Heredity 59 (1987) 151—160 The Genetical Society of Great Britain

THEGENETICAL SOCIETY

(Abstracts of papers presented at the TVVO HUNDRED AND FIFTH MEETING of the Society held on Friday, 14th and Saturday, 15th November 1986 at UNIVERSITY COLLEGE, LONDON)

1. Selection of somatic D. J. Porteous, P. A. Boyd, N. D. Hastie and hybrids with specific V. van Heyningen content for mapping the WAGR MAC Clinical and Population Unit, Western General Hospital, Crewe Road, syndrome Edinburgh EH4 2XU. J. M. Fletcher, H. Morrison, J. A. Fantes, Clonedprobes for a number of available chromo- A. Seawright, S. Christie, D. J. Porteous, some ii assigned were used to define the N. D. Hastie and V. van Heyningen extent of deletions associated with the Wilms' MAC Clinical and Population Cytogenetics Unit, tumour, aniridia, genitourinary abnormalities and Western General Hospital, Crewe Road, mental retardation (WAGR) syndrome. Establish- Edinburgh EH4 2XU. ing reliable dosage studies for a number of different probes has proved difficult. We have therefore WAGR(Wilms tumour, aniridia, genitourinary abnormalities and mental retardation) syndrome concentrated on segregating the deleted chromo- is frequently associated with deletions on the short some 11 from a number of patients in somatic cell arm of . The deletions vary in size hybrids and analysing DNA from these to produce but always include part of band lipl3. To home a consistent map of chromosome lip. At the same in on the Wilms tumour and aniridia loci the end time we have determined the deletion breakpoints points of the different deletion breakpoints need at a molecular level and shown that the results are to be defined at the DNA level. For unambiguous compatible with all the deletions studied having results the deleted and normal 11 arisen as a simple interstitial loss of a contiguous from a number of patients need to be segregated region of the chromosome. from each other, and analysed using as many cloned probes for the region as possible. The probes themselves can be selected from libraries produced from cell hybrids containing only appro- 3. priate parts of chromosome 11. The availability of Restriction fragment length species-specific monoclonal antibodies directed to polymorphisms (RFLPs) in the cell surface markers encoded by genes on chromo- region of the estrogen some ii has allowed us to use the fluorescence receptor activated cell sorter (FACS) to select for a large number of hybrid cells with the appropriate J. NeiIan,' P. Nestor,* L. Hanrahan,t chromosome content. These hybrid cells have been E. Connollyt and F. Gannon* invaluable tools for moving toward the Wilms *Department of Microbiology, University College, tumour and aniridia loci. Galway, Ireland. tDepartment of Pathology, Regional Hospital, Galway, Ireland. 2.Mapping of gene specific DNA Humanbreast cancer is a complex of neoplastic markers on chromosome 11 to diseases that vary in histological appearance and define the WAGR region biological behaviour such as potential for invasive- ness and metastasis. In approximately 40 per cent A. Seawright, J. M. Fletcher, of human breast cancers, a higher than normal 152 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS level of (ER) is present. The development of the majority of the cancers of this 5. Molecular genetic investigations type is blocked by treatment with antiestrogens in colorectal cancer thereby implying that high levels of ER are S. H. Rider, M. B. Davis, K. Tsioupra and necessary for this process. At present, there are no J. D. A. Deihanty explanations at the molecular level for the different levels of ER which are found in different tumours. Ga/ton Laboratory, Department of Genetics, As an initial step towards a greater understanding University College London, London WC1. of these mechanisms, we have initiated an analysis Investigationsare being carried out on a series of of the human ER gene . The experiments we 20 colorectal carcinomas and cell lines. Transfec- report consist of the digestion of panels of 10 tion of N1H3T3 cells with DNA from 17 tumour normal , 10 breast cancer tumour DNAs and samples and 3 cell lines has proved positive in 6 DNA from the cell lines MCF7 and T47D with cases; 3 have so far been shown to contain human the enzymes EcoRI, SstI, McpI or TagI, followed sequences and are being probed for 'ras' and other by Southern blotting and hybridization with radio- oncogenes. Approximately 10-fold amplification active probes (obtained from P. Chambon) which of K-ras sequences has been found in two tumours. correspond to the 5' or 3' portion of the ER. To Restriction enzyme digestion was employed to date, 4 RFLPs have been found in the EcoR I diges- detect changes at the 61st codon of K-ras; no tions, 2 with MspI, 2 with TaqI and one with SstI. positive results were found in 15 carcinomas. Although some of these were peculiar to tumour In order to identify the regions of the human samples, we have no evidence yet which correlates involved in the development of colorectal the RFLPs with an abnormal level of ER or with cancer, we are searching for loss of genetic markers tumour formation. in carcinoma samples by comparison with normal This work is supported by grants from the tissue from the same individual. Eleven tumours Cancer Research Advancement Board of Ireland were investigated using the 10 most polymorphic to F.G. and E.C. enzyme systems; 3 showed loss of gene expression. Changes are also being monitored by the use of polymorphic DNA probes; testing of 8 tumours with 5 probes has revealed loss of one restriction 4.Amplification of c-Ki-ras in fragment to date. testicular teratomas Results are being related to clinical staging where this is known. A. Jonas, M. B. Davis and J. D. A. Dethanty Ga/ton Laboratory, Department of Genetics, University College, London WC1. 6.Regional mapping studies on Teratomasare tumours composed of multiple tissues foreign to the part of the body, usually the gonads, where they occur. Testicular teratomas M. B. Davis,* M. R. Monteiro,* L. F. Westt originate from primordial germ cells. There is and B. WiIIiams evidence that they are characterised by cytogenetic * Department of Genetics and Biometry, and tMRC abnormality, particularly formation of one or Human Biochemical Genetics Unit, Ga/ton multiple copies of a putative l2p Laboratory, University College London. et Cancer Genet. timperial Cancer Research Fund Laboratories, (Delozier-Blanchet al., Lincoln's Inn Fields, London. Cytogenet., 15,375,1985; Atkin and Baker, Cancer Genet. Cytogenet., 10, 199, 1983). The c-Ki-ras Chromosome19 is currently the best mapped of oncogene has been mapped to chromosome 12p the smaller human chromosomes. More than 30 (Popescu et aL, Somatic Cell and Molecular Genet., distinct gene loci, including disease loci, red cell 11, 149, 1981). Southern blotting of genomic DNA enzymes, serum and blood groups have from a solid tumour and a tumour cell line detects been assigned, together with a number of anony- amplification of this sequence. By autoradiography mous DNA segments. A large linkage group, and scanning densitometry this is estimated to be spanning virtually the whole length of chromo- between five and eight fold. These data support some 19 has been defined and recently described the proposal that the small marker isochromosome (Shaw et a!., J. Med. Genet., 23, 2, 1986). Using observed in these teratomas is at least in part only family data, Sherman, Ball and Robson derived from chromosome l2p. (Ann. Hum.Genet., 49,181, 1985) suggested a THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 153 possible order LDLR-LE-C3-L W-PEPD-DM- This research was funded by the Medical [Se, HJ-APOC2-APOE-LU with LDLR the most Research Council. distal marker on the short arm. We have used a \'ariety of gene mapping methods to obtain a regional localisation for a 8.Linkage and dominant number of gene loci on chromosome 19. Human- spinocerebellar ataxia hamster somatic cell hybrids have been constructed using lymphocytes from individuals carrying bal- Vijayalaxmi,* A. E. Harding,t 0. Dumar,*, anced translocations involving chromosome 19, B. PentIand, N. MacDonald,* J. CIayton, with breakpoints at l9pl32, l9cen, 19q131 and S. Bhattacharya* and H. J. Evans* l9q133. The human chromosome content of these * MRC Clinical and Population Cytogenetics Unit, hybrids has been determined using biochemical Western General Hospital, Crewe Road, Edinburgh and DNA markers, and karyotyping by G and EH4 2XU. G-11 banding. A panel of hybrids has been tUniversity Department of Clinical Neurology, The obtained which contain the translocation products National Hospital, London. of chromosome 19 as the only region of chromo- Centre for , Sheffield. §Astley Ainslie Hospital, Edinburgh. some 19 present. ¶The Surgery, Tarbert, Argyll. Family studies using two point lod score analy- ses are also being carried out on families segregat- Autosomaldominant inheritance has been demon- ing for PEPD and other polymorphic markers on strated for a group of conditions referred to as chromosome 19. Data obtained so far have shown spinocerebellar ataxia (SCA). SCA however may PEPD to be closely linked to CYPJ in males, with comprise an heterogeneous group, in some of a maximum lod score of +269 at O=001. The which there is evidence for linkage with the major hybrid and family data combined have provided complex (HLA), on the short a revised map of chromosome 19. arm of . We are studying seven families with this disor- der and are using a range of recombinant DNA 7.Cell killing and mutagenesis probes from the short arm of chromosome 6 to detect linkage and to identify the sites of recombi- in wild-type and deoxycytidine nation in this region. Preliminary data suggest that kinase-deficient Friend the locus for the HLA linked subgroup of SCA leukaemia cells lies distal to the HLA locus. B. A. Boullier and P. G. McKenna Biomedical Sciences Research Centre, University of 9.Cystic fibrosis (CF) : an Ulster, Coleraine, Northern Ireland BT52 iSA. alternative approach to the basic Wild-type(clone 707) Friend leukaemia cells and defect in CF two deoxycytidine kinase-deficient (DCK) sub- clones were compared for sensitivity to cell killing J. R. Dorm, C. Hayward,* D. J. H. Brock,* and mutagenesis following exposure to ultra-violet M. Novak,t D. S. Sechert and irradiation (UV), ethyl methane sulphonate (EMS) V. van Heyningen and methyl methane suiphonate (MMS). Four UV *MRC Clinical and Population Cytogenetics Unit, doses were utilized; 12, 24, 36 and 48Jm2. In Human Genetics Unit, Western General Hospital, the case of chemical mutagens, three 16-hour doses Crewe Road, Edinburgh EH4 2XU. of EMS (100, 200 and 300 hour g ml) and four IMRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH. 16-hour doses of MMS (5,10,15 and 20 g mF1) were utilized. A clear dose-related response was CFantigen is a 12,000 Mwt found to be observed for both cell killing and mutagenesis (to elevated in the serum of CF patients and of clini- 6-thiguanine resistance) with each mutagen and cally normal obligate heterozygotes. The protein each cell type. There was no clear pattern of altered is synthesised in normal peripheral granulocytes mutagen-sensitivity in the DCK subclones rela- and in chronic myeloid leukaemia (CML) cells. tive to the wild-type cells. This implies that in this The gene for CF antigen has been assigned to cell line, DCK does not play a key role in those by analysis of somatic cell hybrids events significant for the normal functioning of capable of expressing this differentiated function DNA repair processes. (van Heyningen et aL, Nature, 315, 513, 1985). It 154 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS is not therefore the product of the aberrant CF T cell proliferation. Further genetic change is gene on . Knowledge of its identity required for transformation to full malignancy. and biological function may, however, give clues to the nature of the basic defect. We have produced monoclonal antibodies to CF antigen (Hayward 11.A cDNA clone coding for et a!., J. Immunol. Methods, 91, 117, 1986). These human sucrase-isomaltase have been used for immunopurification of the pro- tein. Partial amino acid sequencing permitted the F. R. Green,* V. H. Edwards,* H-P. Hauri,t synthesis of an oligonucleotide probe which was S. Povey,* M. W. Ho1 and D. M. Swallow* used to isolate a cDNA clone from a AgtlO CML *MRC Human Biochemical Genetics Unit, The cell cDNA library. Sequencing of this clone Galton Laboratory, University College London, revealed that CF antigen has strong homology with Wolfson House, 4, Stephenson Way, London NW1 2HE. intestinal and brain calcium binding proteins. The tBiozentrum der Universität Basel, Abteilung basic defect in CF is now thought to be at the level Pharmakologie, C. H. -4056 Basel, Switzerland. of control of chloride channel activity in exocrine -tBiology Discipline, Open University, Milton Keynes glands. Calcium binding proteins participate in MK7 6AA. signal transduction in such control pathways. Sucrase-isomaltaseis one of a family of enzymes found in jejunal microvillar membranes. The 10.Localisation of chromosome restricted tissue distribution of this enzyme has up to now precluded conventional genetic analysis by breakpoints in ataxia family studies or by use of somatic cell hybrids telangiectasia lymphocytes using and much of the biochemistry of sucrase-isomal- tase has been elucidated using animal models such in situ hybridisation as the rat and rabbit. In order to investigate the A. M. R. Taylor. S. V. Butterworth,R.Hollis, biochemical genetics of sucrase-isomaltase in man A. Heppell and A. A. Kennaugh we have made use of monoclonal antibodies both Department of Cancer Studies, Medical School, to study the protein in human post-mortem tissues Birmingham B15 2TJ. and to isolate a cDNA clone from a Agtll expression library. Variouschromosomal translocations have been The recombinant cDNA encodes about half described in peripheral lymphocytes from patients the human sucrase/isomaltase protein and has with ataxia telangiectasia often involving the sites been used in preliminary studies of the sucrase- of the immunoglobulin superfamily genes (Aurias isomaltase mRNA. It has also allowed the chromo- et a!., Hum. Genet., 72,210,1986). Clones of cells somal assignment of the human sucrase-isomaltase with some of these translocations may appear in gene and since it recognises more than one restric- the blood, most being very small. We have studied tion fragment length polymorphism in human two large non-leukaemic clones (70 per cent of T DNA more precise localisation by family studies cells) with translocations t(14; 14)(qll; q32) and should be possible. t(X; 14)(q28; qil) respectively. Using in situ hybridisation of probes on the t(14; 14) clone we have shown that the 14q32 break-point lies outside 12.Detection of 13-thalassemia the IgH locus and proximal to it with respect to mutations occurring in different the . The 14q11—l4qter segment of the DNA haplotypes in Greece by the homologous 14 carrying the constant gene region of the T cell receptor achainlocus is translocated use of synthetic DNA probes to this l4q32 site (Kennaugh et a!,, Hum. Genet., A. Athanassiadou, A. Papachatjopoulou. 73, 254, 1986). Tn the t(X; 14) the constant region I. Zarkadis and G. M. Maniatis of the a chain locus is translocated to Xq28 and although a reciprocal translocation is likely from Department of Biology, Medical School, University X to 14, the break-point appears distal to the site of Patras, Patras, Greece. of the RFLP St14 and to the 0-6-PD gene. Wehave recently determined the range of DNA Both clones therefore appear to involve translo- haplotypes occurring in a sample of 69 /3- cations of a single T cell receptor gene to the site thalassemia patients in S. W. Greece and estimated of an unidentified gene. Involvement of the achain their relative frequencies. /3-thalassemia mutations gene may be required for successful non-malignant have been shown (Orkin et a!., Nature, 296, 627, THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 155

1982) to be linked to specific DNA haplotypes and aberrations (McKenna and Yasseen, Leukem. Res., this finding has been used in the prenatal diagnosis 9, 501, 1985). of /3-thalassemia by restriction enzyme analysis of Wild-type Friend leukaemia (clone 707) cells fetal DNA. In Greece the majority of the j3- and two thymidine kinase-deficient subclones thalassemia mutations are single base substitutions 7O7BUE and 7O7BUF, having thymidine kinase which are not directly detectable by cloned DNA activities of 14 and 07 per cent that of wild-type probes. We report here the results we have obtained cells respectively (McKenna and McKelvey, on the delineation of the /3-thalassemia mutations Somatic Cell Mo!. Genet., 12, 325, 1986), were and haplotypes occurring in our sample of patients, compared for sensitivity to killing and the induc- by the use of synthetic DNA probes. Four synthetic tion of cytogenetic aberrations following gamma- oligoprobes (nonadecamers) were used each of irradiation. Three doses of gamma-irradiation were them detecting one of the following mutations: (1) used; 150, 300, and 450 cGys, and cells were har- f3 IVS-1 110, (2) po..39 nonsense mutation, (3) vested for metaphase spreads after 4, 8, 12, 15, 29 IVS-1 1(G-A) frame-shift (/3°) and (4) IVS-1 6(p). and 43 hours. Increased sensitivity to the induction (Oligoprobe hybridisations were carried out as of cytogenetic aberrations for all three doses of described by Pirastu M. et a!., N. Eng. J. Med., gamma-irradiation used was apparent in the two 309, 284, 1983). We have checked in total 52 /3- thymidine kinase-deficient subclones at 15, 29 and thalassemia chromosomes of haplotypes I, II, V, 43 hours. Considerably increased sensitivity to VI and IX and it was found that haplotype I carries gamma-radiation-induced cell killing was also in 81 per cent of the cases the mutation /3-110; observed in the two thymidine kinase-deficient haplotypes II and IX carry mutation /3°-39 in 85 subclones relative to wild-type cells. Subclone per cent of the cases; haplotypes V and VI were 7O7BUE, having twice the thymidine kinase 100 per cent linked to mutations IVs-1 1 and IVS-1 activity of subclone 7O7BUF, consistently 6 respectively. These five haplotypes represent the exhibited greater resistance to gamma-irradiation 89 per cent of all haplotypes detected in 138 /3 than did the lower thymidine kinase activity sub- thalassemia chromosomes analysed in a previous clone 7O7BUF. study. It is apparent that detection of the corre- In the light of these and earlier reported results sponding mutations with synthetic DNA probes is the significance of thymidine kinase for accurate of particular importance for the prenatal diagnosis DNA repair is discussed. of the condition in Greece. Our results also con- tribute to the basis for offering first trimester pre- 14. natal diagnosis to childless couples, something Redefining the components of that is not possible by haplotype linkage and gene conversion analysis. S. A. Zwolinski and B. c. Lamb 13.Increased sensitivity to cell Department of Pure and Applied Biology, Imperial College, London SW7 2AZ killing and cytogenetic aberrations Inthe past it has been possible to express gene in thymidine kinase-deficient conversion in terms of an algebraic model where Friend leukaemia cells following the event is broken down into its component gamma - radiation parts such as y, the probability of hybrid DNA forming between two non-sister V. J. McKelvey, P. G. McKenna and (Kalogeropoulos and Thuriaux, Genet. Res. T. L. Frew Camb., 40,1,1982). However in the absence of Biomedical Sciences Research Centre, University of outside markers it is not possible to test the model Ulster, Colerain, BT52 iSA, Northern Ireland and because of the lack of degrees of freedom. Northern Ireland Radiotherapy Centre, Belvoir Park In an attempt to increase the number of recom- Hospital, Belfast. binant types and therefore to provide the degrees Deficiencyof thymidine kinase in Friend of freedom necessary, some component variables leukaemia cells leads to increased sensitivity to were redefined and the model extended to allow cell killing and mutagenesis by UV-irradiation the analysis of chromatids) so that the wider ratio (McKenna and Hickey, CelL BioL mt. Reps., 5, octads could be included when testing the model. 555, 1981), chemical mutagens (McKenna and Ascobolus immersus seems au ideal candidate to Yasseen, Genet.Res.,40,207,1982) and enhances test the new model because of the ability geneti- the frequency of mitomycin C-induced cytogenetic cally to alter the frequency and spectrum of gene 156 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS conversion at the locus wI coding for ascospore Some of the methods used to distinguish colour (Helmi and Lamb, Genetics, 104, 23, 1983). restrictions from substitutions are very dependent For nearly all the data tested an acceptable good- on the validity of various assumptions. These ness of fit was discovered, suggesting that the assumptions will be discussed and relevant data mixed (single and dual hybrid ) model (Zwolinski and Lamb, unpublished) from of gene conversion was sound. Ascobolus on their validity will be presented. From these results it is evident that the proba- Implications for recombination models will be dis- bility (s) of a certain specific hybrid being cussed. repaired to wild type varies with the type of genetic factors (ccfs) present. This variation of s appears 16.Prospect of recombinant DNA to be independent of other components showing technology in human genetics that some of the simplifications made by previous workers in testing the model were inapplicable. It A. E. H. Emery may be significant in the search for the mechanism Medical School, Teviot Place, Edinburgh EH8 9A G. of recombination that the value of 6 tends to one Thefirst application of DNA technology in human or zero indicating large differences in frequency genetics was in the prenatal diagnosis of i- of invasion by strands of opposite polarity (5'3' thalassaemiain 1976, and the following year saw versus 3'5') of a double helix. the first biosynthesis of a human protein (somato- statin) and the establishment of the first genetic engineering company to develop recombinant 15.Restorations and substitutions DNA methods for making medically important in correction of mispairs in hybrid drugs (Genentech Inc. in the United States). DNA during recombination In 1978 a close linkage between a disease- producing-gene (in this case sickle cell anaemia) B. C. Lamb and S. A. Zwolinski and a restriction fragment length polymorphism Department of Pure and Applied Biology, Imperial (RFLP) was demonstrated, and this approach to College, London SW7 2BB. the detection of carriers of X-linked disorders and the preclinical and prenatal diagnosis of genetic Inmost current models, part of the recombination diseases has been widely exploited since. process involves the replacement of part of one Four years ago successes in characterizing cer- strand of a DNA double helix by a corresponding part of one strand from a homologous but non- tain human cancer genes (oncogenes) were pub- lished, and finally, and very recently, gene therapy sister chromatid. Sites of heterozygosity in the is attracting serious consideration. These various heteroduplex give mispaired or unpaired bases. developments auger well for the future of recom- Any enzymic correction of a mispair or non-pair binant DNA in human genetics. will either be a restoration to the original base pair present on the invaded chromatid, or a substitu- tion, with correction to the base pair of the 17.The molecular pathology of chromatid from which the invading strand came. single gene disorders Different conversion classes may have different origins, with respect to production from restor- D. J. Weatherall ations and/or substitutions, depending partly on MRC Molecular Haematology Unit, Nuffield whether they come from asymmetric or symmetric Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford. hybrid DNA, or from double-strand gap repair. Kitani and Olive (Genetics, 62, 23, 1969), using Cloningand sequencing of mutant genes from heteroallelic crosses in Sordaria fimicola, found patients with single gene disorders is providing a that restrictions equalled or exceeded (often sig- wealth of information about the molecular nificantly) substitutions, with the amount of excess pathology of these conditions. So far, most infor- being allele-specific. Hastings (Cold Spring Harb. mation has been derived from the inherited Symp. Quant. BioL XLIX, 49, 1984 and other haemoglobin disorders, particularly thalassaemia. papers) found that restorations and substitutions Apart from the obvious point mutations which can were equally frequent for alleles E1/E2 at the b2 give rise to premature stop codons or frameshifts locus of Ascobolus immersus, but that in yeast a number of more subtle and interesting lesions there were more substitutions than restorations for have been found, There are several mutations in all six alleles studied at the hisl locus. the conserved regulatory boxed 5' to the human THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 157

globin genes, a whole cluster of point mutations sequence divergence is between 2 and 4 per cent involving intron/exon junctions or within introns per million years and that all the lineages in this and exons which interfere with normal splicing, tree trace back to one woman, who lived 150 to and another family of mutations which involve the 300 thousand years ago, probably in Africa. poly A addition site. In addition there are a large Evidence from diverse studies of nuclear DNA family of deletion disorders at least some of which agrees with the idea of an African origin for have important relevance to the regulation of the anatomically modern Homo sapiens. globin gene cluster. Indeed, work carried out over Nonmolecular evidence obtained by studying the last few years in the thalassaemia field has the remains of ancient bones and tools can also probably given us a good idea of the total repertoire be fitted with this idea. The molecular and fossil of human gene mutations. data together warrant the suggestion that a big- As other genes are cloned a similar pattern is brained population emerged from Africa within emerging. For example the factor VIII genes of the past 200,000 years. The mitochondrial DNA patients with haemophilia are showing a remark- results indicate that there was no mixing of this able diversity of defects and similar heterogeneity population with the Homo erectus populations rep- is turning up in most genetic diseases. resented in Asia by such famous fossils as Java Finally, the haemoglobin disorders have pro- Man and Peking Man seem not to have contributed vided a new and recently described family of muta- any mitochondrial DNA lineages to the present- tions which seem to be involved directly with day gene pool of our species. changes in the developmental pattern of globin gene expression. This group of conditions is pro- 19. Oncogenes viding extremely valuable information about their potential sites of protein! DNA interaction which K. Silora may be involved in the activation and suppression Department of Clinical Oncology, Royal of genes at different developmental stages. Postgraduate Medical School, Hammersmith This talk will review each of these classes of Hospital, DuCane Road, London W12 OHS. mutations and describe their relevance in under- The central problem in cancer therapy is the poor standing gene regulation. selectivity of current systemic agents against the common solid tumours. The demonstration that unique segments of DNA, constant in location and 18. Human evolution conserved in evolution are involved in growth con- A. Wilson trol, opens new avenues for basic and clinical research. The functions of the products of these Department of Biochemistry, University of genes needs to be elucidated. Examples of growth California, Berkeley, California, U.S.A. control functions include homology to growth fac- My talk will deal with the evolutionary insights tors, surface receptors, protein kinases and cell emerging from molecular comparisons of human cycle control proteins. From DNA sequence data beings with one another. The evidence on which peptides predicted to be exposed within intact I shall centre attention comes from mitochondrial molecules can be constructed and used to produce DNA. Every child gets his or her mitochondrial monoclonal antibodies to oncogene products. DNA from the mother exclusively. This simple Such antibodies have now been successfully used mode of inheritance makes mitochondrial DNA a to demonstrate the intracellular localisation of marvellous genealogical tool. A second notable gene products as well as the cell cycle regulatory feature of this molecule is its high rate of evolution. role of the c-myc protein. By having a battery of Mutations accumulate unusually fast in this kind antibodies against the different gene products, their of DNA. So, it gives a magnified view of our direct clinical application for diagnosis and prog- evolutionary history. nosis has become a reality. Immunohistology and With the aid of restriction enzymes my flow cytometry permit the geographical and quan- coworkers have compared the mitochondrial titative analysis of function in normal and neoplas- DNAs of many people around the world and built tic tissues. Furthermore, by purification and bio- a genealogical tree. Several points of branching in chemical analysis the molecular basis for their this tree have been associated with the times at action can be elucidated. It is likely that by the which human beings first colonised Australia, New end of the decade, new drugs that inhibit Guinea and the New World. These calibration oncoprotein function will be available for clinical points agree in implying that the average rate of trial. 158 THE GENETICAL SOCIETYABSTRACTS OF PAPERS 20. Molecular characterisation of muscular dystrophy locus were isolated from numerous laboratories. Southern analysis of human chromosome aberrations Lesch-Nyhan and Duchenne muscular dystrophy M. A. Ferguson-Smith males indicate that a significant percentage of cases occur on the basis of de novo deletional events (15 Duncan Guthrie Institute of Medical Genetics, per cent Lesch-Nyhan and 8 per cent Duchenne University of Glasgow, Glasgow, Scotland. muscular dystrophy). The majority of Lesch- Nyhan males (85percent) have no major gene Identificationof gross chromosome aberrations is rearrangements and have mRNA of normal based on an assessment of chromosome length and molecular weight and size. We have modified the banding patterns with the light microscope. RNAse method of Myers and Maniatis for deletion Defects involving the equivalent of less than 10 of point mutations. In 7 of 14 Lesch-Nyhan males million base pairs are not easily resolved and most studied, a unique RNAse A cleavage site was of these will be missed in screening for human identified. chromosome abnormalities. Two methods hold In our study of over 60 Lesch-Nyhan families, promise for improving the detection and charac- all mutations thus far identified are unique. The terisation of small aberrations. DNA measurement current estimates for the Duchenne muscular by flow cytometry may sometimes help to resolve duplications and deficiencies between I million dystrophy gene are large (5-10) and its putative transcript and organisation incomplete. Neverthe- and 10 million bp. However, family studies are less, the RFLP linkage study of new mutation required to distinguish aberrations from normal families has been successful in the identification chromosome variation in the flow . The development of chromosome specific DNA of the gametic origin of new mutation in families. markers allows the detailed analysis by in situ Only deletional gametic events at Duchenne mus- cular dystrophy and Lesch-Nyhan are comparable hybridisation and Southern blotting of pre- since the Duchenne muscular dystrophy gene is sumptive chromosome deletions and duplications and the definition of their breakpoints in terms of incompletely characterised. The carrier and prenatal diagnostic experience the chromosome map. The extent by which these of recombinant DNA methods for the disorders methods can improve the resolution of chromo- of Lesch-Nyhan, Duchenne muscular dystrophy, some analysis will be illustrated in a series of and cystic fibrosis (Dr A. L. Beaudet from the structural abnormalities of the human X and Y lnstitute of Molecular Genetics) will be reviewed. chromosomes. This experience provides guidelines for recom- binant DNA application to heritable disease diag- nosis and its relative accuracy. 21.Recombinant DNA diagnosis of new mutation diseases— 22.Molecular genetics applied to Lesch-Nyham and Duchenne the development of vaccines muscular dystrophy K.Murray C. Thomas Caskey Department of Molecular Biology, University of Howard Hughes Medical Institute, Institute for Edinburgh, Edinburgh EH9 3JR. Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030, U.S.A. Viralvaccines have traditionally been based upon attenuated or killed strains of the virus. Modern Geneticlethal diseases were predicted by Haldane methods of molecular biology enable viral to occur frequently on the basis of new mutational to be produced in microbial or other cells for events. Two X-linked lethal disorders have been development as subunit vaccines and, in principle, studied in our laboratory by molecular genetic can also be used to make attenuated strains. methods. For Lesch-Nyhan syndrome the gene Hepatitis B virus antigens made by recombinant structurefor hypoxanthine guanine phos- DNA methods have been formulated successfully phoryltransferase EC 2.4.2.8 was determined in into vaccines and have provided useful diagnostic ourlaboratory.For Duchenne muscular reagents. Recent results show that internal viral dystrophy, anonymous junctional and deletion antigens, as well as coat components, have poten- molecular probes which flank the Xp2 1 Duchenne tial for vaccination purposes. THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS 159 23. Prospects for human gene of unknown function, identified by monoclonal antibodies also map to chromosome 19. therapy We have assembled a panel of rodent human W. French Anderson somatic cell hybrids which provide a comprehen- National Institutes of Health, Bethesda, Maryland, sive breakdown of this chromosome. These lines 20892 U.S.A. have been used to order many of the markers and along with multipoint linkage analysis to establish Humangene therapy is defined as the correction the position of the myotonic dystrophy (DM) gene. of a genetic disease by the insertion of a functional Some of available methods for isolating the DM gene into the patient's defective cells. The likely gene, given its precise localisation relative to other initial disease candidate for this new therapeutic genes, will be discussed. approachisadenosine deaminase (ADA) deficiency, a cause of severe combined immuno- deficiency (a profound genetic disorder which 26.Karyotyping of various often results in death before the age of two). Pro- yeasts by field inversion gress towards gene therapy is proceeding along gel etectrophoresis two pathways: scientific and sociolethical. A num- ber of laboratories have been able to use retroviral J. H. Johnston,* C. R. Contopoulou,t vectors to insert a functional gene into the H. K. Mortimert hematopoietic cells of mice. We have now suc- *Department of Bioscience and Biotechnology, ceeded in transferring the human ADA cDNA into University of Strathclyde, Glasgow. the hematopoietic cells of nonhuman primates tDepartment of Biophysics/Medical Physics, using an autologous bone marrow transplanta- University of California, Berkeley, Ca 94720, U.S.A. tion/gene transfer protocol. In the most positive Wehave shown by use of OFAGE that many monkey, approximately 1 cell in 200 of the laboratory and industrial strains of Saccharomyces hematopoietic mononuclear cells appears to be (spp. cerevisiae, uvarum, caribergenesis, bayanus) carrying and expressing the human ADA gene. In produce generally similar patterns of chromosomal addition, we have demonstrated the feasibility of DNA (12-17 bands, the majority of which are gene transfer in utero using fetal lambs. On the 100 kb approximately) (Johnston & Mortimer, mt. socioethical front, mechanisms are now in place J. Syst. Bact., 36,569,1986). However, because of for evaluating in a public forum human gene chromosome polymorphism, most of these strains therapy proposals. In addition, Points to Consider can be identified by specific OFAGE . (i.e., "guidelines") have been published by the U.S. We have also shown that strains of Saccharomyces Government to inform investigators what scientific kluyveri, Candida albicans,Candidautilis, and ethical criteria need to have been met at the Kluyveromyces lactis, Pichia (Hansenula) time a clinical protocol is submitted. canadensis, Saccharomycopsis fibuligeraand Schwanniomyces occidentalis produce only a few 24. slowly-migrating bands, indicating that these Professor D. A. Hopwood yeasts possess a smaller number of larger chromo- somes (1000 kb). We speculate that "domesti- John Innes Institute, Colney Lane, Norwich NR4 2UH. cated" species of Saccharomyces may have evolved from a progenitor wild species, such as S. kluyveri, by selection for sugar fermentation genes, e.g. 25. Human chromosome 19 and MAL, SUC, and their consequent transposition the myotonic dystrophy gene along with telomeric sequences. Similar results for a wide range of yeasts have recently been obtained J. D. Brook, D. J. Shaw, H. Hartley, by Jonge et a!. (Yeast, 2, 193, 1986). M. Sarfarazi and P. S. Harper The demonstration that, under certain condi- Section of Medical Genetics, University of Wales tions, the largest chromosomes of S. cerevisiae College of Medicine, Heath Park, Cardiff CF4 4XN. migrate at a faster rate than medium-sized chromo- Humanchromosome 19 is currently the most fully somes during FIGE (Carle, Frank and Olson, mapped of the smaller chromosomes, with over 40 Science, 232, 65, 1986) suggests an improved loci assigned to it. cDNAs are available for genes method for examination of the large chromosomes and at least 10 random DNA segments have been of S. kluyveri and other yeasts. We have shown cloned. Four genes encoding cell surface antigens this to be the case and obtained greater resolution 160 THE GENETICAL SOCIETY—ABSTRACTS OF PAPERS of chromosomal bands of these yeasts. Our current University of Belfast, 97, Lisburn Road, estimates are that most of these yeasts probably Belfast BT9 3BL, N. Ireland, U.K. possess 4-6 chromosomes, and Saccharomycopsis HumanDNA isolated from a genomic library probably only 2 or 3. We have also shown that the enriched for sequences was 3 large chromosomes of Schizosaccharomyces digested with Sau3A, subcloned into pUC12 and pombe, estimated to contain between 3 to 6 Mb used to transform E. coil JM83 cells. On screening DNA, migrate at faster rates than chromosomes the resulting transformants with a trisomy 21 foetal estimated at 2-25 Mb under particular FIGE con- liver cDNA probe, several positively hybridising ditions. Resolution of the 3 chromosomal bands recombinants were detected. A comparison of the of S. pombe has been obtained with shorter run expression of 6 of these human chromosome 21 times of 10-20 hours. DNA sequences in normal and Down Syndrome We have probed Southern blots of FIGE gels fibroblast and foetal liver samples and performed and identified chromosomes III (LEU2), IV by dot blot analyses followed by densitometer (TRP1), V (URA3,RAD24), VI (TUB2) and poss- studies. The results of these comparisons have ibly XII (SIR3) of S. cerevisiae. Under conditions revealed one sequence which is expressed in Down of high stringency, no hybridisation was observed Syndrome foetal liver but not in normal foetal liver between TRP1, RAD24, URA3, TUB2, and at an equivalent stage development, one sequence chromosomal bands of several other yeasts. which is expressed more than ten times as much However, with reduced stringency, homology with in Down Syndrome tissues and cell lines as in the S. cerevisiae probe pRB 129 carrying URA3 normal tissues and cell lines and one sequence and TUB3 has been observed, generally involving which shows no difference in expression levels in 2 FlOE bands of these yeasts. the trisomy state. Other sequences show a 3 :2 ratio The resolution of these larger chromosomes of expression between the Down Syndrome and suggests the application of FlOE to karyotyping the normal samples. These results suggest that the other eukaryotic microbes, such as filamentous multiple phenotypic effects observed in trisomy 21 fungi and protozoa and to separating large DNA subjects may be due to changes in the develop- fragements of chromosomes of higher eukaryotes. mental regulation of transcription, loss of tissue- specific gene expression and widely varying dosage 27.The expression of effects of different DNA sequences. Although it is widely believed that the phenotypic expression of chromosome 21 specific Down Syndrome is due to a 15-fold increase in sequences in normal and Down the expression of most, if not all, chromosone 21 Syndrome cell lines and tissues specific sequences, this study indicates the need to examine the expression of individual sequences in L. A. J. Stefani, A. V. Palmer and N. c. Nevin different tissues prior to considering their role in Department of Medical Genetics, The Queen's the pathogenesis of Down Syndrome.