43, PO:08:44 Hacihanefioglu, S. PO:08:51, PO:08:56, PO:08:57, Imreh, S. L144, L147 PO:08:77 Inan, M. PO:08:56, PO:08:57 Hadlaczky, G. L144, L158, PO:08:37 Isfahani, F. PO:08:59 Hajfathali, A. PO:08:59 Ishihara, T. L186 Hajkova, P. L74 Ishijima, J. PO:06:53 Hall, L. L97 Isobe, T. L65 15th ICC: Author Index 175

Iturra, P. PO:06:12 Kim, J. L191 Iwano, M. L14 Kim, S.-Y. PO:06:80 Kimura, E. L156 Jacobs, P. L02, L176 Kimura, M. L125 Jahanzad, E. PO:06:25 King, I. PO:06:78 Jamet, D. PO:08:91 King, V. L62 Jamilena, M. PO:08:09 Klatte, M. L45, L84, PO:06:28 Janda, J. L79 Klinger, H. L108 Jankovic, G. PO:08:39, PO:08:45 Kobayashi, S. L186, PO:06:10 Javadi, H. PO:08:49 Kobori, T. PO:08:80 Jeffreys, A. L178 Koch, A. L42 Jellen, E. PO:06:81 Koch, M. PO:06:48 Jelska, A. L135 Koczkodaj, D. PO:08:88, PO:08:89 Jenkins, G. PO:06:77, PO:06:78 Ko¨hler, D. L76 Jenny, W. PO:08:30 Kohn, M. L98 Jenuwein, T. PO:06:69 Kolano, B. PO:06:81 Jeong, T. PO:08:58 Kolia, S. PO:08:03 Jesdapatarakul, S. PO:08:60 Koo, D. PO:06:82 Jessberger, R. L122 Koo, S. PO:08:58 Jiang, J. L90 Koolen, D. L133 Jiang, R. L126 Koornneef, M. L88 Jin, J. PO:06:87 Korniszewski, L. PO:08:06 Johansson, B. L172 Korver, C. M. L100 Johnson, V. L22 Korzeniowska, J. PO:08:88 Jones, R. L12 Koskin, A. L181 Jones, R. C. L106 Kotzamanis, G. L112, L118, PO:08:17, PO:08:19 Josette, C. L104 Koukalova, B. PO:06:47 Jovicic, D. PO:08:70 Kouzarides, T. L73 Julia, S. PO:08:30 Kovacevic, R. PO:08:70 Juliusson, G. L172 Kovarik, A. PO:06:47 Kovarova, P. L79, PO:06:83, PO:06:92 Kozubek, M. PO:06:70 Kai, Y. L125, PO:08:71 Kozubek, S. PO:06:70 Kaluzewski, B. PO:06:16, PO:08:06 Krasikova, A. L13, PO:08:07 Kalz, L. L135 Kraus, J. L188 Kamimura, E. PO:06:44 Kreth, G. L45, PO:06:28 Kanale, S. PO:08:47, PO:08:48 Krijtenburg, P.-J. PO:08:87 Kang, J. PO:08:58 Krishnamurthy, D. S. PO:08:46 Kaplan, T. L157 Krutilina, R. PO:06:63 Karamisheva, T. PO:06:64 Kubalakova, M. L79, PO:06:83, PO:06:92 Karpen, G. L24 Kubickova, S. PO:08:04 Katnoria, J. K. PO:06:79 Kubisiak, T. PO:06:76 Kato, N. L45, PO:06:29 Kukalev, A. PO:06:17 Katoh, M. PO:08:18 Kulikova, T. L13, L160 Kauppi, L. L178 Kunicka, Z. L184 Kauppinen, S. L181 Kurganova, A. L13, PO:06:37 Kawabe, A. L83 Kurihara, D. L83 Kawle, M. PO:08:72, PO:08:90 Kuroiwa, A. L65 Kazmierczak, W. PO:08:82 Kurt, H. PO:08:77 Kazuki, Y. L125, PO:08:71 Kurth, I. L68 Kearney, L. L171 Kuru, D. PO:08:56, PO:08:57, PO:08:77 Kehrer-Sawatzki, H. L60, L98 Kuwae, A. PO:06:10 Kejnovsky, E. PO:08:05 Kuznetsova, I. , Kelleher, C. L68 PO:06:20 PO:06:59 Kelly, J. L28 Kwon, K. C. PO:08:58 Kempski, H. L171 Keymolen, K. L49 La Starza, R. PO:08:94 Khoshbin, E. PO:06:25 Labaere, C. PO:08:36 Kianian, S. PO:06:83 Lachowska-Cierlik, D. PO:06:11 Kill, I. L30 Ladurner, A. L165 176 15th ICC: Author Index

Lam, A. L24 Lozano, R. PO:08:09 Lam, E. L45, PO:06:29 Lucretti, S. PO:06:83 Lam, N. PO:06:12 Lukina, N. PO:06:31 Lam, S. L139 Lysak, M. A. L45, L64, PO:06:28, PO:06:48 Lam, W. L139, L152 Lyusikov, O. PO:08:28 Lamb, N. L151 Laner, A. L190 Macas, J. PO:08:05 Langan, J. PO:06:25 MacAulay, C. L139 Lange, J. L100 MacDonald, N. L119, L158 Langerak, A. L174 Mach, J. PO:06:87 Langowski, J. L35 Machev, N. L102 Lanzone, C. L63 MacLeod, R. L174 Lape`gue, S. PO:08:65 Madon, P. PO:08:72, PO:08:90 Larin-Monaco, Z. L115, PO:08:20 Mahadevaiah, S. L101, PO:08:35 Lassen, C. L172 Mahdavinia, M. PO:08:54 Latypov, A. PO:08:74 Maleki, F. L77 Laureys, g. L142 Malekzadeh, R. PO:08:54 Law, S. L157 Malla, S. L116 Lawrence, J. L97 Maluszynska, J. PO:06:81 Le Baccon, P. PO:06:68 Mancini, M. PO:08:94 Lebris, M.-J. PO:08:85, PO:08:91 Mandahl, N. PO:08:62 Ledebur, Jr., H. L119, L158 Manjunatha, R. K. PO:08:10 Lee, E. L119 Lee, G. L119 Manteuffel, R. PO:06:18 Lee, W.-K. PO:06:80 Marasek, A. PO:06:78 Lee, Y.-K. PO:06:82 Marec, F. L105, PO:08:04, PO:08:13, PO:08:14 Lees, M. L133 Marra, M. L139 Leese, H. L11 Marseglia, G. PO:06:61 Leibovitch, I. L157 Marshall-Graves, J. L96, L106, L107 Leita˜o, A. PO:06:13, PO:08:65 Martin-Lluesma, S. L189 Leitch, A. L184 PO:06:47, PO:06:62 Martinez Ramirez, A. L171 Leitch, I. PO:06:38, PO:06:45, PO:06:46 Martins, C. L17, PO:06:07 Lemeunier, F. PO:06:50 Marynen, P. PO:08:36 Lengerova, M. PO:08:05 Marzin, Y. PO:08:91 Lenzi, M. L123 Masabanda, J. L09, PO:06:53 Leonhardt, H. PO:06:66 Mascarenhas, J. L95 Lermontova, I. PO:06:18 Mashkina, E. PO:06:94 Leung, G. L91 Masumoto, H. L114 Levy, N. L30, L102 Mateos-Langerak, J. L37 Lichter, P. L162 Matsubara, K. PO:06:49 Liebaers, I. L49 Matsuda, Y. L65, PO:06:44, PO:06:49, PO:06:52, Liebe, B. L122, PO:08:36 PO:06:53 Lightfoot, D. L187 Matsunaga, S. L14, L83, L186, PO:06:08, PO:06:10 Lim, J. PO:08:58 Mattei, M.-G. L102 Lim, K. PO:06:47 Mattes, J. L76 Lim, K. Y. L184 Mauvieux, L. PO:08:92 Lim, Y. PO:06:62 Maxwell, A. L158 Linc, G. L88 May, C. L178 Lindenbaum, M. L158 May, D. PO:08:69 Lingner, J. L68 Mayya, S. PO:08:47 Lisi, E. PO:06:35 McCombie, H. PO:08:65 Liso, A. PO:08:94 McFarlane, R. PO:08:30 Liso, V. PO:08:84 McQueen, H. L99 Livingstone, K. L128 McQueen, P. L38 Loidl, J. PO:08:30 Meaburn, K. L30, L111 Lonergan, K. L139 Mehran Nejhad, R. PO:06:05 Lorenz, A. PO:08:30 Meister, A. L45, PO:06:28, PO:06:29 Lorite, P. PO:06:27 Menten, B. L142 Lostao, C. PO:08:52 Menzel, G. PO:06:75 Loubser, M. PO:08:03 Merico, V. L44 15th ICC: Author Index 177

Mertens, F. PO:08:62 Naik, N. PO:08:72 Mestrovic, N. PO:06:19 Nakagawa, K. L83 Meyer-Arendt, K. PO:06:06 Nakano, M. L114 Michiels, A. L49 Nakashima, H. L114 Midro, A. T. L135 Nakayama, S. PO:06:84 Mieloo, H. L159, PO:06:36 Nannetti, G. PO:06:35 Milacic, S. PO:08:70 Naranjo, T. PO:08:26, PO:08:27 Mileham, A. PO:08:83 Nasmyth, K. L121 Miles, M. L182 Navajas-Pe´rez, R. PO:08:08, PO:08:09 Mills, J. L47 Navarro, A. L102 Mills, K. L119 Navarro, J. L54, L188, PO:06:34 Mills, W. PO:06:22 Nelson, C. PO:06:76 Mills, W. E. PO:08:22 Neplechova, K. PO:06:62 Minafra, L. PO:06:60 Nesina, I. PO:08:88 Minelli, S. PO:06:23 Neumann, R. L178 Minina, J. PO:06:64 Neusser, M. L42 Misteli, T. L38 Nevo, E. L80, L85, PO:06:72 Mitchell, M. L102 Newbold, R. L110, L111 Mitelman, F. L145, L172 Nicodemo, D. PO:06:04 Miyakawa, S. PO:06:08 Niessen, S. L19 Mizuno, S. PO:08:07 Nirasawa, T. L186 Mogatusi, L. PO:08:03 NISC Comparative Sequencing, L180 Mohaddes, S. M. PO:08:73 Nishida-Umehara, C. L65, PO:06:44, PO:06:53 MohamadzadehGhobadloo, S. L77 Noom, M. PO:08:01 Mohanty, S. PO:08:72 Noori Daloii, M. PO:06:25 Mohseni, J. PO:08:73 Norikane, S. PO:08:18 Mokros, P. PO:06:91 Nourmohammadi, Z. PO:06:85 Monier, K. L29 Nucaro, A. L. PO:06:60 Montchamp-Moreau, C. PO:06:50 Nutini, A. PO:06:61 Monti, M. L44 Monti-Dedieu, L. PO:06:50 Obado, S. L28 Moorman, A. L171 O’Brien, P. L09, L41, L61, L106 Moosavi, H. PO:08:49 O’Brien, S. L57, L62 Moosavi, Z. PO:08:73 Oda, M. PO:06:08 Mora, L. PO:06:51 Oegema, K. L19 Moralli, D. L36, PO:08:20 Oei, S.-L. PO:06:63 Moran, P. PO:06:09, PO:06:14 Ogur, G. L49 Morel, F. PO:08:85, PO:08:91 Ohtani, T. PO:08:80, PO:08:81 Morel, S. PO:08:92 Ohzeki, J.-I. L114 Morice, P. PO:08:85, PO:08:91 Okita, C. L125, PO:08:18, PO:08:71 Morris, W. PO:08:83 Olaru, D. PO:08:92 Morrow, C. L22 Olejek, A. PO:08:82 Movafagh, A. L77, PO:08:49, PO:08:59 Oliveira, C. L17, PO:06:07 Mravinac, B. PO:06:19 Oliver-Bonet, M. L188 Mu¨ller, S. L42, L58, L60, PO:06:40 O’Neill, R. L185 Munne´,S.L54, PO:06:34 Ortega, B. PO:08:50 Mun˜oz, M. PO:06:27 Ortiz, E. PO:08:52 Murakami, T. PO:06:52, PO:06:53 Oshimura, M. L109, L125, PO:08:18, PO:08:71 Murata, M. L89, PO:06:21, PO:06:93 Owen, R. L182 Murcia, V. L188 Ozdemir, N. PO:06:26 Murdoch, A. L51 Ozyurt, E. PO:08:56, PO:08:57 Murphy, W. L62 Muß, B. L134 Pacchierotti, F. PO:06:55 Padma, S. PO:08:10 Na, Y. G. PO:08:58 Page, D. L100 Nadal, N. PO:08:92 Pagenstecher, C. L134 Nadalin, N. PO:06:35 Palomeque, T. PO:06:27 Nagpal, A. PO:06:79 Pa˚lsson, E. PO:08:62 Naik, D. PO:08:72 Panagopoulos, I. PO:08:62 178 15th ICC: Author Index

Pannunzio, A. PO:08:84 Prigmore, E. L176 Parada, L. L38 Probst, A. PO:06:29 Parekh, S. PO:08:90 Puizina, J. PO:06:91 Parikh, F. PO:08:72, PO:08:90 Park, J. W. PO:08:58 Raimondi, E. L36 Parker, J. L59 Rajcan-Separovic, E. PO:06:30 Parriego, M. PO:08:23 Rao, N. PO:08:86 Parrini, D. PO:06:61 Rapalini, E. PO:08:76 Parris, C. L110 Rapp, A. L161 Pasantes, J. PO:06:09 Raskina, O. L80, L85, PO:06:72 Pasantes, J.-J. PO:06:14 Rechavi, G. L157 Pasaro, E. PO:08:68 Reckviashvili, N. PO:08:64 Pascale, C. L104 Redi, C. A. L44 Pastwinska, A. PO:08:93 Reichart, M. L157 Patel, S. L25 Ren, X. PO:08:18 Patkin, E. L78 Rens, W. L41, L61, L106 Paulis, M. L36 Repping, S. L100 Paulsson, K. L172 Repucci, D. PO:06:03 Pearks Wilkerson, A. L62 Rerkamnuaychoke, B. PO:08:60 Pecinka, A. L45, L64, L84, PO:06:28, PO:06:29, Resing, K. PO:06:06 PO:06:48 Revenkova, E. L122 Pecon-Slattery, J. L62 Ribeiro, E. PO:08:15, PO:08:16 Pena, B. L10 Rieseberg, L. L128 Pen˜agarikano, O. PO:08:50, PO:08:52 Riha, K. PO:06:91 Percipalle, P. PO:06:17 Risk, J. PO:06:25 Perez, C. L119, L158 Rizzoni, M. PO:06:55 Perez-Garcia, C. PO:06:14 Roberti, M. G. PO:08:84 Perez-Luz, S. L112, PO:08:21 Robinson, W. L126, L152 Perkins, E. L158 Robles, P. PO:08:32, PO:08:33 Peterlin, B. L53, PO:08:12 Rocchi, M. L172, PO:08:62, PO:08:84, PO:08:94 Peters, A. L124, PO:06:69 Rodionov, A. PO:06:31 Peters, J. L88 Rodrı´guez-Criado, G. PO:08:75 Petitpierre, E. PO:06:11 Rogel-Gaillard, C. L10 Peyachoknagul, S. PO:08:78, PO:08:79 Roig, I. PO:08:32, PO:08:33 Pezer, Z. PO:06:19 Rona, R. L157 Pfleghaar, K. PO:06:69 Rosek, M. PO:06:11 Pfundheller, H. L181 Ross, F. L170, L171 Pichler, S. PO:08:11 Rossino, R. PO:06:60 Pichon, B. L46, PO:08:40 Rotkova, G. L184 Pienkowska-Grela, B. PO:08:93 Rowell, D. L131, L137 Pierce, K. PO:06:06 Roy, S. PO:08:10 Pierozzi, N. PO:06:86, PO:08:31 Rozen, S. L100 Pinter, I. L130 Rubtsov, N. PO:06:64 Pinto Leite, R. PO:08:15, PO:08:16 Ruchaud, S. L18 Pinto Maglio, C. A. F. PO:06:86, PO:08:31 Rudd, M. L117 Pirzadeh, Z. PO:08:49 Rudgers, G. PO:06:87 Platzer, M. L60 Ruiz Rejo´n, C. PO:08:08, PO:08:09 Plohl, M. PO:06:19 Ruiz Rejo´n, M. PO:08:08, PO:08:09 Plowas, I. PO:06:16, PO:08:06 Ruiz-Herrera, A. PO:06:54, PO:06:57 Podgornaya, O. L43, PO:06:17, PO:06:20, PO:06:59 Rumsey, F. L130 Poeaim, S. PO:08:60 Rupps, R. L126 Pollack, J. L169 Russell, S. L130 Pombo, A. L39 Rygier, J. Pommer, C. PO:06:86 PO:08:93 Ponsa`,M.PO:06:51,PO:06:54 Poot, M. L154 Sabapara, R. PO:06:15 Porter, A. PO:06:22 Safar, J. L79 Possoz, C. L92 Saglio, G. PO:08:94 Poulsen, T. L174 Sahara, K. L16, L105, PO:08:14 Preuss, D. PO:06:87 Saifitdinova, A. L13, L160, PO:08:07 15th ICC: Author Index 179

Saito, T. L65 Shibusawa, M. PO:06:53 Salehi, M. PO:08:61 Shichiri, M. PO:08:81 Salehi, R. PO:08:61 Shimada, Y. PO:06:53 Sa¨ll, T. L145 Shimizu, R. D. PO:06:07 Samoylova, L. PO:08:74 Shimizu, T. PO:06:08 Sanchez, A. PO:06:52 Shipacheva, O. PO:08:74 Sa´nchez-Garcı´a, J. L54, PO:06:34 Shirayoshi, Y. L125 Sandig, C. L60 Shoji, M. L153 Sandstrom, R. L176 Sibson, M. L27 Santalo, J. PO:08:23 Sidhu, M. C. PO:06:90 Santos, J. L63 Siezen, A. L133 Santos, M. L129, PO:08:75 Silahtaroglu, A. L181 Santos, S. PO:06:13 Siljak-Yakovlev, S. L59 Sarquella, J. L188 Simi, P. PO:08:76 Sarrate, Z. PO:08:23, PO:08:34 Simons, A. PO:06:36 Sarri, V. PO:06:23 Simpson, K. PO:08:20 Satija, C. K. PO:06:90 Siripatanadilok, S. PO:08:78, PO:08:79 Sato, H. L89 Siroky, J. PO:06:91, PO:08:05 Saunders, S. L73 Skaletsky, H. L100 Saut, N. L102 Skalicka, K. PO:06:47 Sauter, G. L163 Skalnı´kova´,M.PO:06:70 Sbalquiero, I. PO:06:40 Sklenickova, M. PO:06:62 Scascitelli, M. PO:06:55 Slijepcevic, P. L70 Schermelleh, L. PO:06:66 Slingsby, T. L178 Schertan, H. PO:08:33 Slominska, N. L78 Scherthan, H. L122, PO:08:36 Slupphaug, G. L25 Schindelhauer, D. L190 Smeets, D. PO:06:36 Schinzel, A. L133 Smirnova, A. PO:06:63 Schlenk, R. L169 Smith, J. L09 Schlesier, B. PO:06:18 Smith, M. L116 Schmidt, R. L64 Solanki, J. V. PO:06:15, PO:06:33 Schmidt, T. PO:06:75 Solovei, I. L76, PO:06:32, PO:06:66, PO:06:71 Schmitt, E. L161 Soltis, D. PO:06:46 Schneeweiss, G. M. PO:06:88 Soltis, P. PO:06:46 Schneeweiss, H. L59, PO:06:88 Sone, T. L14, L186 Schneider, H. L130 Sopariwala, A. PO:08:72, PO:08:90 Schober, H. L35 So´tonyi, P. PO:08:37 Schoenmakers, E. PO:08:36 Souto, M. PO:08:15, PO:08:16 Schubert, I. L45, L64, L84, L88, PO:06:18, PO:06:28, Specchia, G. PO:08:84, PO:08:94 PO:06:29, PO:06:48 Speicher, M. PO:06:69 Schubert, V. L45, L84, PO:06:18, PO:06:28 Speicher, M. R. L188 Schwanitz, G. L134, L135 Speleman, F. L142 Schwarz, T. L190 Spence, J. PO:06:22, PO:08:22 Schwarz, V. L42 Sperisen, P. L68 Schwarzacher, T. PO:08:08 Spirito, F. PO:06:55 Schweizer, D. PO:06:91 Srebniak, M. PO:08:82 Searle, J. L127 Staessen, C. L49 Sebastio, L. PO:08:94 Stafne, A. PO:08:55 Seidel, A. PO:08:69 Stanyon, R. L63 Serra, L. L129 Stein, S. L161, PO:06:32 Seyed Rassaghi, S. PO:06:05 Stephenson, J. PO:08:83 Shahabi, M. PO:06:25 Stevens, M. R. PO:06:81 Sham, S. T. PO:08:63 Stewart, S. L119 Sharp, H. L137 Stodola, T. L119 Shatrova, A. PO:06:20 Stokes, P. L11 Shay, J. L69 Storlazzi, C. T. L172, PO:08:84 Sheidai, M. PO:06:85, PO:06:89 Strefford, J. L170 Sherman, S. L151 Strombeck, B. L172 Sherratt, D. L92 Stuessy, T. L59 Shibata, F. L89, PO:06:21 Sturmey, R. L11 180 15th ICC: Author Index

Suchankova, P. L79, PO:06:83, PO:06:92 Traut, W. L105, PO:08:04 Suchkova, I. L78 Tremetsberger, K. L59 Suda, T. PO:08:18 Tsao, M. L139 Sugiyama, S. PO:08:80, PO:08:81 Tsend-Ayush, E. L106 Sullivan, B. L24 Tsuchiya, K. PO:06:44, PO:06:49 Sullivan, K. L29 Turner, J. L101, PO:08:35 Sullivan, S. L74 Tyson, C. PO:06:30 Sumer, H. L27 Sumner, A. L03 Uchiyama, S. L14, L83, L186, PO:06:08, PO:06:10 Suputtitada, S. PO:08:79 Ugarkovic, D. PO:06:19 Surace, C. PO:08:62 Umehara, C. PO:06:52 Surani, A. L74 Umesono, Y. PO:06:52 Sykorova, E. L184, PO:06:62 Uppala, R. PO:06:15, PO:06:15, PO:06:33, PO:06:33 Szamalek, J. M. L60 Urien, C. L10 Szeles, A. L144 Usatov, A. PO:06:94 Sznajer, Y. PO:08:40 Ushiki, T. L156, L182 Szuhai, K. L141 Uzan, M. PO:08:56, PO:08:57

Tada, T. L153 Vagnarelli, P. L21 Tagavi, S. PO:08:73 Valetto, A. PO:08:76 Taghikhani, M. PO:06:25 Valverde, L. PO:08:50 Takagi, N. L153 Vamos, E. PO:08:40 Takao, T. L186 Van Assche, E. L49 Takata, H. L186, PO:06:08 van Daalen, S. L100 Takeyasu, K. L191, PO:08:80 van Dam, M. L154 Tani, A. PO:06:93 van der Kraan, I. L37 Tanic, N. L15 van Dongen, J. L174 Tanke, H. L138 van Driel, R. L37 Ta¨nzer, S. L60 van Drunen, E. L173 Tarkan-Argu¨den, Y. PO:08:51, PO:08:56, PO:08:57, van Erp, F. PO:06:36 PO:08:77 van Noort, J. L93 Tarui, H. L65 van Ravenswaaij, C. L133, L159 Taylor, M. L28 van Roy, N. L142 Taylor, S. L22 Van Steirteghem, A. L49 Tease, C. PO:08:29 van Zutven, L. L173, L174 Teixeira, T. L68 Vanderbyl, S. L119 Telenius, A. L119 Vandesompele, J. L142 Te´lez, M. PO:08:50, PO:08:52 Vankerckhove, S. L46 Teller, K. PO:06:71 Varma, N. PO:08:59 Tempest, H. L50 Veale, R. PO:08:55 Terauchi, A. PO:06:08 Veble, A. PO:08:12 Terriou, P. L102 Velthuizen, S. L173, L174 Tewes, A. PO:06:18 Veltman, J. L159 Thanananta, N. PO:08:78, PO:08:79 Venkataswamy, E. PO:08:10 Thirakhupt, K. PO:06:02 Venkatesh, N. H. PO:08:10 Thomas, A. PO:06:78 Verbeeck, J. PO:08:36 Thomas, S. L176 Verbrugge, S. L93 Thorogood, N. L97 Verdoliva, C. PO:06:04 Thorsson, A. PO:08:53 Verheyen, G. L49 Tibshirani, R. L169 Vermeesch, J. PO:08:36 Timme, S. PO:06:32 Verschure, P. L37 Tinelli, F. PO:08:76 Vershinin, A. L81, PO:06:56 Tjia, W. M. PO:08:63 Vetrie, D. L179 Tomilin, N. PO:06:63 Vidal, F. PO:08:23, PO:08:34 Tomizuka, K. L125 Vignal, A. L09 Tommerup, N. L181 Visser, A. L29 Torricelli, F. PO:06:35, PO:06:61 Vitkova, M. L105, PO:08:04, PO:08:13 Toure, A. L101 Voet, T. PO:08:36 Trakhtenbrot, L. L157 Vogel, J. L130 15th ICC: Author Index 181

Volkov, A. L95 Wojcierowski, J. PO:08:88, PO:08:89 Volkov, R. PO:06:47 Wolstenholme, J. L51 Voronin, A. PO:06:59 Wolvers-Tettero, I. L174 Vujosevic, M. L15 Woroniecka, R. PO:08:93 Vukomanovic, D. PO:08:39 Wright, A. L91 Vyskot, B. PO:08:05 Wright, W. L69 Writzl, K. L53, PO:08:12 Wade-Martins, R. PO:08:20 Wu, L. J. L94 Wakayama, T. L125 Wuite, G. PO:08:01 Wakefield, J. PO:08:03 Wako, T. L82 Xu, Z. L116 Walch, A. L146, PO:06:32 Waldor, M. PO:08:02 Walker, L. PO:06:65 Yamada, K. L65 PO:06:44 Wang, X. L92 Yang, F. L41 Warbuton, D. L155 Yasukochi, Y. L16, PO:08:14 Ward, D. L04 Yates, J. L19 Wasik-Szczepanek, E. PO:08:88, PO:08:89 Yilmaz, A. PO:08:51 Watanabe, N. PO:06:83 Yilmaz, S. PO:08:51, PO:08:56, PO:08:57, PO:08:77 Watson, S. L152 Yokokawa, M. L191 Wawrzkiewicz, A. PO:08:82 Yoneda, A. L83 Webb, A. L178 Yoshido, A. L16, L105, PO:08:14 Weber, B. PO:06:75 Yoshimura, S. H. L191 Wells, D. L47, L54, PO:06:34 Yoshino, T. PO:08:81 Wenke, T. PO:06:75 Yosunkaya-Fenerci, E. PO:08:77 Wennekes, J. L88 Young, B. L177 Werner, M. L146, L161, PO:06:32 Yuksel, A. PO:08:77 Western, P. L74 Whalley, K. L47 Zagaria, A. PO:08:84, PO:08:94 White, C. I. PO:06:58, PO:08:24 Wiczkowski, A. PO:08:82 Zajac, E. PO:06:16 Wiech, T. L146 Zastavna, D. PO:08:67 Wienberg, J. PO:06:01 Zeitlin, S. L25 Wiernik, P. PO:08:45 Zhai, X. L50 Willard, H. F. L05, L117 Zhdanova, N. PO:06:64 Willem, P. PO:08:03, PO:08:55 Zieler, H. PO:06:87 Willem Belot, P. PO:08:64 Zinner, R. PO:06:32, PO:06:69 Willemen, J. L159 Zitzelsberger, H. L146 Wilton, L. L52 Zorn, B. L53, PO:08:12 Winter, R. L133 Zou, Y. L69 Wise, D. L189 Zuccotti, M. L44 Witkowska, A. PO:08:93 Zuyderduyn, S. L139