Use of irradiation hybrids in gene mapping on human chromosome 11
Godfrey Tregelles Gillett
October 1998
A thesis submitted for the degree of Doctor of Philosophy at the University of London
The Galton Laboratory MRC Human Biochemical Genetics Unit Department of Biology, University College London ProQuest Number: 10609004
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The original objective of the work described in this thesis was the development of genetic and physical mapping resources on human chromosome llq23 in order to facilitate the positional cloning of a putative locus for Tuberous Sclerosis in that region of the chromosome. To this end I constructed a panel of high dose irradiation hybrids using as a starting point a human - hamster somatic cell hybrid retaining chromosome 11 as its only human material.
Forty-seven of the hybrids were characterised in detail by molecular methods and also in some cases by fluorescent in situ hybridisation. A promising hybrid (Jol2) was subcloned further to derive a hybrid containing only llq23 as its human component. By this time it had become clear that most cases of Tuberous Sclerosis can be accounted for either by mutations at TSC1 (9q34) or TSC2 (16pl3) and that a locus on chromosome 11, if it exists, must account for very few cases of the disease, making an attempt at positional cloning impractical.
The hybrids were therefore used in collaboration with others for the development of region specific probes, some of which were directly useful in the mapping and ultimately the cloning of genes causing ataxia telangiectasia (llq23) and multiple endocrine neoplasia type 1 (llql3). In particular I was able to show that Alu -PCR products from well - characterised hybrids could be used in a rapid and reliable way to obtain regionally defined cosmid clones from the gridded chromosome 11 library available from ICRF.
In the course of this work I made use of these and other hybrids to provide new chromosomal localisations for several other genes including those coding for cofilins (CFL1 and CFL2), a fucosyl transferase (FUT4), a retinoic receptor (RXRB) and a mitochondrial NAD - dependent malic enzyme.
Abstract 2 Table of contents
Abstract 2
Table of contents 3
List of figures 7
List of tables 10
Abbreviations 12
Contributors 13
Acknowledgements 14
C h a p te r 1: Introduction 15
Clinical aspects of tuberous sclerosis 16
Positional cloning 21
Structural abnormalities 22
Meiotic mapping methods: linkage analysis 25
Physical methods: isolation of chromosomes or chromosome fragments 31
Physical methods: localising the chromosome fragments - FISH 42
Isolation and identification of transcribed sequences 46
Positional-candidate cloning strategies 53
Analysis of candidate genes 57
Positional cloning of TSC 59
C h a p te r 2: Materials and Methods 73
Materials 73
Methods 75
Table of contents 3 Chapter 3.1: Radiation hybrids derived from a chromosome 11-only parent 93
Introduction 93
Results 94
Discussion 100
Chapter 3.2: Isolation of a hybrid containing llq23 only, J12.1A 111
Introduction 111
Results 111
Analysis of subcloned hybrids 111
Analysis by Southern hybridisation and PCR 111
Analysis by FISH 112
Isolation of cosmids from regions of human chromosome 11 contained in selected hybrids 114
Discussion 116
Chapter 3.3: Isolation of hybrids containing human chromosome llql3 127
Introduction 127
Results 129
Discussion 130
Chapter 3.4: The identification of genomic cosmids from the MEN1 critical region, using hybrid Jo2' 134
Introduction 134
Methods 135
Results 136
Discussion 138
Table of contents 4 Chapter 3.5: Localisation of a simple sequence repeat polymorphism, D11S614 144
Introduction 144
Results 145
Discussion 148
Chapter 3.6: Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 152
Introduction 152
Methods 162
Results 166
Discussion 167
C h a p t e r 3.7: Use of resources generated: mapping of FUT4 207
Introduction 207
Methods 210
Results 211
Discussion 213
C h a p t e r 3.8: Resolution of chromosome 11 - related mapping issues: Mitochondrial NAD+ -dependent malic enzyme 216
Introduction 216
Methods 218
Results 219
Discussion 220
Table of contents 5 C h a p t e r 3.9: Resolution of chromosome 11 - related mapping issues: assignment of RXRB to chromosome 6p21.3 228
Introduction 228
Methods 232
Results 234
Discussion 235
C h a p t e r 4: Discussion 241
Bibliography 257
Appendix 1: 297
Diagnostic criteria for TSC 297
Appendix 2: 305
Exploitation of selected irradiation hybrids to isolate markers in the regionof the AT locus 305
Appendix 3: 310
Standard solutions , buffers, media andprimer sequences 310
Standard solutions and buffers 310
Bacterial cell culture 311
Mammalian cell culture 312
Solutions for FISH 314
Primer sequences 315
Publications arising from this thesis 318
Envoi 319
Table of contents 6 List of figures
1.1 Techniques involved in positional cloning 23
1.2 A cartoon of hybrid irradiation - fusion gene transfer 35
3.1.1 Lactate dehydrogenase (LDH) starch gel electrophoresis 106
3.1.2 Adenosine deaminase (ADA) starch gel electrophoresis 107
3.1.3 Use of TYR4 amplification and MspI digestion of product to score radiation hybrids for TYR and TYRL 108
3.1.4 Alu-PCR of selected irradiation hybrids 109
3.1.5 (a, b, d) FISH of hybrid Jol2 using total human DNA as probe 110
3.1.5 (c) FISH of human metaphase chromosomes using Alu-PCR products from hybrid Jol2 as probe 110
3.2.1 Alu-PCR of irradiation hybrid Jol2 subclones 122
3.2.2(a-d) FISH of hybrid subclones J12.1A using biotinylated total human DNA as probe 123
3.2.3(a-d) FISH of hybrid subclones J12.1A and J12.1B using biotinylated total human DNA as probe 124
3.2.4(a-d) FISH of hybrid subclones J12.4 and J12.5 using biotinylated total human DNA as probe 125
3.2.5(a-d) FISH of hybrid subclones J12.7 and J12.9 using biotinylated total human DNA as probe 126
3.3.1(a, b) FISH of human metaphase preparations probed with biotinylated Alu-PCR product from radiation hybrids (a) Jo2', and (b) Jo48' 133
3.4.1 Autoradiograph of genomic cosmid filter ICRF cl07 (set 12) probed with 32P-oligo-labelled AZw-PCR product from hybrid Jo2' 143
3.5.1 FISH of metaphase chromosomes from cell line GM6229A using biotinylated 8D11 cosmid DNA as probe 151
List of figures 7 3.6.1 Nucleotide and predicted aminoacid sequence of human promyelocyte (non-muscle) cofilin cDNA, CFL1 175
3.6.2 CFL1 5' probe, Staden proportional algorithm comparison 176
3.6.3 CFL1 3' probe, Staden comparison 177
3.6.4 Cofilin NM-type, Staden comparison 178
3.6.5 Cofilin NM-type 3'UTS, Staden comparison 179
3.6.6 Cofilin M-type & NM-type, Staden comparison. 180
3.6.7 Cofilin NM-type, Staden comparison 181
3.6.8 Cofilin M-type & NM-type, Staden comparison 182
3.6.9 Cofilin M-type 3'UTS, Staden comparison 183
3.6.10 Cofilin NM-type & M-type, Staden comparison 184
3.6.11 Cofilin NM-type & M-type 3'UTS, Staden comparison 185
3.6.12a Cofilin NM-type 3'UTS sequence alignment, GCG "Lineup" 186
3.6.12b Cofilin NM-type 3'UTS sequence alignment, Mammalian cDNAs, GCG "Lineup" 187
3.6.13 Cofilin NM-type 3'UTS, Staden comparison 188
3.6.14a Cofilin NM-type 3'UTS sequence alignment, Human cDNAs, GCG "Lineup" 189
3.6.14b Cofilin NM-type 3'UTS sequence alignment, Human cDNAs, GCG "Lineup" 190
3.6.14c Cofilin NM-type 3'UTS sequence alignment, Human cDNAs, GCG "Lineup" 191
3.6.14d Cofilin NM-type 3'UTS sequence alignment, Human cDNAs, GCG "Lineup" 192
3.6.15a PCR amplification of CFL1 in human, rodent and somatic cell hybrid DNA 193
3.6.15b PCR amplification of CFL1 and SEA in cosmid glycerol stocks from the ICRF human chromosome 11 library, number 107 (L4/FS11) 193
List of figures 8 3.6.15c PCR amplification of CFL1 in human, rodent and somatic cell hybrid DNA (chromosome 11 mapping panel) 195
3.6.15d Autoradiograph of genomic cosmid filter ICRF cl07 (set 12) probed with 32P-oligo-labelled CFL1 product 196
3.6.16 Human metaphase chromosomes showing FISH of biotinylated cofilin cosmid probe localised to chromosome llql3 197
3.6.17 Alignment of predicted aminoacid sequences of M- and NM-type Cofilins. 198
3.6.18a Cofilin M-type 3'UTS sequence alignment, GCG "Lineup" 199
3.6.18b Cofilin M-type 3'UTS sequence alignment, GCG "Lineup" 200
3.6.18c Cofilin M-type 3'UTS sequence alignment, GCG "Lineup" 201
3.6.19 PCR amplification of CFL2 in human, rodent and somatic cell hybrid DNA 202
3.6.20 Cofilin M-type & Gamma-Actin, Staden comparison 205
3.6.21 Cofilin M-type & Beta-Actin, Staden comparison 206
3.7.1 Human metaphase chromosomes showing FISH of biotinylated FUT4 cosmid probe localised to chromosome Ilql4-q22 215
3.9.1 PCR of RXRB in human , rodent and somatic cell hybrid DNA, 131 bp product 239
3.9.2 PCR of RXRB in human, hamster and somatic cell hybrid DNA, 141 bp product 239
3.9.3 Human metaphase chromosomes showing FISH of biotinylated RXRB cosmid probe localised to chromosome 6p21.3 240
List of figures 9 List of tables
1.1 Inherited disease genes identified by positional cloning 24
1.2 Representative genes isolated by exon trapping 48
1.3 Representative inherited disease genes identified by positional candidate methods 54
3.1.1 Markers in those Jo series chromosome 11 radiation hybrids positive for at least one marker 105
3.1.2 Scoring of hybrids for TYR and TYRL using the TYR4 PCR plus MspI digestion and the TYR1 PCR 97
3.2.1 Markers in Jol2 radiation hybrid subclones 121
3.2.2 Human chromosome content of Jo 12 subclones, J12.1B-J12.9 113
3.2.3 Human chromosome content of the Jo 12 hybrid subclone, J12.1A 113
3.2.4 Localisation by FISH of ICRF cl07 cosmids which hybridise to Alu-PCR products from J12.1A and/or Jo48' 115
3.3.1 Markers in the "llql3 subset panel" of radiation hybrids 132
3.4.1 ICRFcl07 cosmids which hybridise to labelled A/w-PCR product from Jo2' 137
3.5.1 D11S614 and other llq markers in hybrid lines containing translocations in llq 150
3.5.2 Pairwise LOD scores for D11S614 v.s. four llq23 markers, in 35 families 147
3.5.3 LOD scores for TSC v.s. D11S614, in 26 families 147
3.6.1 Segregation of CFL1 in a somatic cell hybrid mapping panel 194
3.6.2 Segregation of CFL1 in a hum an chromosome 11 mapping panel 195
3.6.3(a, b) Segregation of CFL2 in a somatic cell hybrid mapping panel 203-4
List of tables 10 3.7.1 Segregation of FUT4 in a hum an chromosome 11 somatic cell hybrid mapping panel 211
3.8.1 Segregation of ME3-3'89 in a somatic cell hybrid mapping panel: the initial ME3 mapping study 223
3.8.2(a, b) Segregation of ME3-3'89 in a somatic cell hybrid mapping panel 224-5
3.8.3(a, b) Segregation of ME3-3'89 in a somatic cell hybrid mapping panel 226-7
3.9.1 Segregation of RXRB in a somatic cell hybrid mapping panel 238
3.9.2 Segregation of RXRB in a human chromosome 6 mapping panel 234
4.1 Type of mutation seen in patients with TSC1 and TSC2 252
4.2 Examples of articles identifying 5'UTS or 3’UTS homology 257
A. 1.1 Diagnostic criteria for TSC 298-9
List of tables 11 Abbreviations
A adenine or adenosine M Mol BAC bacterial artificial h micro chrom osom e Pg m icrogram bp base pairs pi m icrolitre C cytosine or cytidine pM m icrom olar °c degrees Centigrade Mb megabase pairs cDNA complimentary DNA MRI magnetic resonance cM centiMorgan imaging CT computerised (axial) Mspl restriction enzyme tomography cutting at C / CGG DAPI 4,6-diamidino-2- MTT tetrazolium salts, phenylindone thiazolyl blue dATP deoxyadenosine NAD nicotinamide adenine triphosphate dinucleotide dCTP deoxycytosine PAC Pl-derived artificial triphosphate chrom osom e ddH20 deionised and distilled PBS phosphate buffered water saline dGTP deoxyguanosine PCR polymerase chain triphosphate reaction DMEM Dulbecco's modified PEG polyethylene glycol Eagles medium PFGE pulsed field gel DMSO dimethylsulphoxide electrophoresis DNA deoxyribonucleic acid PMS phenazine dNTP deoxynucleoside methosulphate triphosphate RFLP restriction fragment DTT dithiothreitol length polymorphism dTTP deoxythymidine RNA ribonucleic acid triphosphate rpm revolutions per EDTA ethylene diamine m inute tetraacetic acid s second EST expressed sequence tag SDS sodium dodecyl FCS fetal calf serum sulphate FISH fluorescence in situ SSC saline sodium citrate hybridisation STS sequence tagged site g gram T thymidine or thymine HAT hypoxanthine Tris tris(hydroxymethyl) aminopterin aminomethane thym idine TSC tuberous sclerosis HPRT hypoxanthine complex (guanine) U uracil or uridine phosphoribosyl u units transferase v /v volume for volume kb kilobase pairs w /v weight for volume 1 litre, ml millilitre YAC yeast artificial m m inutes chrom osom e
Abbreviations 12 Contributors
Frances Benham instigated the project together with Sue Povey, supervised Debbie Hunt, screened the ICRF library 107 L4/FS11 with labelled Alu-PCR product from irradiation hybrid Jo48' and performed the D11S614 linkage analysis.
John Boyle, Ben Carritt, Carol Jones, David Markie, Sue Povey and Veronica Van Heyningen provided the human chromosome 11-only somatic cell hybrid J1C14 (CJ), the Galton Somatic Cell Hybrid Mapping Panel (SP), additional somatic cell hybrids (BC) and translocation hybrid mapping panels for human chromosome 6 (JB), 11 (W H ) and 18 (DM).
Mari-Wyn Burley, Debbie Hunt tested the Galton TSC family DNAs with chromosome 11 markers (M-WB, DH) and characterised the radiation hybrid DNAs by Southern hybridisation (DH).
Jude Fitzgibbon
Suggested mapping RXRB and isolated the RXRB cosmid.
Margaret Fox, Sue Povey, Lynne West and Karen Woodward
Shared the culture of the radiation hybrids (MF, SP), performed all the fluorescent in situ hybridisation of the hybrids (MF, SP), the RXRB cosmid (KW) and those isolated from the ICRF library (MF, LW).
Peter Goodfellow supervised the irradiation of J1C14 at ICRF and gave much helpful advice.
Steve Jeremiah determined the retention of LDHA and ADA in the irradiation hybrids by starch gel enzyme electrophoretic assay, and managed the Galton Somatic Cell Hybrid Mapping Panel.
Philip Johnson suggested mapping FUT4 on the radiation hybrid panel.
John Osborne and David Webb provided the clinical support to the Galton TSC mapping effort and I assisted them in the clinical examination and venesection of some of the families.
Contributors 13 Acknowledgements
Many people have given their time, expertise and companionship to enable me to begin to learn some rudiments of genetics and to bring this thesis to fruition.
The coincidence at Wolf son House of the MRC Human Biochemical Genetics Unit, the MRC Blood Group Unit and the UCL Department of Genetics and Biometry (as it then was) provided a unique environment in which to enjoy a MRC Training Fellowship and I thank the present or former heads of each for their interest and support: Profs David Hopkinson (MRC HBGU), Pat Tippett (MRC BGU), Bette Robson and Steve Jones (UCL). I thank the MRC for the providing the opportunity to escape from the routine for a while and David Brenton who ensured that the escape was accompanied by relevant clinical practice.
Sue's team in Rooms 115 and 208 tried to remedy ignorance and tolerate eccentricity with good grace and ensured a very happy workplace. They offered help and advice open-handedly; would that I had accepted it as willingly! Especial thanks to Margaret Fox, Mari-Wyn Burley, Steve Jeremiah and Robin Ali. The initial project was Frances Benham's idea; her and Debbie Hunt's contributions were invaluable.
Unlike Dr Samuel Johnson, Sue Povey not only found me an argument but provided me with an understanding as well and I am profoundly grateful to her. If the principal reward of science is "the widening of the horizon as one climbs"1, it will at times have been frustrating for her to have me tag along but I am very glad to have shared in part of the journey and glimpsed the next range of mountains. I apologise for keeping her waiting so long for this small record of an ascent.
I thank Elisabeth and my parents (without whom this would not have been possible) for their encouragement and patience, despite the many distractions the "jetfoiling Englishman" discovered to avoid task in hand.
1 Celia Payne Garoschkin Acknowledgements 14 C h a p t e r 1: Introduction
Historical Review
Tuberous sclerosis or TSC, the tuberous sclerosis complex, is an autosomal dominantly-inherited syndrome of hamartomas affecting many organs of the body. Hamartomas are benign growths which occur in normal tissue, i.e. masses of mature cells or tissue indigenous to the particular site but which do not have the normal architecture of the surrounding tissue. The organs in which these occur most commonly in TSC are the brain and retina, skin, heart, and kidney, but almost any tissue may be affected including spleen, liver, stomach, pancreas, intestine, adrenal, testis, thyroid and aorta. The mechanisms by which these lesions occur are unknown, but it has been suggested that TSC is a disorder of cellular migration, proliferation or differentiation.
The disease has been recognised for over 130 years. The German pathologist von Recklinghausen gave the first description of a case to the Obstetrical Society of Berlin in 1862 (references in Gomez, 1988a ). The condition was first described as a syndrome by Bourneville in 1880, who found "tuberous sclerosis of the cerebral convolutions" at post-mortem examination of a teenage girl. She had suffered from epilepsy since infancy, and in her teens developed a facial skin rash (thought by Bournville to be acne rosacea). The following year Bourneville and Brissaud found similar sclerotic cerebral tumours in a boy of who had developed seizures in infancy, who also had a loud heart murmur. At post-mortem examination, the cardiac ventricular walls were hypertrophied, and the kidneys contained small tumours. The authors concluded that the association of cerebral and renal tumours was significant.
Ch. 1 Introduction, Clinical 15 Von Recklinghausen in 1862 described a neonate who died shortly after birth in whom he found cardiac "myomata" and "a great number of scleroses in the brain". This report antedates those of Bourneville and is the earliest pathological description of the disease but the reference to the cerebral masses is brief and there is no suggestion that the concurrence of the two abnormalities constituted a syndrome.
In the 1880's there were reports by Hartdegen (1881, who suggested that cerebral "glioma" were neoplastic) and by Balzer and Menetrier (1885, who named the facial skin rash, "adenoma sebaceum"). The classical triad of diagnostic features of TSC, epilepsy, mental retardation and adenoma sebaceum was recognised by Vogt (1908). He diagnosed TSC in a patient with adenoma sebaceum and the diagnosis was confirmed at post-mortem. He recognised that cardiac and renal tumours were features of the disease, as had Perusini (1905).
Clinical aspects of tuberous sclerosis
Diagnostic Criteria
The variable expression of phenotype in TSC has necessitated the development of criteria to aid clinical diagnosis, of which those of Gomez are the most widely accepted (Gomez, 1988a; Gomez, 1988b; Gomez, 1991). The criteria, as amended by a National Tuberous Sclerosis Association (NTSA) Professional Advisory Committee (Roach et ah, 1992), are summarised in Table A. 1.1, (Appendix 1). Signs present on examination or investigation are classified according to the support which each lends to a diagnosis of TSC, as primary (or definitive), secondary and tertiary. Primary criteria are signs which are characteristic of TSC and the presence of any one is sufficient to establish the diagnosis firmly. Secondary NTSA criteria were termed "provisional" (or "presumptive" or "probable") in previous classifications and tertiary features "suggestive" (or "suspect" or "suspicious"). Secondary signs are more significant than the tertiary. Secondary and tertiary features are not diagnostic, in so far as they may
Ch. 1 Introduction, Clinical 16 occur in normal individuals (not thought to have a mutated TSC gene) or in other pathological conditions. In the presence of one or more of the primary features, they may support a diagnosis of TSC. As the authors of the NTSA report point out, the criteria should be sufficiently sensitive to lead to a diagnosis of TSC in those patients with subtle or uncommon features of the disease, but should also be rigorous enough to exclude individuals with inadequate evidence of the complex. Confident exclusion of the disease is difficult, even in the absence of primary features on physical or radiological examination, and there have been several examples of apparent incomplete penetrance which have confounded clinical counselling and gene mapping studies, see below (Wilson et ah, 1978; Lowry et ah, 1979; Baraitser et ah, 1985; Webb et ah, 1991).
Ideally, these facets of the individual's history, clinical examination and investigations (radiological and molecular) should be combined in a Bayesian analysis to give an overall risk of the individual having TSC, as has been achieved with other dominantly-inherited conditions (e.g. myotonic dystrophy and Huntington disease). This will not be possible, however, until population-delimited studies of prevalence of the clinical features of TSC have been carried out in groups of patients with TSC and in the general population.
Clinical features:
TSC is the commonest dominantly inherited condition causing epilepsy and learning disability. These are the most serious clinical features (at least in childhood). A population prevalence study in the South-West of England identified 131 individuals with TSC 78% of whom had suffered seizures (70% under the age of one year). 87% of the 68 patients whose degree of handicap was assessed needed some supervision of the activities of daily living and 65% had little or no language (Webb et ah, 1996). Any form of epilepsy may occur in TSC and tonic-clonic, akinetic, myotonic and atypical absence seizures have been noted. Infantile spasms, complex partial- and myoclonic- seizures are particularly common. TSC may
Ch. 1 Introduction, Clinical 17 present in infancy with spasms and there may be delay in diagnosis if paediatricians do not recognise the parents' description of the attacks (JP Osborne, personal communication). Seizures during infancy seem to be associated with subsequent mental retardation: if epilepsy does not develop during the first few years of life mental handicap is rare (Gomez et ah, 1982; Simmons, 1984; Webb et ah, 1996). Conversely, some patients develop normally until the onset of seizures, whereupon their progress slows or they lose developmental milestones. Children with larger and more numerous cortical tubers tend to have an earlier age of onset of epilepsy, seizures which are less easily controlled and more severe mental retardation (Gomez, 1991). There is an increased prevalence of learning disorder in males with TSC but this is not accounted for by any difference between males and females in the number, size or site of ependymal or parenchymal lesions (Clarke et ah, 1996; Webb et ah, 1996). Other possible determinants of neurological outcome are addessed in the Appendix.
Inheritance and prevalence:
Gunther and Penrose identified that TS is inherited as a mendelian autosomal dominant trait (Gunther et ah, 1935) following Berg's suggestion that TSC could be inherited (Berg, 1913). Penetrance is high, but there is great variability in expression, and the majority of cases result from new mutations. The prevalence in the general population has been estimated at between 1 /5,800 and 1 /150,000, and the percentage which are sporadic, 50-86%. Estimates of cases due to new mutations (made prior to the identification of the TSC genes) are between 60-70% (Sampson et ah, 1989a; Fryer, 1991). Accurate estimates of prevalence and incidence require complete ascertainment within a given population. The West of Scotland prevalence study (Sampson et ah, 1989a) determined whether parents were affected by means of clinical signs including fundoscopy and skin examination with UV (Woods) light and reached an estimate of 60% of cases due to sporadic mutations, a minimum prevalence (of both inherited and sporadic TSC) of 1/12,000 in children under ten, and an overall prevalence of 1/27,000. These results are consistent with the other
Ch. 1 Introduction, Clinical 18 prevalence studies attempting complete ascertainment using clinical signs (Hunt et al, 1984; Wiederholt et al, 1985; Osborne et ah, 1991; Webb et ah, 1996). Taken together, the overall prevalence is approximately 1 in 30,000, 1 in 15,000 in children under 5 years and at birth incidence of 1 in 10,000; the new mutation rate was estimated to be 60-70%. More recent prevalence studies have suggested a higher prevalence of 1 in 6,800 among teenagers aged 11-15 and by implication, in younger children (Ahlsen et ah, 1994). These studies may have had a selection bias towards inclusion of those individuals with more severe manifestations of TSC, especially epilepsy and mental retardation. Individuals without these classical presenting features in childhood may have been omitted because they do not come to the attention of the specialist physicians who are canvassed. Given this bias in favour of ascertainment of patients who are more severely affected the true at birth incidence may be even higher. The Avon study attempted to take this into account, and reached an estimate of 1 in 5,800 for the true at birth incidence (Osborne et ah, 1991).
It is difficult to imagine how more thorough ascertainment studies, using ultrasound and magnetic resonance imaging could gain ethical committee approval, however, since the investigations required, though not physically invasive are intrusive, time consuming and costly. More accurate estimates may not be possible until the genetic defect can be easily characterised and the at birth prevalence of mutations can be estimated from banked DNA, mRNA or protein resources such as those of ALSPAC (the Avon Longitudinal Study of Pregnancy and Childhood) or Regional Neonatal Screening Service Guthrie cards (Golding et ah, 1996).
Penetrance
The early prevalence studies were neither complete ascertainments of affected individuals in a region of known population nor did they address the effect of incomplete penetrance on the assessment of whether an isolated case was a true sporadic, new mutation or inherited from a clinically unaffected parent who was a gene carrier. Penetrance of TSC is
Ch. 1 Introduction, Clinical 19 high but is age-dependent and expression is very variable even among members of the same family. True incomplete penetrance is rare but it is conceivable that ascertainment studies may have overestimated the proportion of cases due to new mutations (because of clinically inapparent disease in the gene-carrying parent). There have been a number of reports of the phenomenon dating back to 1935 but it is not known whether any of the earlier cases have been re-examined using modern imaging techniques (Gunther et al., 1935; Wilson et ah, 1978; Lowry et ah, 1979; Baraitser et ah, 1985). Cardiac echosonography (in children) and renal ultrasound and cranial MRI (in children and adults) have become mandatory in the full assessment of at-risk family members who are clinically unaffected or who have tertiary diagnostic signs. Some obligate gene carriers may fail to display any of the abnormalities suggestive or characteristic of TS when first evaluated and may do so only in later life (Webb et ah, 1991). This is a particular problem in counselling of at-risk unaffected individuals, especially with regard to their chance of having offspring with TSC. A related issue is the recurrence risk estimates given to parents of a child with TSC where the parents are normal on detailed clinical examination and there is no family history. If the child's TSC were the result of a new mutation, then the risk estimate would be very low. However, if either parent is a germline mosaic for a TSC mutation the the recurrence risk estimate could be as high as 50%. A composite risk of between 1-5 % is given for affected future pregnancies. Since the identification of the TSC genes, germline mosaicism has been demonstrated by molecular means (Yates et ah, 1997).
TSC has been diagnosed prenatally by the demonstration of multiple cardiac rhabdomyomas by ultrasound examination (Muller et ah, 1986) or subependymal nodules by MRI (Revel et ah, 1993) but neither technique has sufficient positive predictive value to be used to exclude TSC in an apparently unaffected fetus. Ultrasound is unreliable in this context before about 26 weeks of gestational age and so if a fetus was shown to be affected therapeutic termination would be legally and emotionally fraught.
Ch. 1 Introduction, Clinical 20 Resolution of these clinical issues depends on the understanding of the genetic basis of TSC. Without an obvious candidate protein or enzyme, this necessitated the use of the positional cloning techniques described in the next section.
Positional cloning
Introduction
Positional cloning is a strategy for mapping a phenotype and cloning the gene or genes responsible when there is no prior knowledge of their function or localisation in the genome. It is a multi-component process the first step of which is the localisation of the condition to a chromosome or chromosomal region. This has traditionally achieved by identification of chromosomal abnormalities in affected individuals, linkage analysis or comparative mapping. This initial step is followed by detailed molecular analysis of the candidate region including finer genetic mapping (identification of critical recombinants to narrow the candidate region), linkage disequilibrium studies, physical mapping, isolation of transcribed sequences expressed in relevant tissues, and finally the identification of mutations in candidate genes. Techniques which have been used or considered in identifying genes involved in TSC are summarised below.
In the late 1980's positional cloning was known as "reverse genetics" in contradistinction to "orthograde" approaches to gene cloning. The latter requires the prior identification of the protein encoded by the gene, limited protein sequencing, synthesis of minimally redundant oligonucleotides from the back-translated sequence, and isolation of cDNA by hybridisation of the oligomer probes to a suitable cDNA library. This approach was used in the first example of the cloning of a gene in humans (Shine et al., 1977). The term "reverse" has been abandoned in the context of gene mapping for several reasons. It may be confused with the terminology used to describe the "central dogma" of the expression of information encoded in the genome. The phrase is also used to describe a technique in which a
Ch. 1 Introduction, Clinical 21 gene is modified in vitro and re-inserted into host to define or confirm a novel mutated phenotype. It also implies a less commonly used technique, whereas the majority of mapping successes in recent years have exploited positional cloning and positional candidate methodologies.
Structural abnormalities
The chromosomal locations of the majority of disease genes successfully identified by positional cloning prior to 1995 were indicated by cytogenetic rearrangements or deletions which disrupt the locus. These are summarised in Table 1.1, adapted from Collins, 1995. Of the 42 inherited diseases listed the isolation of 26 (62%) was aided by a chromosomal rearrangement. Early examples to be reported include the X-linked genes chronic granulomatous disease CGD (Royer-Pokora et al, 1986b; Royer- Pokora et al., 1986a) and Duchenne muscular dystrophy DMD (Monaco et al., 1986; Koenig et ah, 1987), and retinoblastoma (13ql4 (Friend et al, 1986)). Subsequently, smaller-scale structural aberrations have been detected by more detailed cytogenetics, including FISH (Prader Willi syndrome (Butler et al., 1976); Angelman syndrome (Magenis et al., 1987)) or by Southern hybridisation (expansion of trinucleotide repeats, FRAXA (Verkerk et al, 1991)). One male, patient B. B. with an interstitial deletion who presented with DMD, CGD, the McLeod red cell surface antigen and a retinitis pigmentosa, provided a particularly useful resource for the isolation of three of these genes (Francke et al., 1985).
Other examples include 17qll.2 translocations in NF1 (Fountain et al., 1989; Ledbetter et al, 1989b; Menon et al, 1989; O'Connell et al, 1989); a interstitial deletion in lq32-41 localising van der Woude cleft lip/palate syndrome (Bocian et al, 1987); and translocations leading to 17pl3.3 microdeletions in Miller-Dieker lissencephaly syndrome (Ledbetter et al, 1989a).
Ch. 1 Introduction, Positional Cloning 22 Disease or mutant phenotype
Structural Genetic linkage Comparative abnormality analysis mapping
Chromosome localisation
Fine genetic mapping, linkage disequilibrium, physical mapping
Contig construction (YAC, BAC, PAC, cosmid)
GENE ISOLATION Traditional approaches: Conservation on zoo blots CpG islands Northern blots cDNA library screening with cloned genomic DNA
Newer approaches: cDNA selection exon amplification regionally mapped candidate genes and ESTs "bulk" genomic sequencing and computer analysis
Mutation analysis Analysis of gene function
Fig. 1.1: Techniques involved in positional cloning. (Monaco, 1994)
Ch. 1 Introduction, Positional Cloning 23 Year Disease Cytogenetic rearrangement
1986 Chronic granulomatous disease + Duchenne muscular dystrophy + Retinoblastoma + 1989 Cystic fibrosis - 1990 Wilms tumour + Neurofibromatosis, type I + Testis determining factor + Choroideraemia + 1991 Fragile X syndrome + Familial polyposis coli + Kallmann syndrome + A niridia + 1992 Myotonic dystrophy Lowe syndrome + Norrie syndrome + 1993 Menkes disease + X-linked agammaglobulinaemia + Glycerol kinase deficiency + Adrenoleukodystrophy + Neurofibromatosis, type II Fluntington disease - von Hippel-Lindau disease - Spinocerebellar ataxia I - Lissencephaly + Wilson disease - Tuberous sclerosis, TSC2 + 1994 McLeod syndrome + Polycystic kidney disease, PKD1 + Dentatorubral pallidoluysian atrophy - Fragile X type E, FRAXE + Achondroplasia - Wiskott Aldrich syndrome - Early-onset breast/ovarian cancer, BRCA1 - Diastrophic dysplasia - Aarskog-Scott syndrome + Congenital adrenal hypoplasia + Emery-Dreifus muscular dystrophy - Machado-Joseph disease - 1995 Spinal Muscular atrophy - Chondrodysplasia punctata + Limb-girdle muscular dystrophy - Ocular albinism +
Table 1.1: Inherited disease genes identified by positional cloning, taken from Collins, 1995. Key: Cytogenetic rearrangement + present, - absent.
Ch. 1 Introduction, Positional Cloning 24 Only 11 of the 42 in Collins' list were cloned without the aid of chromosomal aberrations. These include myotonic dystrophy and cystic fibrosis. Cytogenetic data may occasionally provide false leads, however (Fryns et al, 1984).
Balanced translocations of somatic cells may be associated with neoplasia, notably in hematological malignancies. Examples include chronic myeloid leukemia t(9;22)(q34;qll)(ABL;BCR) and Burkitt lymphoma (translocations involving the MYC gene, 8q24, and the lambda (22q) or the kappa (2p) light chain or heavy chain (14q32) immunoglobulin genes). Characterisation of the translocations has led to the isolation of the genes involved (de Klein et al, 1982; Lenoir et al, 1982; Konopka et al, 1985).
Meiotic mapping methods: linkage analysis
Introduction
Where there is no cytogenetic clue to the chromosomal position of a gene, pedigrees in which the gene is segregating may be used to localise it by meiotic linkage mapping. Genetic linkage is the non-random assortment during meiosis of alleles at loci which are neighbouring on the same chromosome. Two loci which are close to one another on the same chromosome will tend to be inherited together. If the two loci are far apart, recombination between the homologous chromosomes at meiosis will tend to separate the loci, creating new combinations of alleles. The frequency with which this recombination occurs is measured by estimating the number of recombinant offspring of a mating expressed as a proportion of all offspring (recombinant and non-recombinant), and is called the recombination fraction, theta (0). It increases with increasing distance between the loci up to a maximum of 0.5, where the frequency of recombinant and parental combinations of the alleles is equal, at which linkage beween widely-spaced loci on the same chromosome cannot be distinguished from independent assortment due to the loci being on separate chromosomes.
Ch. 1 Introduction, Positional Cloning 25 Testing families for linkage using the likelihood method involves the comparison beween the probability of the occurrence of the observed distribution of disease and marker in a mating under the assumption of linkage at a chosen recombination fraction less than 0.5, and the null hypothesis, the probability assuming no linkage between the disease and the marker locus, recombination fraction 0.5 (Haldane et al., 1947; Morton, 1955; Morton, 1956; Elston et al, 1971; Ott, 1986). This comparison takes the form of an odds ratio, L(9)/L(0.5), the probability assuming linkage at the given recombination fraction, L(0), being the nominator, and the probability of the null hypothesis, L(0.5), the denominator. Odds ratios from different families may be multiplied to give an overall probability, but for convenience the decimal logarithm of the odds ratio
(loglo(L(0)/L(O.5)) at several values of 0, the "lod score" Z(0), is usually calculated for each family and the scores at each recombination fraction added. Lod scores are of particular value when combining odds ratios from phase known and phase unknown families. The maximum value of the lod score obtained after the scores from the available families has been added together indicates the most likely value for the recombination fraction between the disease gene and the marker locus. Recombination fractions often chosen for estimation of the odds ratio and lod score are zero, 0.001, 0.05, 0.1, 0.2, 0.3, and 0.4, although in practice, very large samples are required to differentiate recombination fractions greater than 0.25 from non-linkage (0.5).
This maximum lod score value is a measure of the statistical significance of the result of the linkage study. Traditionally, a value equal to, or greater than three is required to assert the presence of linkage. This corresponds to an odds ratio of 1000:1. The prior probability that any two random loci are linked at a recombination fraction of =< 0.3 is about 0.02 or 1:50. Given a lod score of 3.0, the posterior odds for linkage is the product of the prior odds of linkage, 0.02 and the odds ratio, 1000, that is 20:1. The posterior probability of linkage is therefore 20/21, 95%, and that of no linkage (the probability of a false positive result) is 1/21, 5%, which corresponds to the
Ch. 1 Introduction, Positional Cloning 26 level of significance used in many conventional statistical tests. In practice, linkage studies asserting linkage with lod scores of above three subsequently prove to be false in less than 2% of cases (Rao et al., 1978).
Negative lod scores indicate absence of linkage, and scores more negative than -2 are usually accepted as excluding linkage at the stated recombination fraction value. Intermediate values (between -2 and 3) are inconclusive, indicating that the study needs to be extended by adding to the number of families or by increasing the information content of the existing families by the use of more informative, highly polymorphic markers.
Use of multiple markers throughout the genome in an attempt to map a locus increases the posterior probability of linkage and it could be argued that a higher lod score should be required to assert linkage. However, where many markers are used the prior probability of linkage increases so that a LOD score of >3 is still a reasonable indication of linkage in a monogenic disorder.
Linkage analysis: genetic mapping
The construction of high resolution linkage maps on which to localise disease genes has been greatly facilitated by the discovery of simple or short tandem di- (AC/TG) or tri- nucleotide repeat sequences or microsatellite markers (Weber et al., 1989). Microsatellite markers occur frequently throughout the human genome at approximately 30-60 kb intervals (Stallings et al., 1990). In contrast to restriction fragment length polymorphisms they are highly polymorphic: the average heterozygosity of microsatellite markers in the 1996 Genethon human linkage map is 0.7 whereas the maximum heterozygosity of a bi-allelic RFLP is 0.5 (Dib et al., 1996). They also have the practical advantage that they may be detected by PCR rather than by the more laborious technique of Southern hybridisation. Mapping of these markers and scoring of pedigree members may be semi-automated. The combination of advantages of microsatellite
Ch. 1 Introduction, Positional Cloning 27 frequency, heterozygosity and automation has contributed to the development of increasingly dense maps of the human genome (Weissenbach et al., 1992; Gyapay et al., 1994; Dib et al., 1996). The 1996 Genethon human linkage map has been constructed using 5264 microsatellite markers with an average interval size of 1.6 cM, 59% of the map is covered by intervals of 2 cM at most and only 1% remains in intervals above 10 cM (Dib et al., 1996). The 1994 CEPH map makes use of 3617 short tandem repeat polymorphisms of a total of 5840 polymorphic markers, giving an average marker density of 0.7 cM (Murray et al., 1994). Similar approaches are expediting the mapping of other organisms including mouse, v.i. (Dietrich et al., 1996), rat (Yamada et al., 1994; Toyota et al., 1996) and pig (Johansson et al., 1995). Meiotic linkage mapping using these highly polymorphic markers may now permit localisation of a disease gene in a single large family pedigree (Kandt et al., 1992; Weber et al, 1994). Analysis of critical recombinants in affected and non-affected individuals can help to narrow the candidate interval to around 1 cM (in a linkage study of mutiple pedigrees), roughly approximating to a physical interval of 1 Mb. Combined linkage and physical maps incorporating linkage, radiation hybrid, YAC and transcript data now offer a very valuable tool for identification and isolation of candidate genes within the region of the genome identified by initial linkage studies (Hudson et al, 1995).
Linkage analysis: Fondation Jean Dausset - CEPH
Jean Dausset founded the Centre d'Etudes du Polymorphism Humain in 1984 to provide the scientific community with resources for human gene mapping (Dausset et al, 1990). The "CEPH Reference panel" of genomic DNA's from individuals in 61, large, three-generation French families was the first to be released. DNA's from about 40 of these families have been widely used for the genetic mapping of polymorphic markers, initially RFLP's and since the late 1980's, microsatellites. The panel has proved to be one of the most useful tools for the generation of chromosome maps, and has been at the core of the activities of most chromosome mapping
Ch. 1 Introduction, Positional Cloning 28 committees. Consortia have been set up for the mapping of individual chromosomes and high quality maps produced. Several whole genome maps have been produced from reference panel genotypes including the Genethon maps of 1992, 1994 and 1996 (Buetow et ah, 1994; Gyapay et al, 1994; Murray et al, 1994; Spurr et al, 1994; Dib et al, 1996). Breakpoint maps identifying recombinant chromosomes in the panel have also been constructed and used to map new markers with minimal typing and data analysis (Attwood et al, 1994; Litt et al, 1995; Cox et al, 1996).
The Centre, now named the Fondation Jean Dausset - CEPH, maintains a database of the markers that have been used in tested on the panel. Version 8.1 (release date January 1997) has data on 11,932 markers (more than 2.5 million genotypes). More than 8,900 of these are microsatellites, over half of which are highly polymorphic. The average heterozygote frequency of loci in the current version is 0.64 (http://www.cephb.fr/).
Linkage analysis: linkage disequilibrium
Linkage disequilibrium is the phenomenon of divergence of haplotype frequencies from those predicted by the hypothesis of random assortment of alleles. It may occur for two principal reasons: an alteration to create a novel allele may have occurred recently so that there has not been sufficient time to establish equilibrium or there may be selective pressure to conserve a particular haplotype. It may be seen in well-mixed populations (Feder et al, 1996) but is most evident in genetic isolates showing founder effect (descended from a small founding group in which one or a few "ancestral" mutations have led to an inherited disease) such as the French Canadians and Finnish. In the absence of abnormal, highly- recombinant, adjacent "hot-spots", markers near the disease gene will tend to be co-inherited, forming a common haplotype associated with the disease in different individuals. Evidence of linkage disequilibrium helps to indicate the region within which the disease gene must lie and can narrow the candidate region obtained by meiotic linkage strategies. It has been observed in the mapping of several genes including cystic fibrosis
Ch. 1 Introduction, Positional Cloning 29 (Estivill et al., 1987), myotonic dystrophy (Harley et al., 1992), torsion dystonia (Ozelius et al., 1992; Rischet al, 1995) and neurofibromatosis type I (Jorde et al, 1993). It was effectively used to limit the candidate region for diastrophic dysplasia on chromosome 5q32-q33.1 to 0.06 cM and to aid the isolation of the DTD gene, a sulphate transporter (Hastbacka et al, 1992; Hastbacka et al, 1994). Another example is the mapping of the gene for Fanconi's anaemia type 1, FA1, by classical linkage analysis and allelic association in a South African Dutch founder population (Pronk et al, 1995). Linkage disequilibrium is unlikely to aid gene mapping where most cases of the disease are due to recent mutations, such as TSC.
Linkage analysis: comparative linkage mapping
The relative localisation of genes may be conserved between differing mammalian species, despite their evolutionary distance. This preservation of gene content and order is known as synteny. Knowledge of linkage of two loci in one species may assist in the mapping of the loci in a second species. This strategy is dependent upon the existence of high resolution linkage maps of at least one of the species (Daniels et al, 1987).
Mouse genome research is of particular significance to the human because of many biological, pathological and genetic similarities between the two species. The mouse linkage map, based initially on phenotypic and enzyme markers, has led to many insights into human biology. The mouse provides a useful model for generating these maps since it can be bred to genetic homogeneity, it reproduces at frequent and predictable intervals and the litters are large. Laboratory strains may interbreed with wild mouse species (e.g. Mus spretus) to produce fertile FI females which may be back-crossed with either of the parental strains (Avner et al, 1988). The evolutionary distance between the two parental species increases the likelihood of detecting sequence variations in different alleles in the progeny.
Ch. 1 Introduction, Positional Cloning 30 Some panels, such as the European Collaborative Interspecific Backcross (UK Human Genome Project Resource Centre - Institut Pasteur), contain DNA from over 1,000 F2 progeny, which theoretically permits the generation of a map of the mouse genome at a resolution of 0.1-0.3 cM (Breen et al, 1994; Rowe et al, 1994; McCarthy et al, 1995). 6,580 MIT Genome Center mouse microsatellites and 797 RFLPs have been mapped on DNA from the EUCIB progeny with an average inter-marker distance of about 0.2 centimorgans or 400 kilobases (Dietrich et al, 1996). This has permitted the development of a detailed, ordered linkage map of the mouse genome and provided the foundation for a STS YAC contig of the mouse genome (Dietrich et al, 1995). Concurrently, Interspersed Repetitive Sequence-PCR products are being mapped by linkage on the EUCIB panel, and mouse YAC libraries are then screened to identify cross hybridising YACs to increase the density of the physical map (McCarthy et al, 1995).
Correlative mapping studies have proved especially fruitful and have identified conserved syntenic groups of loci, candidate regions and genes for homologous human diseases. Mapping by synteny is discussed below {("Candidate by cross-species phenotype correlation and synteny (syntenic candidate)").
Physical methods: isolation of chromosomes or chromosome fragments
Introduction
Isolating a chromosome or chromosomal region of interest is an important step in physical mapping since it facilitates the positioning of known markers within the cloned region and the isolation of novel markers from the region. At the outset of this project in 1989 there were a limited number of means of obtaining these resources from specific
Ch. 1 Introduction, Positional Cloning 31 chromosome regions. Techniques in use included fluorescence activated cell sorting, FACS, radiation-fusion gene transfer and chromosome microdissection. FACS had already proved useful for isolation of individual chromosomes or chromosomal fragments (Collard et al., 1980; Lebo et al., 1986; Seawright et al., 1988). The then novel use of flow-sorted chromosomes was the preparation of monochromosomal clone libraries, e.g. RLDB ICRF, National Laboratory Gene Library Project (Nizetic et al., 1991).
Chromosome microdissection
In contrast to the large-scale cloning strategy of FACS-sorting of chromosomes, the technique of microdissection of banded chromosomes provided a more focussed approach for the isolation of markers in a specific region of interest. This methodology was first used to clone regions of the Drosophila genome (Scalenghe et al., 1981). It was applied to human chromosomes when chromosomal banding techniques permitted sufficient cytogenetic resolution and PCR allowed amplification of the cloned dissected fragments. The technique involves the precise excision of single bands and cloning of the digested DNA fragments. These clones may then be used to screen genomic libraries to identify cosmids and YACs from the region of interest (Ludecke et al, 1989; Buiting et al, 1990; Davis et al, 1990; MacKinnon et al, 1990). A later development of this technique, preparative in situ hybridisation (Prep-ISH) involves a form of cDNA selection. Products from PCR amplification of a cDNA library are hybridised to denatured metaphase spreads which are then stained. The region of interest can then be microdissected, and the excised chromosomal fragments amplified to form a sub-library of regionally- assigned cDNAs for further analysis (Hozier et al, 1994).
Ch. 1 Introduction, Positional Cloning 32 Radiation hybrids
Introduction
The generation of mammalian somatic cell hybrids by irradiation and fusion was first proposed by Pontecorvo, developing experiments he had performed to make Drosophila hybrids using X-irradiation in the early 1940's (Pontecorvo, 1971). Irradiation of one parent of a mouse-hamster somatic cell hybrid (the "donor") followed by fusion (to a "recipient" cell line) led to the preferential elimination of the irradiated parent's chromosomes from the daughter hybrid. The radiation doses used were low, 600-1000 rad, and Pontecorvo noted that with higher doses, 1600 rad, very few clones were established, and the chromosomal component of these was almost entirely from the non-irradiated parent.
This technique was later adapted by Goss and Harris to determine the relative position of four genes (PGK, alpha-GAL, HPRT and G6PD) on the long arm of the X chromosome (Goss et al, 1975; Goss et al, 1977a; Goss et al, 1977b). Lymphocytes from a human male were irradiated to fragment the chromosomes and then immediately fused with hamster cells. They were able to estimate the order and relative distances between four loci (one of which, HPRT, was the selectable marker) from the frequency with which each of locus was co-transferred in the daughter hybrids. After a further ten years two groups realised independently that the technique could be used to map regions of the genome that do not contain selectable markers, and that the absence of any selection for retention of any human material permitted ordering of loci and estimates of distance between the loci (Benham et al, 1989; Cox et al, 1989). It has subsequently been exploited to generate mapping resources and to construct maps both within delimited regions such as individual chromosomes (Richard et al, 1991; Bouzyk et al, 1996) or across the whole genome (Walter et al, 1994; Gyapay et al, 1996; Schuleret al, 1996a; Schuleret al, 1996b).
Ch. 1 Introduction, Positional Cloning 33 The technique of mapping by radiation-induced gene segregation combines features of analysis of linkage in families with tests for the presence of human genomic sequence in somatic cell hybrids. The principle of the technique is that the further apart two markers lie on a chromosome the more likely a given dose of radiation will fragment the intervening DNA, resulting in separation of the markers on different fragments. Conversely, co-retention of two markers in a majority of hybrids indicates that the markers lie close to one another. This presumes that loss of fragments from the hybrids occurs randomly (in the absence of any selection), independent of size of fragment. In an example of this approach, a human-rodent hybrid containing a single human chromosome is lethally irradiated with X or Gamma rays. The irradiation leads to double stranded breaks in the DNA and the fragmentation of both human and rodent chromosomes. Doses commonly used in such experiments lead to the death of the irradiated cells. The cells may be rescued by fusion to a "recipient" rodent cell line. If this latter line is deficient in TK or HPRT (as in the original Goss and Harris experiment) then HAT selective medium will kill any recipient cells not fused to a donor cell containing either the rodent TK1 or HPRT genes (originating from the donor hybrid), respectively. Irradiated cells are fused with an HPRT-deficient rodent parent, and hybrid clones containing an active rodent HPRT gene are selected by growth in medium containing HAT, Figure 1.2.
Ch. 1 Introduction, Positional Cloning 34 X-rays Donor hybrid retaining human Recipient chromosome & rodent cell selectable marker line
Fusion
Selection
Unfused donor cell: Unfused recipient lethally irradiated rodent cell: selected against
Radiation hybrid: human chromosome fragments integrated into rodent chromosomes or reconstituted as independent fragments
A "donor" hybrid containing a single human chromosome or chromosome fragment (and a selectable marker) is irradiated with a lethal dose of X-rays and is then fused with a "recipient" rodent somatic cell line. Selection is applied to eliminate any unfused rodent cells. All daughter hybrids contain the selected marker (e.g. HGRP) and may also contain non-selected human chromosome fragments. Modified from Walter et al., 1994. Fig. 1.2: A cartoon of hybrid irradiation - fusion gene transfer.
Ch. 1 Introduction, Positional Cloning 35 Selection
Retention of a specific region of the human chromosome may be achieved by the imposition of selection of an expressed gene within that region. However, even in the absence of selection for retention of human chromosomal material, hybrids do contain randomly integrated fragments of the chromosome from the irradiated parent, in which case the size of the fragments are related to the radiation dose (Benham et al, 1989; Cox et al, 1990; Benham et al., 1992). The crucial feature of this technique is that survival of the daughter hybrids is dependent on the retention of a rodent not a human gene, and so the presence of any human fragments (in the daughters) is a result of random co-integration. There is no selection for the retention of any human chromosomal material.
Uses of radiation hybrids
Radiation hybrids may be exploited in two principal ways. A hybrid panel generated without selection may be used in mapping (since retention of human chromosomal fragments is random). An individual hybrid containing a fragment of the irradiated parent human chromosome which is well-characterised may be used to generate or identify clones from that region. Both of these properties of radiation hybrids were used in this project and are described below.
Radiation hybrid mapping: single chromosome donor parent hybrid
Panels of radiation hybrids have been generated using donor parents containing a single human chromosome (or part) and these have proved useful mapping tools (Benham et al., 1989; Goodfellow et al., 1990; Burmeister et al, 1991; Florian et al., 1991; Abel et al., 1993; Bouzyk et al, 1996). The size of the chromosomal fragments produced is determined by the dose of radiation applied to the donor hybrid. The initial studies of Cox used radiation doses of approximately 6000 rads. If sufficient hybrids are generated in a single experiment, the panel may be used to provide mapping data. The approach is analogous to meiotic mapping, in so far as it is the frequency of chromosomal breakage between loci, and the
Ch. 1 Introduction, Positional Cloning 36 dispersion of fragments into separate daughter hybrids which provides a measure of the distance between them. The resolution of the panel is dependent largely on the radiation dose used and the number of hybrids analysed: a high X-ray dose will separate two closely physically associated sites sufficiently frequently to provide an indication of the distance between them, but a large number of hybrids have to be analysed.
The stochastic analysis of segregation of markers in a hybrid panel produces estimates of the distance between markers expressed, in one form of analysis, in centiRay units (for a given radiation dose) where 100 cRayxooo corresponds to one expected obligate chromosome break after exposure to x,000 rads (Cox et al, 1990). The statistical approaches used to determine marker order and distance may be grouped into three categories, non-parametric, maximum-likelihood and Bayesian; each give similar results (Boehnke et al., 1991; Falk, 1991; Barrett, 1992; Bishopet al., 1992). Analysis of radiation hybrid mapping data is a compromise between rapid, simple methods (e.g. an initial two-point statistical analysis to generate groups of loci linked with odds of > 1000:1) and accurate but computationally-intensive techniques (detailed maximum likelihood approaches to order loci within the groups). Maps derived using these methods have proved to be congruent with those produced by other physical mapping techniques and by meiotic mapping (Cox et al., 1990; Burmeister et al., 1991; Richard et al, 1991; W arrington et al, 1991; Altherr et al, 1992; Ceccherini et al, 1992; Rothschild et al, 1992; Tamari et al, 1992; Warrington et al, 1992; Bouzyk et al, 1997). Distances between peri- centromeric markers obtained by radiation hybrid mapping do not correlate closely with those obtained by other means but marker order is consistent.
Radiation hybrid mapping: whole genome donor parent
Whole genome radiation hybrid panels have been generated using a 46 XY diploid human fibroblast cell line as the irradiated parent (Walter et al, 1994). The thesis underlying this development was largely based upon that of Goss and Harris, published almost twenty years earlier (Goss et al,
Ch. 1 Introduction, Positional Cloning 37 1975). A panel of only 44 hybrids was sufficient to make an ordered map of 40 markers on chromosome 14, consistent with existing maps. The same group, together with Genethon, have constructed a further panel of 128 hybrids which has been typed with 404 mapped microsatellite markers. This has generated a high-resolution framework map of the human genome (Gyapay et al., 1996). These panels are described in more detail in the Discussion.
A current major application of these and similar panels is to map EST sequences (v.i.) with a single set of PCR reactions to within a few cRays, equivalent to as little as a few tens of kb (Foster et al, 1996; Gyapay et al, 1996). This approach was used by the EST Mapping Consortium to map more than 16,000 ESTs to intervals related to a framework of known genetic markers (Schuler et al, 1996a; Schuleret al, 1996b). Currently, over 48,000 ESTs are listed on the European Bioinformatics database as mapped to one or more of five whole genome panels, (http: / / www.ebi.ac.uk/ RHdb/STATS/HUMAN/rhdb_stat.html) although there are likely to be many duplicate ESTs among these.
Integrity of hybrid fragments
The close comparability of radiation hybrid data with other maps indicates that the fragments generated during irradiation do not consistently contain large deletions or inversions (which would introduce errors into the maps). The integrity of fragments has also been shown by comparison of the structure of the human DNA contained in hybrids with that of the parent hybrid or human genomic DNA by field-inversion gel electrophoresis (Cox et al, 1989) and in sets of fragment hybrids derived in separate experiments from the same region (Glaser et al, 1990). This is in contrast to a previous technique for reducing somatic cell hybrids, chromosome-mediated gene transfer (McBride et al, 1973), in which DNA rearrangements frequently occurred (Bickmore W et al, 1988; Glaser et al, 1990). However, much of the strength of radiation hybrid mapping lies in the statistical approach which does not place undue reliance on any one hybrid.
Ch. 1 Introduction, Positional Cloning 38 Hybrid selection
In a radiation-fusion experiment using a monochromosomal hybrid without selection only a few daughter hybrids may be suitable for generation of new markers within a specific area of the chromosome. Where a suitable selectable marker exists in the region of interest (a cell surface antigen or an enzyme) selection may be applied to increase the number of hybrids from that region (Glaser et al, 1990; Brook et al., 1992; Henske et al., 1992). However, the daughter hybrids cannot be used to provide direct mapping data based on co-retention of markers as described above. Selectable markers are not evenly dispersed throughout the genome and consequently a disease locus of interest may not be near a suitable marker. Artificial selectable markers may be introduced to the region to solve this (Doucette-Stamm et al., 1991).
Retention of chromosomal material from the irradiated parent
The retention of fragments of the irradiated parent appears to be dependent on the integration of fragments into the recipient, non irradiated parent's chromosomes as insertions or translocations, or by retention of rodent or human centromeric sequences (Benham et al., 1989; Cox et al., 1990). Multiple fragments of the irradiated donor are retained after fusion (Benham et al., 1989; Goodfellow et al., 1990; Florian et al., 1991). This is of little consequence where hybrids are to be used for cRay radiation hybrid mapping, but it limits the usefulness of hybrids as a cloning resource within a region of interest. The stability of fragments in the daughter lines is variable, and some will be lost during culturing. Consequently, hybrids used for a cRay mapping project must all be obtained in one radiation-fusion experiment and be subjected to the same culture and harvesting conditions. The progressive loss of fragments during more prolonged culturing may be an advantage where a radiation hybrid containing a delimited region of the parent genome is required as a cloning resource (Benham et al., 1992). This is a rather hit-and-miss affair, however, and purification of one donor fragment from other, unwanted fragments may be achieved by a further fusion of the hybrid to a rodent
Ch. 1 Introduction, Positional Cloning 39 cell line, followed by limiting dilution of the daughters into multi-well plates, such that approximately a single cell is pipetted per well (Altherr et ah, 1992; Benham et al., 1992). This approach is dependent upon the random loss of chromosomes that occurs after fusion resulting in the generation of one or more hybrids containing the fragment of interest alone. This strategy was used by us in this project; it was also used by Altherr et al., (op. cit.) to isolate a hybrid with a fragment of approximately 2Mb surrounding the Huntington disease gene.
Generation of novel clones front radiation hybrids
Prior to the introduction of PCR, an approach for isolation of human DNA from somatic cell hybrids was to make a genomic library from the total hybrid DNA, and to isolate clones of human origin by hybridisation with a human repetitive sequence probe. This technique was used to isolate novel markers in the region of the Huntington, Mytonic Dystrophy and Tuberous Sclerosis TSC1 loci (Pritchard et al, 1989; Brook et al, 1992; Fitzgibbon, 1993). Unfortunately, if the fragment is small, very few clones are isolated.
Interspersed repetitive sequence (inter-AZw ) amplification of radiation hybrids
A more efficient strategy has been to amplify between conserved regions of repetitive elements unique to Homo sapiens, such as LI or Alu to generate PCR product which includes human intervening sequence. The Alu family consists of more than a million copies of a sequence about 300 bases in length which are widely dispersed throughout the human (and other primate) genomes, but are absent from rodent genomes. Rodent B1 SINEs are closely related and both have a common origin in a 7SL RNA-derived proto -Alu element (Smit, 1996). There is 50-fold under-representation at the centromeric regions which may be relatively gene-poor (Moysis et al, 1989). Sequence differences exist, but there is sufficient homology to identify families of related sequences and regions of consensus at the termini of the sequence from which PCR primers may be designed. Use of a single primer in a reaction permits amplification of sequence in between two Alu inserts which lie in opposite orientation to each other. Repeats
Ch. 1 Introduction, Positional Cloning 40 are present on average every 7 kb, so amplification of a hybrid containing a 2 Mb fragment would be expected to generate many different sized products, which when electrophoresed through agarose or acrylamide produce a pattern of bands which is a unique "fingerprint" of the human DNA contained within the hybrid. These fingerprints may be used to compare the human DNA content of hybrids generated from the same parent: hybrids with similar banding patterns may have overlapping human DNA content (Benham et ah, 1992; Fitzgibbon, 1993).
Uses of inter-A/w PCR product
The pooled product may also be hybridised to DNA from known monochromosomal hybrids in order to identify the source of the DNA in the amplified material without recourse to cytogenetic techniques (Ledbetter et al., 1990). Inter-Alu PCR product may also be biotinylated for use as probe for FISH (v.s.), a potent method of characterising somatic cell and radiation hybrids, used by our group (Woodward et al., 1995). Recombinant libraries may be generated from bulk product or individual bands may be picked, purified and cloned to generate novel anonymous markers from the human DNA contained within the hybrid (Nelson et al., 1989; Brooks-Wilson et al, 1990; Cotter et al, 1990). The purified, cloned or uncloned product may also be used to identify cosmid and YAC clones from genomic reference libraries either by sequencing of product to identify sequence tagged sites and isolation of homologous clones by PCR (Cole et al, 1991), or more directly by hybridisation of labelled product to the plated or gridded library (Monaco et al, 1991; Chumakov et al, 1992; Cole et al, 1992; Nahmiaset al, 1995).
Coincidence sequence cloning from somatic cell hybrids
Sequences shared between two separate sources of genomic DNA may be isolated by a the technique of "coincidence sequence cloning" (Brookes et al, 1991). The first application of this method combined DNA from a human-mouse somatic cell hybrid and human DNA. Products recovered proved to be of human origin (not rodent) and all were present in the hybrid source. This approach has been modified (end ligation-CSC) to
Ch. 1 Introduction, Positional Cloning 41 permit rapid enrichment of DNA that is present in both sources by more than lO^-fold, and has been used to isolate single copy sequences from a whole genome and to identify gene fragments in 260 kb of cloned genomic DNA (Brookes et al., 1994).
Physical methods: localising the chromosome fragments - FISH
Introduction
Physical mapping includes strategies in which DNA clones may be ordered to form a linear map by non-meiotic means. These techniques operate over different scales of resolution. At low resolution this may involve the ordering of cloned DNA of over 1 Mb in yeast artificial chromosomes YACs by pulsed field gel electrophoresis and Southern hybridisation or by fluorescence in situ hybridisation. At the opposite end of the scale, DNA sequencing gel-reads of 350 bp in length may be assembled into contigs in a shot-gun sequencing project to obtain sequence from a genomic clone of a few kilobases. Low resolution techniques have been transformed in recent years by novel methods for generating, characterising and exploiting YAC- based physical maps covering physical regions of 1 - 5 Mb to construct fine- scale cosmid-based maps for further analysis (Zuo et al., 1993; Nizetic et ah, 1994; W apenaar et al., 1994). Two low resolution techniques have been used in this project. The radiation hybrid approach fulfils two functions, isolation of chromosome fragments and marker mapping and is described in the preceding section. The second technique is fluorescent in situ hybridisation, FISH, which has played a very important role in mapping advances of the last decade.
Ch. 1 Introduction, Positional Cloning 42 Fluorescent in situ hybridisation
FISH enables the visualisation of hybridisation between two complementary DNA sequences, the probe (genomic DNA cloned in plasmids (Lichter et ah, 1988), cosmids (Lichter et ah, 1990b) or YACs (Driesen et ah, 1991)) and the target (metaphase chromosome spreads, interphase nuclei or DNA preparations). Hybridisation onto metaphase spreads permits the rapid localisation of unmapped sequences (Wiegant et ah, 1991). FISH of complex DNA probes to human chromosome preparations became possible with the introduction of fluorescent methods of detection (Albertson et ah, 1988; Bhatt et ah, 1988) and the technique of chromosomal in situ suppression (Lichter et ah, 1990b). Large and complex probes contain repetitive sequences which, if labelled without competition, produce unwanted, non-specific hybridisation. The addition of excess unlabelled total human DNA or DNA enriched for repetitive sequences, Cot-1 DNA, competes out the majority of the these sequences in the probe DNA, and a more specific signal is obtained. Lichter mapped cosmid clones from chromosome 11 onto extended (prometaphase) chromosomes and confirmed the localisation of the clones as assigned by Southern hybridisation of mapping somatic cell hybrid DNA. Hybridisation of two or more cosmids simultaneously enabled deduction of gene order (Lichter et ah, 1990a). High resolution cytogenetic maps of chromosomes 3, 12 and 5 have been constructed by visualisation of fluorescent signal on R-banded chromosomes (Takahashi et ah, 1992; Takahashiet ah, 1993a; Takahashiet ah, 1993b). FISH can facilitate the integration of physical, meiotic and cytogenetic maps since the FISH- mapped clones can include polymorphic markers or sequence tagged sites. For example, it has been used to order YAC or cosmid contigs on a chromosome, and to associate loci mapped by meiotic means to physical markers and chromosomal bands (Gingrich et ah, 1993; Green et ah, 1994). If decondensed, interphase nuclei are used as target, resolution may be increased by one to two orders of magnitude, permitting separation and ordering of markers 50 kb - 2 or 3 Mb apart (Lawrence et ah, 1988; Trask et
Ch. 1 Introduction, Positional Cloning 43 al, 1989; Lawrence et al., 1990; Trask et al, 1991). Ordering may be achieved either by comparing the average distance between pairs of probes or by the use of more than one fluorescent label (Trask et al., 1991), exploiting multiple colour FISH techniques (Nederlof et al, 1989; Nederlof et al, 1990). Multicolour FISH using three fluorophores together with digital image analysis permits the differentiation of as many as seven different probes (Ried et al, 1992) applied simultaneously, or the painting of chromosomes or chromosome fragments using paints constructed from fluorescent-labelled chromosome-specific cosmid libraries (Dauwerse et al, 1992). Markers separated by more than 1 Mb may be difficult to order using this technique because of the complex tertiary structure of some DNA in interphase nuclei (Den Dunnen et al, 1992). This complication may be avoided by techniques which make use of free chromatin extracted from the nucleus in vitro. This can be hybridised with multi-colour labelled markers to resolve sequences separated by only 21-350 kb (Heng et al., 1992). Even higher resolution may be achieved by the techniques of halo FISH or DIRVISH. In the former, DNA extrusion results in extended loops of chromatin around the nuclear envelope in the form of a halo (W iegant et al., 1992) which can be used to resolve markers less than 10 to 200 kb apart. Similar discrimination (<5->700 kb) is obtained with a technique of stretching DNA followed by FISH, DIRVISH (Parra et al., 1993). These methods complement alternative physical mapping techniques for generating long-range maps, and may rapidly identify gaps between contigs, visualise overlaps in cosmids and YACs and demonstrate YAC chimerism (Florijn et al, 1995). A novel technique "molecular combing", controlled extension of bare DNA on a silanised surface followed by FISH, is a development of these techniques. It has been used to make precise measurements of 10-150 kb, to identify and localise gaps within contigs and microdeletions in TSC2 in affected patients (Michalet et al, 1997).
The principal use of FISH in patient diagnosis has been the exploitation of the technique to delineate individual chromosomes and the chromosomal component of translocation- and marker- chromosomes (Pinkel et al.,
Ch. 1 Introduction, Positional Cloning 44 1988). Label "paints" have been obtained by inter -Alu repeat amplification of individual human chromosomes separated by fluorescence-activated sorting of preparations in metaphase (Cotter et al., 1991). A similar exploitation of the FISH technique has also been used to characterise the chromosomal component of hybrid cell lines by labelling with biotinylated total human DNA (Pinkel et al., 1986). Conversely, the human component of a hybrid may be used to probe normal metaphase spreads, either by labelling total hybrid DNA or by inter -Alu amplification of the hybrid, techniques known as reverse in situ hybridisation (Pinkel et al., 1988; Lichter et al., 1990a; Sinke et al., 1992).
Spectral karyotyping SKY or multiplex fluorescent in situ hybridisation M- FISH are recent developments of FISH which permit the differentiation of each of the 24 human chromosomes (Schrock et al, 1996; Speicheret al, 1996a). DNA probes for a given chromosome are all labelled with a combination of fluorescent dyes specific for that chromosome and are hybridised (together with similarly labelled markers for each of the other chromosomes) to metaphase spreads. By combinatorial probe-labelling, five fluors can generate 2^-1 different "paints", more than sufficient to label each chromosome with a unique spectral identifier. The spectral characteristics of each chromosome viewed through a charge-coupled device camera are decoded by a combination of optical fiters or interferometry and software to produce an image of the spread in which each chromosome pair is ascribed a unique false colour. These essentially similar techniques, SKY and M-FISH, are revolutionising tumour cytogenetics (where tumour chromosomes can be obtained) by facilitating the rapid identification of each chromosome in the karyotype, including the components of marker chromosomes. Previously this was not possible using cytogenetic banding and is laborious using conventional single fluor FISH (Speicher et al, 1996b).
Ch. 1 Introduction, Positional Cloning 45 Isolation and identification of transcribed seq u en ces
In the genome only 3-5% of genomic sequence is transcribed, and the identification of the needle of transcribed sequence in the haystack of intervening sequence and repetitive DNA is one of the essential steps in isolating genes on the basis of their chromosomal location. Many strategies have been devised to facilitate this process.
Traditional approaches
Early attempts used interspecific cross-hybridisation of genomic clones from candidate regions to identify interspecies-conserved segments or expressed sequences, which were then used to probe appropriate cDNA libraries to isolate candidate cDNAs (Monaco et al., 1986; Call et al, 1990). Adrian Bird's observation that undermethylated CpG-rich islands lie at the 5' end of many genes (especially those which are ubiquitously expressed, "housekeeping'' genes) provided another means of identification of potentially transcribed regions (Bird, 1986). This phenomenon was exploited to identify genes in the region of CFTR (Estivill et al, 1987; Rommens et al, 1989). Unfortunately, tissue-specific genes may not have associated islands, and where an island exists it may be distant from the associated gene.
Novel vectors such as yeast artificial chromosomes, YACs were developed in the late 1980's to clone very large segments of genomic DNA of several hundred kilobase pairs (Burke et al, 1987). Techniques were developed to suppress the contribution of repetitive sequences to filter hybridisation. These enabled the use of inserts from these vectors to probe cDNA libraries (Elvin et al, 1990; Snell et al, 1993). This approach led to the identification of the genes mutated in Norrie disease and Neurofibromatosis, type 1 (Wallace et al, 1990; Chen et al, 1992a; Chen et al, 1992b) and additional cDNAs from the HLA-A and Huntington's candidate regions (el Kahloun et al, 1993; Snell et al, 1993). A variation of
Ch. 1 Introduction, Positional Cloning 46 this strategy involves the construction of single chromosomal genomic libraries by identification of clones from monochromosomal somatic cell hybrids containing repetitive sequences unique to the donor parent. Careful depletion of repetitive sequences permits organ-specific cDNA libraries to be used as probes to identify clones from the genomic library containing cross-hybridising sequences (Hochgeschwender et al., 1989). This technique is particularly relevant where a disease locus has been mapped to a chromosome or chromosomal region from which such a genomic library has been made.
Some attempts have been made to isolate human heteronuclear RNA directly from somatic cell hybrids by selecting for RNAs containing repetitive sequences specific to H. Sapiens (Liu et al., 1989; Corbo et ah, 1990). Human cDNA synthesis is primed by oligonucleotides derived from Alu repeats (Corbo et al., 1990) or consensus 5' splice sequences (Liu et al, 1989). A subtractive hybridisation strategy has also been developed, based on the recognition that cDNA fragments from non-coding segments of mature human transcripts will not form stable heteroduplexes with their rodent homologues under high-stringency hybridisation conditions. This permits enrichment of human cDNAs from the region of non overlap of the human components within two sister hybrids, followed cDNA isolation by hybridisation of the enriched probe to a human cDNA library (Jones et al, 1992). The technique was used to clone the Rl-alpha regulatory subunit of cAMP-dependent protein kinase, the product of the tissue-specific extinguisher-1 TSE1 locus on human chromosome 17 (Jones et al, 1991). The efficiency of each of these approaches is limited by the low level of expression of many tissue-specific genes in hybrid cell lines.
Exon trapping
The classical hybridisation-based techniques for the identification of transcribed sequences in cloned genomic DNA are labour intensive and are limited by the difficulty of ensuring adequate suppression of repetitive sequences. In the last decade, approaches have exploited the specificity of
Ch. 1 Introduction, Positional Cloning 47 PCR to isolate sequences containing splice sites (Auch et al., 1990; Duyk et al, 1990; Buckler et al, 1991; Hamaguchi et al, 1992; Ozawa et al., 1993; Nehls et al, 1994) or 5' terminal exons (Krizman et al., 1993; Nisson et al., 1994). Exon trapping can be used to screen large regions of genomic DNA and it does not depend on expression for exon identification; it has been used to isolate numerous genes in the last few years, some of which are listed in Table 1.2, below.
Hyper glycerolaemia GK1, glycerol kinase (Walker et al, 1993) Neurofibromatosis, central, II NF2 (Trofatter et al, 1993) Huntington's chorea HD, Huntingtin (MacDonald et al, 1993) Menkes disease ATP7A (Vulpe et al, 1993) Usher syndrome, type IB MY07A, Myosin 7a (Gibson et al, 1995) ATP50 (Chen et al, 1995c) SIM (Chen et al, 1995b) GABPA (Chrast et al, 1995) MAGEL1 (Muscatelli et al, 1995) TIAM1 (Chen et al, 1995a) VAV2 (Henske et al, 1995) IFP35 (Brown et al, 1995) NUP98, Nucleoporin (Nakamura et al, 1996)
Table 1.2: Representative genes isolated by exon trapping.
In addition, the technique has been used to isolate other genes from the HD region (Ambrose et al, 1992; Taylor et al., 1992; Duyao et al, 1993), exons from regions of human chromosome 6, the MHC locus (North et al., 1993), chromosome 9q (Church et al, 1993; Churchet al, 1994) and chromosome 17q21, the BRCA1 locus (Brody et al., 1995; Brown et al, 1995).
Ch. 1 Introduction, Positional Cloning 48 cDNA selection
This technique attempts to select cDNAs showing some homology to cloned genomic DNA from a candidate region. The genomic clone may be in the form of cosmids, or YACs containing inserts of up to a megabase of DNA. As originally described the genomic clone or clones are immobilised on filters to which are hybridised cDNA clones from an appropriate cDNA library (Lovett et al., 1991; Parimoo et al., 1991). cDNAs which show non-specific binding are eliminated by washing from the filter leaving the specific hybridising cDNAs which are then eluted separately. Inserts from these cosmids are amplified by PCR and the procedure repeated using the amplified inserts to give further enrichment. Repeat sequences in either of the probe cDNA (Lovett et al., 1991) or target genomic DNA (Parimoo et al, 1991) have to be blocked in order to prevent non-specific hybridisation. A single round of selection-amplification may produce a 1000-fold enrichment of a cDNA known to be present in the cDNA library (Parimoo et al., 1991). Exploitation of biotin-streptavidin capture systems in solution has further increased the enrichment possible using this technique, up to 105-fold after two cycles of enrichment (Korn et al, 1992; Morgan et al, 1992; Tagle et al, 1993). Pseudogenes and low copy number repeat sequences in the genomic template are also selected and the resulting cDNA libraries demand careful characterisation. cDNA selection has been used in many attempts at gene isolation. One early success was the isolation of the Bruton agammaglobulinemia tyrosine kinase, BTK gene, mutated in children with X-linked agammaglobulinaemia (Vetrie et al, 1993, Vorechovsky, 1993 #423). Similar hybridisation techniques have been used between species to generate collections of sequences conserved in mouse and pig using human cosmids from Xq28 (Sedlacek et al, 1993).
Transcription maps
Using selection techniques it is possible to isolate a significant proportion of the expressed sequences in a region of interest. These cDNAs may be localised relative to one another to form a transcription map (Gecz et al,
Ch. 1 Introduction, Positional Cloning 49 1993, Sedlacek, 1993 #382). Large scale transcription mapping projects have often formed part of long-standing gene isolation projects where the gene has eluded conventional mapping techniques. Rommens et al. localised 58 transcripts from the Hunting Ion disease candidate region at 4pl6.3 to 9 aligned transcription units (Rommens et al, 1993). Totaro et al. integrated 14 transcription units in a 1.2 Mb region around HLA and isolated 13 novel cDNA fragments in an attempt to isolate the haemachromatosis gene on 6p (Totaro et al., 1996). Developments permitting FISH mapping of cDNAs at reasonably high resolution (2-5 Mb) may facilitate the generation of transcript maps (Korenberg et al., 1995).
cDNA Sequencing
STSs, Sequence Tagged Sites, are short regions of sequenced DNA, from a larger section of cloned and mapped DNA. The utility of these segments was recognised in the late 1980's and they have since become standard markers in physical mapping. Coding sequence represents the most immediately useful information content of the genome, however, and several groups adopted a strategy of partial sequencing of cDNA clones selected at random from cDNA libraries rather than of random genomic DNA. The partial sequences are known as ESTs, expressed sequence tags. Automation of DNA sequencing has facilitated this approach and made the strategy economically feasible. The sequence data is incomplete and not error-free, but it is sufficiently accurate for the sequences to be used as markers in gene mapping, or in positional-candidate cloning projects (Wilcox et al, 1991; Berry et al, 1995; andiu.).
An early example of this technique was that of Adams et al. who partially sequenced over 600 randomly selected human brain cDNA clones. 289 represented novel genes, without homology with other sequences in the GenBank database. 48 had significant similarity to genes from other organisms. 46 of the ESTs were mapped by PCR of somatic cell hybrid DNA (Adams et al, 1991; Polymeropoulos et al, 1992). Subsequent papers from this group have reported the sequencing of increasing numbers of
Ch. 1 Introduction, Positional Cloning 50 novel ESTs: 2375 (Adams et ah, 1992), 3400 (Adams et ah, 1993a), 1600 (Adams et ah, 1993b). In the most comprehensive review, 174,472 ESTs comprising over 52 megabases of DNA were sequenced. These were combined with a further 118,406 ESTs from the database dbEST. Of the total, 87,983 were distinct, and 77,769 identified previously unknown genes (Adams et ah, 1995). It is estimated that approximately half of all human genes had been sampled as of 15 June, 1996 (http://www.ncbi.nlm.nih.gov/SCIENCE96/) and that there were a total of about 800,000 ESTs (mostly unmapped) available in public databases in 1997 (Rowen et ah, 1997). This strategy has also been exploited in the mapping of other organisms, such as Caenorhabditis and Schistosoma (McCombie et ah, 1992a; Franco et ah, 1995).
In order to facilitate the positional-candidate cloning of genes, each EST (or EST contig) should be mapped at a resolution of 0.5 - 5.0 Mb. Mapping at this scale may most easily be achieved with large insert clone libraries, such as gridded-array YACs, PACs and BACs, or by using radiation hybrids (v.s.). The EST Mapping Consortium of the Universities of Stanford, Oxford, Cambridge and MIT is using these mapping resources to map to <0.5 Mb intervals up to 50,000 ESTs (Schuler et ah, 1996a; Schuleret ah, 1996b).
Genomic DNA sequencing, PI recombinants and BACs
The traditional technique for the analysis of genomic DNA is "shot-gun" sequencing, where the DNA is broken down into fragments of appropriate length to be subcloned into a sequencing vector, such as bacteriophage M13. Computer programs e.g., Staden and GCG facilitate the matching of sequence to form regions of overlapping sequence (Gleeson et ah, 1991; GCG, 1994). Several groups have used sequencing strategies to analyse human genomic DNA cloned in cosmids, e.g. 58 kb in the Huntington disease region on chromosome 4pl6.3 (McCombie et ah, 1992b), 116 kb of 19ql3.3 (Martin-Gallardo et ah, 1992) and 180 kb at the retinoblastoma locus (Toguchida et ah, 1993). Latterly, the complete T cell receptor beta locus has
Ch. 1 Introduction, Positional Cloning 51 been sequenced from a cosmid contig amounting to 685 kb, the largest continuous stretch of human DNA analysed at the time (Rowen et al., 1996). More recently, a third generation of clone resources has been used to support mapping and sequencing projects, Pl-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) libraries (Shizuya et al., 1992). The usefulness of BAC and PAC clones was illustrated in 1994 and 1995 when the first breast cancer susceptibility gene, BRCA1, was identified from a BAC clone after other clone resources had failed (Miki et al, 1994). The following year a PAC contributed to the identification of the second breast cancer susceptibility gene, BRCA2 (Wooster et al., 1995). BAC clones are a preferred cloning vehicle at present because of an average insert size of 120 kb and a small vector-to- insert ratio (<1:20), compared with 75 kb and 1:5 for PACs (Boehm, 1998).
These random techniques result in significant redundant sequencing. In order to reduce this, variations have been introduced: ordered shotgun sequencing of minimally overlapping plasmids (Chen et al., 1993), and genome sample sequencing (Smith et al., 1994). This latter technique requires the construction of an ordered, high-density cosmid, BAC or PAC contig; the ends of each clone is then sequenced. The eventual sequence represents only 30-50% of the complete DNA, but permits the isolation of genes in the region and the amplification of the majority of the intervening, un-sequenced DNA, if desired. The cost of conventional sequencing is falling rapidly, but partial sequencing strategies like these are being adopted not only to reduce cost but also to expedite large-scale sequencing projects. Identification of coding regions within the "haystack" of DNA sequence is facilitated by computer programs, e.g. BLAST, BLASTX and GRAIL (Uberbacher et al., 1991; Gishet al, 1993) reviewed in (Altschul et al., 1994).
Cloning by complementation
In rare cases, cloned DNA containing coding sequence may be identified by its ability to remedy a deficiency present in a cell line from an affected
Ch. 1 Introduction, Positional Cloning 52 patient. Fanconi's anaemia, type C, FAC, was cloned by isolating cDNA from an episomal expression library that complemented the sensitivity to DNA crosslinkers of lymphoblasts from FAC patients transfected with the cDNA (Strathdee et al., 1992; Buchwald, 1995). This approach does not always provide helpful information. In an attempt to clone the Ataxia Telangiectasia gene, Jung et al isolated cDNA that corrected the radiation sensitivity and DNA synthesis defects in fibroblasts from an ATI group D patient by expression cloning, and showed that the cDNA encoded NFKBI, a truncated form of I-kappa-B. Unfortunately, this locus was localised to chromosome 14 and the ATD locus mapped to chromosome 11 and has since been cloned (Jung et al., 1995; Savitsky et al., 1995).
Positional-candidate cloning strategies
Candidate by mapping in the human genome
An increasingly common means of linking causative mutation to disease has been through the identification of mutations in a gene previously mapped to the chromosomal region to which a clinical condition has been located, and where the function or expression-pattern of the gene indicates that it is a possible candidate for the disorder. The identification of the fibroblast growth factor receptors (FGFR) as genes mutated in the craniosynostoses and in achondroplastic dwarfism is a good example. Crouzon's craniosynostosis had been mapped to 10q25-q26 by linkage methods (Preston et al, 1994). The knowledge that FGFR2 had a similar chromosomal location and was expressed in relevant tissues such as cartilage of developing skull led the group at the Institute of Child Health, London to screen selected exons and to identify mutations in exon B in several affected individuals (Reardon et al, 1994).
The molecular characterisation of achondroplastic dwarfism, ACH, followed a similar process: the identification of FGFR3 during the screening of expressed sequences on chromosome 4p in the search for the Huntington Disease locus (Thompson et al, 1991), m apping by linkage of
Ch. 1 Introduction, Positional Cloning 53 ACH to 4p (Francomano et al., 1994; Le Merrer et al., 1994; Velinov et al., 1994) and the demonstration of the Gly380Arg substitution in the transmembrane domain of FGFR3 in all patients studied (Shiang et al., 1994). Subsequently, some forms of thanatophoric dysplasia were shown to be due to mutations in the same gene (Tavormina et al., 1995). Pfeiffer craniosynostosis (FGFR1, 8pl2-pll.2), Jackson-Weiss syndrome (FGFR2) and Aperts acrocephalosyndactyly are further examples of gene identification which have resulted from the co-localisation of a syndrome (by linkage) and a candidate FGFR gene. Knowledge of the chromosomal location of members of the FGFR family has been an essential prerequisite for these to be considered as candidate genes for these disorders of osteogenesis. Further examples are listed in Table 1.3 below (amended from Collins, 1995):
Alzheimer's disease APP, apoE etc. Amyotrophic lateral sclerosis superoxide dismutase, SOD1 Charcot-Marie-Tooth, type 1A (HMSN1A) peripheral myelin protein 22, PMP22 Charcot-Marie-Tooth, type IB (HMSN1B) myelin protein zero, MPZ Familial hypertrophic cardiomyopathy cardiac myosin heavy chain, MYH7 Familial melanoma pl6 (CDKN2) Hereditary haemorrhagic telangiectasia, type 1 endoglin, ENG Hereditary non-polyposis colon cancer MSH2, MLH1, PMSL1, PMSL2 Hyperekplexia glycine receptor, a l subunit, GLRA1 Long QT syndrome SCNSA, MERG cardiac ion channels Malignant hyperthermia ryanodine receptor, RYR1 Marfan syndrome fibrillin 1, FBN1 Multiple Endocrine Neoplasia, type 2A receptor tyrosine kinase, RET Supravalvar aortic stenosis elastin, ELN Retinitis pigmentosa multiple, inc. peripherin, rhodopsin Waardenburg syndrome homeobox gene PAX3
Table 1.3: Representative inherited disease genes identified by positional candidate methods (from Collins, 1995).
Ch. 1 Introduction, Positional Cloning 54 An early example of the exploitation of a mapped EST as a candidate gene was the identification of glycerol kinase, GK1, as the gene mutated in hyperglycerolaemia (Sargent et al., 1993). An EST from a human testis cDNA library was found to have 60% identity to a Bacillus subtilis glycerol kinase gene and mapped to Xp22.1-p21.2, the candidate region within which the hyperglycerolaemia gene was known to lie. The GK1 sequence was found to be deleted in two patients with the disorder. The gene was also isolated by more conventional positional cloning strategies, including exon trapping (Walker et al., 1993). Presuming that the majority of gene transcripts are represented as ESTs, the positional-candidate strategy is likely to become the principal method of cloning genes involved in disease states in future, as the density and resolution of the EST maps increases (Collins, 1995). This approach complements the genomic sequencing strategy: data from clones spanning a critical region may be used to scan the dbEST database at NCBI to identify potential candidate cDNAs rapidly.
Candidate by cross-species phenotype correlation and synteny (syntenic candidate)
Model organisms, such as Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans,Fugu rubipres and M us musculus have served as invaluable models for the development of the strategies needed to analyse the more complex human genome. Study of these organisms has provided much useful information about gene regulation, developmental processes, genetic diseases and evolutionary mechanisms. It has demonstrated that even widely diverged organisms, such as C. elegans and H. sapiens, may show gene conservation which may be exploited to identify functional coding sequence (McCombie et al, 1992a). These similarities are even more apparent in the puffer fish, Fugu, a prototypic vertebrate. It has a representative complement of vertebrate genes but a genome which is only about a seventh the size of the human (Brenner et al, 1993; Elgar et al, 1996). If synteny can be established
Ch. 1 Introduction, Positional Cloning 55 between a region of human and puffer fish genomes, genomic sequencing of the region in Fugu can rapidly produce a fish gene inventory and aid identification of the human orthologues (Trower et al, 1996; Schofield et al., 1997). The extent to whichFugu is representative of the human genome remains to be determined and it is possible that a Fugu inventory from a "syntenic candidate" region may miss genes unique to mammals. However, the exon-intron structures of individual genes isolated from Fugu are closely related to their human counterparts and coding regions with high aminoacid conservation have helped to identify possible functional domains (Maheshwar et al., 1996).
Comparison of human maps and those of other mammals have identified many regions of synteny which have allowed predictions about the localisation of unmapped loci (in the other species). The increasing density of mammalian maps is likely to facilitate this, although detailed studies have revealed small rearrangements within regions of overall synteny (Johansson et al., 1995; Pilz, 1995). Some examples of the mapping of murine genes which have informed human genetics are described below.
Splotch/Waardenburg
Waardenburg syndrome is an autosomal dominant disorder in which there is a defect in pigment synthesis in the hair cells of the cochlea causing deafness, associated with partial albinism, a white forelock and facial dysmorphism. Mutations in Pax-3 were identified in mice from the mutant strain Splotch (Epstein et al., 1991). Splotch and Pax-3 had been mapped to mouse chromosome 1 in a region syntenic to human chromosome 2q. The human homologue of Pax-3 (infromally known as HUP2) was found to map to the same region of 2q37.3 as the locus for Waardenburg syndrome. Recognition of the similarities of phenotype and relative map position led to the screening of HUP2 and the identification of mutations in patients with Waardenburg syndrome (Tassabehji et al., 1992).
Ch. 1 Introduction, Positional Cloning 56 shaker, Shl/Myosin VIIA/USH1B,DFNB2 deafness
Another example of the identification of a gene involved in human syndromic deafness by this approach is the demonstration of mutations in the Myosin VIIA gene as the cause of deafness in both the shaker-1 mutant mouse (chromosome 7) and in individuals with Usher syndrome, typelB which maps to the syntenic region of human chromosome llql3.5 (Kimberling et al., 1992; Gibson et al., 1995; Weil et al., 1995). Further examples of syntenic candidate mapping are the Trembler mouse/PMP22/CMTlA identification (Low, 1976; Lupski et al., 1991; Patel et al., 1992; Suter et al, 1992), FID and Hdh (Nasir et al, 1994) and OB and ob (Zhang et al, 1994). A useful database resource for genome cross- referencing, XREFdb, is available, http://www.ncbi.nlm.nih.gov/XREFdb/, (Bassett et al, 1997).
Analysis of candidate genes
Isolation of coding sequences from a region of interest is a critical - but intermediate - step in the identification of candidate genes. Detailed description of the methodologies employed is outside the scope of this thesis. A brief resume follows.
An important initial screen is to determine the expression pattern of the transcript by Northern hybridisation to ensure that the message is present in organs in which the true gene is likely to be expressed (tissues known to be affected by the disease, for example). Qualitative differences may be seen in patients compared with normal controls (Nellist et al, 1993). However, a disease which appears to be confined to one organ system may be caused by mutations in a gene with widespread tissue expression, as exemplified by huntingtin and emerin (MacDonald et al, 1993; Bione et al, 1994).
Ch. 1 Introduction, Positional Cloning 57 Large-scale rearrangements may be detected in genomic DNA from affected individuals by PFGE or Southern hybridisation using the candidate gene as probe. Identification by PFGE of overlapping deletions at 16pl3.3 in patients with tuberous sclerosis was central to the successful cloning of TSC2. Four genes were isolated from the 120 kb region of overlap, only one of which, TSC2, was interrupted by all five PFGE deletions (Nellist et ah, 1993). Restriction fragment pattern abnormalities were detected in Taq blots of DNA from 8 of 33 families with X-linked agammaglobulinaemia using probes derived from the candidate transcript, BTK (Vetrie et ah, 1993). Triplet repeat expansions may be demonstrated in the same way (Verkerk et al., 1991; Yu et al., 1991) even before the identification of the gene product (Knight et al., 1993; Orr et al., 1993; Banfi et al., 1994).
The majority of gene "hunts" have required the more laborious strategy of a systematic search for mutations in the candidate gene in DNA from patients. In diseases inherited in a dominant manner, the analysis of samples from individuals without a family history (sporadic or new mutations) is critical. Comparison of sequence from the proband and the parents facilitates the differentiation between causative mutations and polymorphisms.
A number of techniques for rapid mutation detection have been devised to avoid the effort of direct sequencing of the complete candidate gene sequence in affected individuals. All current methods are dependent upon PCR and have been reviewed (Rossiter et al., 1990; Cotton, 1993). DGGE, CCM and SSCP are three approaches which have proved to be particularly useful and have been widely used to detect mutation at many loci; an early example is (Kogan et al., 1990). Denaturing Gradient Gel Electrophoresis permits the identification of single base changes in an allele by the alteration of characteristic denaturing patterns when the duplex is electrophoresed (Myers et al., 1985a; Myers et al, 1985b; Myers et al, 1985c). Chemical Cleavage Mismatch uses the chemicals hydroxylamine and osmium tetroxide modify single base pair mismatches
Ch. 1 Introduction, Positional Cloning 58 in duplexes formed between the target and wild-type DNA; any mismatches are then cleaved (Cotton et al., 1988). Single Strand Conformational Polymorphism detects sequence differences by the change in electrophoretic ability in neutral, non-gradient gels (Orita et al., 1989a; Orita et al., 1989b). These techniques identify the approximate site of a mutation or polymorphism within a sequence of 200-600 bases. Once a change has been detected, DNA from other individuals can rapidly be examined and the region sequenced.
Other screening techniques, e.g. the Protein Truncation Test, depend on identification of translation-terminating mutations in in vitro translated mRNA (Roest et al., 1993; van der Luijt et al., 1994). PTT is rapid but the detection of mutations leading to aminoacid substitutions by this technique is more complex (requiring isoelectric focussing of the protein or mass-spectrographic analysis of peptide fragments). However, in many diseases a large proportion of mutations result in truncation and so may be identified using PTT.
Positional cloning of TSC
Linkage analysis
The history of TSC mapping as been chequered by confident assertions of linkage (to four different chromosomes) followed by more cautious reassessment following the discovery on unlinked families. The posters and papers containing mapping data published prior to the onset of this study are presented in detail below.
The first report of significant linkage of a gene for TSC was to the ABO blood group system, which mapped the gene to distal chromosome 9q (Fryer et al., 1987). It confirmed and extended the suggestion of linkage between TSC and ABO detected in Scottish families in which a lod score of 1.46 was found at zero recombination (Connor et al., 1987c). This earlier
Ch. 1 Introduction, Positional Cloning 59 linkage study of TSC had been published the same year (using Scottish families) but had failed to provide evidence for significant linkage to any marker. In the larger Fryer study, which included four of these Scottish families, nineteen families were examined for the presence of symptoms or signs of TSC according to the Gomez criteria and at-risk, unaffected adults were investigated using renal ultrasound, skeletal survey and cranial CT scan. Twenty-six markers (blood groups and protein polymorphisms) were used, of which only two, ABO and AK1, gave lod scores with TSC over 1.0. ABO was informative in eight families, there were no recombinants with TSC and the maximal lod score (Zmax) at zero recombination was 3.85. Confidence limits for the recombination fraction were 0.00-0.10. Sexes were not considered separately in the analysis since there were no recombinants. In one family not informative for ABO, a Zmax of 1.20 was obtained with the nearby protein polymorphism AK1 (at zero recombination, confidence interval 0.00-0.42). One family informative for both ABO and AK1 was excluded from the analysis because the clinical status of a critical family member could not be fully ascertained, due to the presence of a solitary renal cyst. Such solitary cysts are not uncommon in normal, unaffected individuals and if the individual were to have been assumed to be unaffected, the family would have supported linkage of TSC to both ABO and AK1.
The authors raised the possibility of antenatal diagnosis in families informative for ABO, but cautioned against too hasty exploitation of the association because of the need to exclude genetic heterogeneity. AK1 was felt to be a less useful marker since the polymorphism was informative in less than 10% of families.
This caution was shown to be well founded by a report from Hope Northrup's group at Baylor College, published six months later (Northrup et al, 1987). They studied six families of which three were informative with ABO. Two of the three gave negative lod scores with ABO and the overall scores for the three families were negative at recombination fractions between 0.001 and 0.2. One of the two families was considered in
Ch. 1 Introduction, Positional Cloning of TSC 60 detail, because of two phase unknown recombinants in unaffected children who had been fully investigated, including renal ultrasound and cranial CT; non-paternity was excluded. When combined with the Fryer data, the maximum lod score for the eleven informative families was reduced from 3.85 to 2.01 at a recombination fraction of 0.1. It was suggested that the discrepant results might indicate linkage with ABO but at a higher recombination fraction, "diagnostic problems" or locus heterogeneity. The first suggestion was perhaps a polite concession to the original authors since the data the Baylor group presented ruled out linkage in a least one of their three families. The second was unlikely, given the adoption of the Gomez criteria and a very similar strategy for the investigation of clinically normal, at-risk individuals. The third possibility, the presence of more than one locus for TSC, subsequently proved to be the correct explanation.
Later in the same year of publication of the Fryer report, some of the authors (those from Glasgow) described linkage between TSC and a RFLP detected by a 1.9 kb Pstl-digested fragment of v-abl, ABL (Connor et al., 1987b). The abl oncogene is translocated from 9q34 to chromosome 22 in chronic myeloid leukaemia to form the BCR-abl transgene on the Philadelphia chromosome. Six families were studied with this RFLP of which three were informative. In these there were thirteen informative meioses (four phase known). No recombination between TSC and the ABL RFLP was seen, and the maximal lod score was 3.18 at zero recombination. Five at-risk but clinically normal family members were excluded from the analysis because they had not had renal ultrasound, radiological skeletal survey and cranial CT scanning unlike the other unaffected individuals. Had they been included (as normal), there would have been five additional informative meioses and the maximal lod score would have been 4.64 at zero recombination (sexes taken together in both calculations). The Glasgow group commented that this DNA marker would be more useful for early prenatal diagnosis than either of the ABO or AK1 (glyco)protein polymorphisms, and added a first trimester prenatal exclusion had been performed in one of the families. This was confirmed
Ch. 1 Introduction, Positional Cloning of TSC 61 in a letter to the Lancet (Connor et al., 1987a), of which the Baylor group had commented in the light of their own data, "...it is premature to use linkage analysis for prenatal diagnosis of TS until the linkage relation is proven more conclusively and recombination distances are better defined."
The optimism about the use of this RFLP for diagnosis was again shown to be unfounded in the following year, when the London and Leeds groups described the three families which had been found to be informative for ABL of the 30 studied, figures which illustrate the low frequency of the less common allele (Povey et ah, 1988). Two of these three families showed recombination between TSC and ABL. The data when combined with those of Connor 1987 reduced the maximum lod score to 2.36 at a recombination fraction of 0.1. Like the Baylor group the previous year, they suggested two possible explanations for the recombinants, "that TSC is loosely linked to abl in all families or that in some families the mutation for TSC is not in the 9q linkage group at all." Unfortunately, neither of the two families in which recombinants were seen were informative for ABO or AK1, so it was not possible to determine which of the two explanations was the more likely.
In the latter part of 1987 the US NTSA Collaborative Project groups at Irvine, California and Indiana presented two similar posters at HGM9 and the American Society of Human Genetics conference which provided conflicting evidence for linkage to markers on chromosome 9q. The first poster (ASHG) presented data on fifteen families studied using twenty polymorphic blood group, protein and DNA markers (Smith et al., 1987a). Only one marker, an ASS pseudogene ASSP3 previously m apped to 9 q ll- q22, gave evidence of linkage to TSC. It was informative in five families, no recombinants were seen and the maximal lod score was 2.2. Seven of the families were informative for ABO, but the lod scores were negative at theta values between 0.0 and 0.2 (Z/theta-5.34/0.0, -1.73/0.1, -0.425/0.2). In three of the seven families lod scores were positive with a cumulative maximal score of 1.22 at zero recombination. The abstract did not mention
Ch. 1 Introduction, Positional Cloning of TSC 62 whether these positive scores occurred in those families which showed evidence of linkage between ASS and TSC. They concluded that locus heterogeneity was a possible explanation.
The second abstract reported very similar work in twenty-two families (Smith et ah, 1987b). Low positive lod scores were obtained with three markers on chromosome 9: AK1, ASSP3, ABO. Fourteen families were informative for ABO, in seven lod scores were positive and the maximal cumulative lod score in these was 2.43 at a recombination fraction of zero. Recombination in the three other families was such that overall scores in the ten families were negative at recombination fractions less than 0.2 (Z/theta -8.3/0.0, -1.44/0.1, -0.3/0.2). This abstract also raised the possibility of locus heterogeneity as an explanation for the conflicting results and in addition suggested the existence of one or more secondary modifying loci to account for the variability in phenotype within families.
The research group of Raymond Kandt at Duke University provided further evidence consistent with heterogeneity in their abstract presented at the American Society of Human Genetics meeting in 1988 (Kandt et ah, 1988). Eight famililes were studied including two obtained from the Camden Repository (also included in the Collaborative Project analysis; ABO typing was not performed on these). One of the six unique families was a large multigenerational kindred from New Zealand. Three loci were studied, ABO (in 83 individuals), and ABL and DllS10/pMCT136 (in 105 individuals). Multilocus linkage analysis (assuming penetrance at 90%) excluded linkage (lod < -2.00) of TSC within 32 cM around the ABO locus (14 cM centromeric and 18 cM telomeric).
This study was formally reported the following year (Kandt et ah, 1989), with the addition of a further family. Fourteen DNA markers (twelve on chromosome 9, two on chromosome 14) and nine blood group antigens and enzyme markers (chromosomes lp, lq, 2p, 4q, 6, 9q, 16q, and 18q) were used. Four families were informative with ABO, of which three gave negative lod scores (with recombinants in affected individuals in each
Ch. 1 Introduction, Positional Cloning of TSC 63 family). In the fourth, the maximal lod score was 0.83 at a recombination fraction of 0.05 (sexes taken together). Two point analysis of results from all four families excluded linkage within 5 cM either side of ABO (Z/ theta <-2/0.001). Recombinants between TSC and ABL were found in affected members of one family. No markers gave significant positive lod scores in the two point analysis: low positive scores were obtained with loci on 2p, 9p, 14q and three loci on 9q (Zmax with EKZ19.3 0.47, theta 0.15). Multilocus linkage analysis excluded TSC within a 20 cM interval. Exclusion mapping using John Edwards' program Exclude gave a 1% probability that there was a TSC locus on chromosome 9 (and in the light of later developments, probabilities of 5.5% on chromosome 11 and 2.9% on chromosome 16). The authors concluded that, "The only region of chromosome 9 likely to yield a TSC locus is 9p; i.e. the region that was not saturated with probes....the entire long arm has been excluded in this set of families."
In addressing possible reasons for the discrepancy between their results and the original report of linkage to ABO, they considered a founder effect in the UK population reported by Fryer et al., clinical misclassification and heterogeneity. A founder effect in any country is unlikely given the high new mutation rate in TSC (approximately 60% of individuals with TSC are thought to be new mutations). Also, one of the three families informative with ABO but showing recombination beween TSC and the locus was of British ancestry. Misclassification may have occurred in either analysis, but was more likely to have taken place in the Duke study since only one- third of the at-risk, but clinically unaffected individuals (10 of 32) had cerebral CT scans and only three of the thirty-two underwent renal ultrasound, whereas all at-risk family members in the Fryer study had both CT and renal ultrasound. This was partially taken into account by the setting of penetrance in the Duke analysis to 90% (c.f. Fryer 98%). Not having found any evidence of linkage to any of the loci studied, they pointed out that their data did not provide sufficient evidence for rejection of the null hypothesis of homogeneity, and commented that the linkage to ABO in British families might have occurred by chance!
Ch. 1 Introduction, Positional Cloning of TSC 64 The first report of significant linkage to markers on a chromosome other than 9 took place at the Tuberous Sclerosis Association meeting at Nottingham in 1988 where Moyra Smith (Irvine) described preliminary studies using markers on llq. One initial clue to the possibility of a locus on chromosome 11 was the finding of an unbalanced 11/22 chromosome translocation in a neonate with pathognemonic features of TSC who died within an hour of birth. At post-mortem examination the child was found to have multiple cortical tubers, subependymal astrocytomas and cardiac rhabdomyomas. The case was presented later that year at the American Society of Human Genetics meeting and in greater detail at the New York Academy of Sciences and National Tuberous Sclerosis Association meeting in April 1990 (Clark et al., 1988; Clark, 1991). The mother of the child had a de novo balanced translocation t(llq23.3;22qll.2), and the child was trisomic for 22pter-22qll.2 and distal llq (llq23.3-qter). This was the first case of TSC in the family. Two sibs of the proband (one full sib and one half sib, from a different father) shared the same balanced translocation as the mother. Since the mother (nor the father) had any evidence of TSC on intensive investigation and the sibs were also normal, the criticism has been raised that the neonate was a new TSC mutation and the trisomy was an incidental finding, derived from the constitutional reciprocal translocation t(ll;22) in the mother and unrelated to the aetiology of the TSC. The authors discussed this issue in the later report, "If the t(ll;22) disrupted the gene for TSC at either llq23.3 or 22qll.2 other family members carrying the balanced translocation should express the disease. If overexpression or underexpression of the gene caused TSC, other reports of similar individuals with this derivative chromosome 22 should demonstrate the disorder." Somatic mutations involving translocations which led to the overexpression of adjacent genes had been recognised as a mechanism of oncogenesis since the early 1980's (de Klein et al., 1982), but overexpression was at the time a novel mechanism to invoke to account for an inherited disease like TSC. Since then, gene duplication has been recognised as a mutational mechanism, e.g. (Valentijn et ah, 1992). The precise location of the breakpoints on
Ch. 1 Introduction, Positional Cloning of TSC 65 chromosome 11 and 22 in the proband is still unclear, but there is some unpublished evidence to suggest that it is not at the usual site of the constitutional translocation, which might lend some support to Clark's contention that it is pathogenetic and not an epiphenomenon (M. Smith 1991, personal communication).
The association of TSC and a translocation involving llq23 encouraged the local TSC research group, led by Moyra Smith to test for linkage between TSC and polymorphic markers in that region of the chromosome in their families. These results were presented at the Nottingham meeting and in poster form at ASHG and HGM10 (Flodman et al., 1989; Smith et al., 1989); they were published two years later (Smith et ah, 1990).
The poster at ASHG (Flodman op. cit. prepared prior to the HGM10 abstract) presented results on 9 families (104 individuals, 66 affected) using four markers from 9q34 (ABO, AK, ABL, ASSG3) and five from llq22-23 (S144, S351, APOA1, S147, S350). Of the four families which showed recombination with 9q34 loci, three showed "evidence of linkage to llq loci" and there was no data on the fourth. Conversely, there were four families in which there was recombination between TSC and the llq22-23 markers. In three there was linkage with ABO and the fourth was not informative. In one family lod scores were positive for both chromosome 9 and 11 markers. Multipoint linkage analysis with three of the 11 loci (S144, S351, S350) in four families placed TSC on chromosome 11 at D11S144 (zero recombination) with a lod score of 4.558. Analysis of the four "recombinant" families did not provide any evidence for linkage with these loci.
The poster at HGM10 (Smithet al., 1989) gave results of the analysis of 16 families using 35 markers. Cumulative lod scores in all families for each of four chromosome 9 markers were negative. In the six families in which there was linkage to ABO, the Zmax was 1.79 at zero recombination. Ten families were informative for chromosome 11 markers with Zmax for D11S144 of 3.264 at theta 0.08 (D11S351 1.008 at theta 0.09), and in six of
Ch. 1 Introduction, Positional Cloning of TSC 66 these lod scores were positive. Four of the six had negative scores with ABO or AK1. Two of the four families in which scores were negative with chromosome 11 markers had a conditional probability of being ABO- linked of greater than 0.6. When these families were removed from the analysis the Zmax for D11S144 in the remaining eight families rose to 4.56 at theta 0.07 (D11S351 2.17 at theta 0.06). HOMOG tests were consistent with heterogeneity. The authors concluded, "Our study establishes that there is a locus for TSC that maps to llq22-23."
The publication from Moyra Smith's group which appeared in Genomics in 1990 was first submitted in May 1989, before either the ASHG or HGM10 abstracts appeared, which may explain the discrepancies between the article and the later, HGM10 poster (Smith et ah, 1990). 15 families were studied, 14 with chromosome 9 markers (in effect ABO only, since ABL, ASSG3 and AK1 were each performed in 3 or fewer families) and 10 with chromosome 11 markers (D11S351 in 9 families, D11S144 in 8, D11S350 in six, TYR, S147, HBI18P1 in three or fewer families). The reason for the partial analysis was not because probes were not informative in the families which were omitted but because there was insufficient DNA for some later analyses. Overall two-point lod scores for chromosome 9q34 markers did not "support assignment of the TSC gene to this region". There was no detailed analysis of those of the 14 families which showed linkage to ABO ( c.f. the HGM10 poster). Linkage was obtained in the 8 families studied with D11S144 (Zmax 3.26 at theta 0.08) and the three families studied with TYR (Zmax 2.88 at theta 0.00). A multipoint analysis using the marker order centromere-TYR-S144-S350-telomere placed TSC at TYR as four times more likely than the order S144-TSC-S350 and sixty- three times more likely than S144-S350-TSC. A USERM9 MENDEL test on the marker data TYR, D11S144 and D11S350 did not provide any evidence for heterogeneity (a TSC locus not linked to those markers) which is suprising given the conclusions of both posters from the same group described above, (the title of their HGM10 poster being, "Evidence for genetic heterogeneity in tuberous sclerosis: one gene maps to the 9q34 region and a second gene maps in the Ilq22-llq23 region").
Ch. 1 Introduction, Positional Cloning of TSC 67 The article states that there was no evidence for linkage to a locus on chromosome 9, perhaps because of the incomplete analysis of the critical chromosome 9-linked families due to insufficient DNA. With hindsight it is unfortunate that the publication of this article descibing the Californian group's "work-in-progress" at the time of submission in May 1989 should have been delayed until January the following year by which time they and several other groups had presented posters and publications providing evidence in support of heterogeneity, ( v.i.).
The Texas group led by Hope Northrup raised the possibility of further heterogeneity (in addition to loci on chromosomes 9 and 11) in their analysis of 7 families using markers on chromosome 9 (novel ASS polymorphisms and ABO) and chromosome 11 (D11S144 and D11S351) (N orthrup et al., 1989). ASS was informative in all 7 families and linkage with TSC was excluded (Zmax/theta -2.66/0.05, 0.002/0.4). Neither was there linkage with ABO in the 3 families in which ABO was informative (lod scores negative to theta 0.3). Lod scores between TSC and the chromosome 11 markers were also negative for values of theta between 0.05 and 0.4 in the two (D11S144) or three (D11S351) informative families.
Jonathan Haines, Boston presented linkage data from an international collaborative study of over 90 families provided by US and European groups at HGM10 (Haines et al., 1989). There was strong evidence for linkage with ABO (Zmax 3.8, theta 0.2). 60% of families had been typed for at least one chromosome 11 marker and in those in which D11S144 was informative the maximum lod score was 1.46 at theta 0.3. When the scores for D11S144 in those families with a less than 10% chance of being linked to ABO were summated, the Zmax was 1.41 at theta 0.25. The decrement in Zmax as a result of this manouver suggests that this "not chromosome 9" group might have contained families linked to a third locus, a possibility also indicated by several families with negative lod scores for markers on both chromosomes 9 and 11.
Ch. 1 Introduction, Positional Cloning of TSC 68 Sue Povey described a subset of these results obtained in the British families, in a poster at the same conference (Povey et ah, 1989). Six chromosome 9 markers and three chromosome 11 markers (D11S144, D11S29 and D11S351) were scored in the 26 families in which at least one marker was informative. There was no evidence for linkage to markers on either chromosome and the authors concluded, "These data alone would not be sufficient to place a single gene for TSC on any chromosome; they do however provide more support for a locus on chromosome 9 than chromosome 11."
Further evidence for genetic heterogeneity was provided by Julian Sampson who published the results of a similar study later that year (Sampson et ah, 1989b). Eight families were typed with five chromosome 9 markers and three chromosome 11 markers (D11S144, D11S29 and D11S351). Cumulative lod scores were positive for each of the chromosome 9 markers although linkage was only significant with ABL (Zmax 3.01, theta 0.09). Three of the four families in which ABL was informative gave positive lod scores with this marker (Zmax 5.09, theta 0.001). The fourth had two recombinants in affected individuals in four meioses (Z/theta -5.097/0.001). HOMOG tests gave significant evidence for heterogeneity. There were no clinical features to suggest that this family had been misdiagnosed as having TSC. Multipoint analysis of the data excluding this fourth family placed TSC at ABL with a peak lod score of 6.1 (but with a broad confidence interval 8cM proximal to 18cM distal to ABL). There was no evidence of linkage with any of the chromosome 11 markers, although the family not linked to ABL gave weak positive lod scores with D11S144 and D11S351.
This was the evidence for the mapping of TSC at the time the study described in this thesis began. Subsequently, an international collaboration was set up to attempt to resolve the issue of heterogeneity. Initial results from the analysis of pooled data suggested that about one third of families were linked to the 9q34 locus and provided some evidence for a second locus on llq (Haines et ah, 1991; Janssen et ah, 1991; Povey et ah, 1991).
Ch. 1 Introduction, Positional Cloning of TSC 69 Concurrently individual groups reported linkage to 9q34 in selected families (Sampson et al., 1989b; Janssen et ah, 1990; Haines et ah, 1991; K andt et ah, 1991) but the linkage to chromosome 11 was not confirmed (Povey et ah, 1992). Linkage to a third chromosome, 12q22-24, was reported (Fahsold et ah, 1991) which was also not confirmed (Povey et ah, 1992). Several large, multigenerational families did not shown linkage to loci on any of the three chromosomes. Five of these were studied by Raymond Kandt and his colleagues using highly polymorphic microsatellite repeats mapped to the region of chromosome 16, 16pl3, to which autosomal dominant polycystic kidney disease had been mapped (Kandt et ah, 1992). Each family gave positive lod scores with one marker, D16S283 (Zmax 9.52, theta 0.02). This linkage was confirmed by other groups (Pericak- Vance et ah, 1992; Smithet ah, 1992; Povey et ah, 1994). Subsequent re- evaluation of the linkage data indicated that about half of TSC families were linked to each of the loci, one on chromosome 9, TSC1, and the other on chromosome 16, TSC2 (Kwiatkowski et ah, 1993; Povey et ah, 1994). These two loci account for the majority of inherited TSC in families where there is sufficient data to ascertain linkage. There are many families which are too small to indicate linkage to either locus, and a very few families where the disease does not appear to segregate with either chromosome 9 or chromosome 16 markers (Povey et ah, 1994). There is still a possibility that there may be a third locus responsible for disease in these unlinked kindreds.
At the inception of the project in 1989, there were three significant regions of interest on the long arm of human chromosome 11, llql3-14, q22-23 and q25, within which disease-related loci had been mapped. These correspond to positive R bands which appear to be gene rich. Linkage studies in families with Multiple Endocrine Neoplasia type 1, MEN1, had mapped the gene to llql3 close to PYGM and PGA (for references Results chapter {MEN1). Studies of MENl-associated tumours showed loss of heterozygosity for chromosome 11 markers. A gene involved in atopy had also been located nearby, more distally in llql3, linked to the microsatellite marker D11S97 (Cookson et ah, 1989). The second region
Ch. 1 Introduction, Positional Cloning of TSC 70 was llq22-23 where there was further evidence of the close linkage of Ataxia Telangiectasia complementation group A to THY1 (Gatti et al., 1988; Sanal et al, 1990) in the region of the m yeloid/lym phoid leukaemia (MLL) breakpoint t(4;ll)(q21;q23) and the Ewing sarcoma-peripheral neuroepithelioma breakpoint t(ll;22)(q24;ql2).
The proposal to generate irradiation hybrids from a human chromosome 11-only somatic cell hybrid to aid mapping of the TSC gene on chromosome 11 was devised in 1988 following Moyra Smith's report of linkage to the marker D11S144 at the Tuberous Sclerosis Association conference in Nottingham in 1988. The project was funded by the Medical Research Council and I was able to start work in October 1989. It was at this time that the first reports appeared at the ASHG and HGM10 conferences indicating further locus heterogeneity (non-9, non-11 -linked families). Moyra Smith's initial studies in the Irvine families provided good evidence for a locus on chromosome 11 (D11S144 Zmax 4.56, theta 0.07 in selected families) which was supported by the international data collaboration coordinated by Jonathan Haines (D11S144 Zmax 1.46, theta 0.3). There was also circumstantial evidence implicating a locus on chromosome 11 from reports of individuals with both TSC and MEN1 or parathyroid adenoma (Ilgren et al, 1983; Wei et al, 1984). We therefore felt that it was reasonable to pursue the original aim of the project and generate hybrids from chromosome 11 to complement the hybrids which were being prepared concurrently by Jude Fitzgibbon and Jonathan Wolfe from a hybrid containing human chromosome 9q.
Ch. 1 Introduction, Positional Cloning of TSC 71 Aims of the project
The principal aim of this study was to generate a panel of radiation hybrids derived from chromosome 11. We decided to use a high-dose irradiation in the expectation of producing hybrids which would retain small fragments of chromosome 11. There were two goals, firstly to produce daughter hybrids containing one or two fragments from which novel probes could be derived. Secondly, it was our intention that these might also be used as a mapping resource to help order markers from the D11S144 region to which TSC had been linked in some families. It became apparent during the course of the study that positional cloning of a chromosome 11-linked TSC gene (if any) would only be possible once the two principal TSC genes on 9q and 16p had been isolated, due to the very small number of families which show linkage to chromosome 11. Hence the purpose of the project changed to the exploitation of the hybrids in the positional cloning of AT, and MEN1 and the localisation of other genes on chromosome 11. Two hybrids were selected to generate novel markers and clones from the AT- and MEN1- regions to facilitate the mapping efforts of other groups.
Ch. 1 Introduction, Positional Cloning of TSC 72 C h a p t e r 2: Materials and Methods
Materials
Genomic cosmid library high density filters
Human chromosome 11 specific cosmid library filters were the kind gift of Dr Gunther Zehetner of the Reference Library - DataBase RLDB, Imperial Cancer Research Fund (at that time). The library number 107 (L4/FS11) was constructed by Dr Dean Nizetic from digests of DNA from flow-sorted chromosome 11 from the FC11 cell line, ligated into Lawrist 4. FC11 has a deletion in the llq23.1 region. DH5 alpha MCR (BRL) was used as host. Dr Nizetic and Dr Finbarr Cotter advised on the screening of the filters.
Cell culture media
Bacterial cell culture
Bacterial culture media were obtained from Difco laboratories, prepared using deionised water and sterilised by autoclaving at 15 lbs psi, 121 °C for 20-30 minutes. The components of the media are listed in Appendix 3.
Mammalian cell culture
Mammalian cell culture media including RPMI-1640 medium, Eagle's minimum essential medium (MEM), non-essential aminoacids and GPS were purchased from Gibco BRL. The components of the mammalian cell culture media are given in Appendix 3.
Cell lines
The hypoxanthine phosphoribosyl transferase (HPRT) positive cell line J1C14, a hamster-human hybrid containing a single copy of human chromosome 11 (Kao et al., 1976) was given by Prof. Carol Jones. The HPRT- hamster pulmonary cell line Wg3h (derived from DON, (Goss et
Ch. 2 Materials and Methods 73 al, 1975)) and the Galton interspecific hybrid mapping panel were provided by Professor Sue Povey. Hamster-human and rodent-human somatic cell hybrids from this panel are referenced individually in the Results chapters. The chromosome 11 hybrid mapping panel was provided by Professor Veronica van Heyningen (Hunt et al, 1994). The human chromosomal content of the Galton panel and the chromosome 11 hybrids has been characterised extensively by two or more of the following techniques: isozyme analysis, Southern hybridisation, PCR, karyotyping or FISH.
Chemicals
All chemicals were of AnalaR grade and were purchased from British Drug Houses (BDH), Poole unless otherwise stated. Radiochemicals including Redivue© (a-32p) dCTP and (y-32p) dATP were purchased from
Amersham and (a-^S) dATP from NEN/Dupont.
Cosmids
Cosmids encoding RXRB were provided by Dr Jude Fitzgibbon. Chromosome 11 cosmids were isolated by Dr Frances Benham and myself from the ICRF chromosome 11 specific gridded cosmid library (described above). This was provided by Dr Gunther Zehetner and co-workers; coordinate references are given in the text.
Enzymes
Restriction endonucleases were purchased from Gibco BRL or NBL. Taq DNA polymerase was obtained from Anglia, HT Biotechnology Ltd or Promega, Proteinase K from Boehringer Mannheim and pancreatic ribonuclease A (RNase A) from Sigma Chemical Company.
Ch. 2 Materials and Methods 74 Standard solutions and buffers
All solutions and buffers were prepared using distilled water and sterilised by autoclaving at 15 lbs psi, 121 °C for 20-30 minutes. The components for standard solutions and buffers are given in Appendix 3.
Radiation hybrids
See Methods below.
Methods
Standard DNA techniques
DNA extraction from cultured cells
Cells from at least three large flasks (3 x 160 cm^) were washed thoroughly in Hanks' balanced salt solution (Appendix 3). 7 ml lysing solution (Appendix 3) was then added to each flask and incubated at 37 °C for at least 5 hours. Three extractions with phenol:chloroform:isoamylalcohol (25:24:1) were followed by two extractions with chloroform.
Precipitation of DNA
DNA was precipitated by the addition of 0.1 x volume of 3M sodium acetate followed by two volumes of ice-cold ethanol and the spooled DNA was washed in 70 % ethanol, air dried and resuspended in an appropriate volume of TE. When there was insufficient DNA to form a macroscopic precipitate, the solution was centrifuged in a cold-room microfuge for 10 minutes. The supernatant was discarded and the pellet washed in 70% ethanol, air-dried and resuspended in TE buffer (Appendix 3). The concentration of DNA in solution was measured spectrophotometrically in a Ciba Corning 2800 Spectrascan at a wavelength of 260 nm, fluorimetrically (see below) or by comparison of relative intensity of ethidium bromide staining of the DNA with control of known concentration after electrophoresis through agarose.
Ch. 2 Materials and Methods 75 Mini-preparation of cosmid DNA
A 10 ml bacterial culture was grown overnight in LB medium (Appendix 3) with appropriate antibiotic selection (Appendix 3). The culture was centrifuged in a standard bench-top instrument at 3000 rpm for 10 minutes, the pellet resuspended in 200 pi lysis buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris pH 8) and transferred to a 1.5 ml eppendorf tube. After 10 minutes at room temperature 400 pi fresh 0.2 M NaOH/1 % SDS was added and the tube placed on ice for 5 minutes, after which 300 pi sodium acetate pH 5.2 was added and the incubation continued for a further 10 minutes on ice. Centrifugation at top speed in a microfuge for 5 minutes pelleted the cell debris and the supernatant was transferred to a fresh eppendorf. 600 pi isopropanol was added and the tube was chilled at -70 °C for 10 minutes. The tube was then centrifuged in a microfuge for 5 minutes, the pellet washed with 70 % ethanol and then resuspended in 50 pi TE (Appendix 3). Finally 1 pi of 10 mg/ml RNase A (Appendix 3) was added and the eppendorf incubated at 37 °C for 15 minutes. The DNA miniprep was stored at -20 °C until required.
As an alternative to the method above, "Wizard" miniprep DNA purification systems (Promega) were used according to the manufacturers instructions.
Polymerase chain reaction (PCR)
During preparation of the PCR reactions certain precautions were taken to minimise the risk of DNA contamination. The reactions were set up in a preparation room (in which PCR products were not handled) using Gilson pipettes specific for PCR, the tips and eppendorfs were autoclaved and hands washed prior to the procedure.
Latterly, most reaction mixes were prepared using sterile distilled water to give a final concentration of 1 x reaction buffer (Promega), 10 % glycerol, 210 pM of each dNTP (Boehringer Mannheim), and 500 nM of each oligonucleotide primer (Oswell DNA service or HGMP, Hinxton, UK). 23 pi of the mix was aliquotted into each 0.5 ml eppendorf tube, 0.25 pg DNA
Ch. 2 Materials and Methods 76 was added to each tube (except the no-DNA control) and one drop (-25 pi) of paraffin oil overlaid. The DNA was denatured at 95 °C for 5 minutes, 0.8 units Taq DNA polymerase (Anglia, Promega or Advanced Biotechnologies) added to each tube and 30-35 cycles of denaturation (at 93 °C for 20 s), primer annealing (appropriate temperature for 20 s) and extension (72 °C for 20 s) performed. The annealing temperature was determined for each pair of primers from the Oligo or Primer computer programs (Rychlik et ah, 1989; Lincoln et ah, 1991). A Hybaid Thermal Reactor was used for all the reactions. Some amplifications in 1990 and 1991 were performed with final reaction mixes as follows: "Cetus" 210 pM each dNTP, 1.5 mM MgCl2, 50 mM KC1, 10 mM Tris-HCl pH 8.3 (FGF3, CD3D and THY1 were also amplified using a mix containing 2.0 mM MgCl2); "Anglian" 10% DMSO, 1.5 mM each dNTP, 6.7 mM MgCl2, 16.6 mM (NH4)2S 0 4, 67 mM Tris-HCl pH 8.8, 6.7 pM EDTA, 10 mM p mercaptoethanol, 170 mg/1 bovine serum albumin; "Promega" 210 pM each dNTP, 1.5 mM MgCl2, 50 mM KC1, 10 mM Tris-HCl pH 9.0, 0.01% gelatin, 0.1% Triton X-100. Reactions using primers for D11S490, D11S420 included 10% DMSO (Gillett et ah, 1993).
Restriction endonuclease digestion
Between 5-10 pg genomic or hybrid DNA and 0.5-1 pg cosmid DNA was digested to completion with the appropriate enzyme using the buffer and conditions recommended by the enzyme manufacturer. The amount of enzyme used was 1 to 6-fold that required to complete the digestion in one hour under optimal conditions. Due to the inhibitory effect of the glycerol present within restriction enzyme storage buffers, this component formed no more than 10% of the reaction volume. Reactions were incubated for 1-2 hours for cloned DNA and for at least 3 hours for genomic DNA.
Agarose gel electrophoresis
DNA. fragments were size separated on 0.4-2 % w /v agarose gels (Sigma) prepared in 1 x TBE (Appendix 3). 1 x TBE was used as the electrophoretic running buffer. Ethidium bromide (Sigma) was incorporated at a
Ch. 2 Materials and Methods 77 concentration of 0.5 pg/ml in both gels and running buffer. The samples were mixed with 0.1 x volume of loading buffer (Appendix 3) and then loaded into the wells of the gel. The voltage and duration of electrophoresis was dependent on the size of the DNA fragment to be analysed. On completion the DNA was visualised by ultra-violet (UV) transillumination. The DNA fragment length was estimated by comparison to a 1 Kb DNA size ladder or a Haelll digest of DNA from phage (j)X174 (Gibco BRL) electrophoresed concurrently.
Southern blotting
DNA was transferred from agarose gels to nylon membrane by the Southern blotting technique (Southern, 1975). The gel was agitated in 0.25 M hydrochloric acid (HC1) for 10 minutes partially to nick and to depurinate the DNA. Following a brief rinse in distilled water the gel was submerged in denaturing solution (Appendix 3) for 30 minutes, rinsed in distilled water and placed in neutralising solution for a further 30 minutes. Hybond N or N Plus membrane (Amersham) was cut to size placed on the upper surface of the gel and DNA transferred by capillary blotting using 20 x saturated salt solution as the transfer solution. The following day the DNA was fixed to the membrane by exposure to UV (face down on a standard transilluminator for 15 minutes).
Recovery of DNA fragments from agarose gels
DNA fragments were isolated from agarose gels by centrifugation through glass wool. The block of agarose containing the band of interest was excised from the gel and placed above a small plug of glass wool in a mini eppendorf pierced at its base. This was placed within a large eppendorf and centrifuged in a microfuge for one minute. The concentration of DNA in the centrifuged solution was quantitated by fluorimetry and used without any further purification in oligolabelling reactions.
Ch. 2 Materials and Methods 78 Radio labelling of DNA probes
DNA fragments were labelled by means of random oligonucleotide priming, using random hexanucleotides to prime DNA synthesis from a single stranded template (Feinberg et al, 1983). The reaction was performed using a Multiprime™ DNA labelling kit (Amersham) according to the manufacturer's instructions (but at half volumes) using (a-32p) dCTP (3000 Ci/mmol). 25 ng of the DNA probe was denatured at 99 °C for 5 minutes in a volume of 30 pi and then placed on ice. 10 pi buffer 1 (containing dATP, dTTP, dGTP and reaction buffer) and 5 pi solution 2 (containing random hexanucleotide primers) were added together with 2 - 2.5 pi radiolabelled nucleotide dCTP (10 Ci/pl) and 1 unit DNA polymerase I 'Klenow' fragment. Labelling took place at room temperature overnight or at 37 °C for two hours. Once completed, 50 pi 1 x TNE/0.1 % SDS was added to the reaction and it was centrifuged through a 1 ml Sephadex G50 column at 2000 rpm in a bench-top centrifuge (with standard rotor radius) for 5 minutes to remove unincorporated nucleotides.
Hybridisation of DNA probes
Hybond N or N Plus membrane was pre-wetted in 2 x SSC and sealed in a plastic bag with 10-20 ml hybridisation buffer (Appendix 3). The plastic bag was submerged in a 65 °C shaking waterbath for approximately two hours. Except where stated, sonicated salmon sperm DNA (Sigma) was added to the radiolabelled DNA to give a final concentration in the hybridisation buffer of 100 pg/ml. The mixture was denatured by boiling for 5 minutes and then quenched on ice. Flybridisation followed addition of the probe mixture to the hybridisation buffer and continued at 65 °C for at least 16 hours.
Post-hybridisation washes and signal detection
The membrane was initially washed twice in 2 x SSC for 5 minutes at room temperature. More stringent washes followed and after each wash the radioactivity was monitored with a Geiger counter. Further washes
Ch. 2 Materials and Methods 79 were only performed if a significant level of radioactivity could still be detected. In order of increasing stringency, these were: 30 minutes in 2 x SSC/l % SDS 60 °C, 30 minutes in 0.5 x SSC/1 % SDS 60 °C and 30 minutes in 0.1 x SSC/1 % SDS 60 °C. After the final wash excess moisture was removed by blotting with 3MM paper (Whatman) and the damp membrane was wrapped in plastic. The membrane was exposed to X-ray film (Kodak X-omat AR or Fuji) in a light-proof cassette with intensifying screen (unless as stated) for 1-7 days.
Screening the ICRF human chromosome 11 gridded cosmid library
Gridded human chromosome 11 cosmid library filters (ICRF library num ber 107 L4/FS11, v.s. ) were prehybridised in Church hybridisation buffer (Appendix 3) and were screened with random-prime labelled A/wPCR product derived from appropriate irradiation hybrids or with product from a specific locus. The product was passed through a Promega PCR mini-column using the protocol supplied, the concentration of DNA in the eluate estimated by fluorimetry and 25 ng labelled. Post hybridisation washes were performed as above, except that 100 mM NaPi / 0.1 % SDS or 40 mM NaPi / 1 % SDS was used. Filters were initially washed thrice at 65 °C for 5 minutes. Longer washes at higher temperature were used if necessary.
Precise alignment of the filter with the autoradiograph film was mandatory in order to identify the coordinates of each positive cosmid precisely. This was achieved by mounting the film together with the plastic-film wrapped filter onto a backing card using sellotape, through which registration holes were pierced using a pin. By careful alignment of the filter with the autoradiograph, the x-y coordinates of each hybridising clone could be worked out. Since part of the library had been applied in duplicate and in the same relative order as the complete library, it was possible to recognise duplicate hybridising clones, and to use the coordinates of each as a check of accurate identification.
Ch. 2 Materials and Methods 80 Stabs of the corresponding colonies were requested from RLDB, ICRF. A loop of the stab was streaked onto 2 x YT agar plates (see Appendix 3), which were incubated and a single colony picked to inoculate 10 ml LB medium with kanamycin selection. Plates were streaked with the overnight culture and hybridised with the labelled product (saved from the original screen) to confirm the validity of the clone sent by the Database. A glycerol stock was prepared by mixing 0.5 ml culture with 0.5 ml 30 % glycerol and stored at -70 °C. A cosmid miniprep was prepared from the remainder of the culture for use in FISH studies.
Enzymological techniques
Lactate dehydrogenase (LDH)
LDHA in hybrid cells was assayed by horizontal starch gell electrophoresis (Harris et ah, 1976). Cells remaining in 25 cm2 or 75 cm2 culture flasks after trypsinisation were washed in PBS, pelleted and frozen. Lysates were electrophoresed at 3.5 V/cm through 15 by 22 cm 11% starch/0.01 M phosphate pH 7.0 gels in 0.2 M phosphate pH 7.0 bridge buffer for 17 hours. Gels were sliced horizontally in the direction of electrophoresis and the half gel overlaid with the staining system (which leads to the formation of formazan dye). The reaction mixture was composed of
(1) 0.05 M Tris/HCl buffer, pH 8.0, 20 ml, (2) Calcium lactate (pentahydrate): 100 mg (8 mM final concentration), (3) NAD (nicotinamide adenine dinucleotide): 10 mg, (4) MTT (tetrazolium salts, thiazolyl blue): 5 mg in 1 ml water, (5) PMS (phenazine methosulphate): 2.5 mg in 0.5 ml water, (6) Agar 2%: 20 ml.
LDH is a tetramer and isozymes may contain polypeptides derived from LDHA (chromosome lip) or LDHB (chromosome 12). If present in the irradiation hybrids (derived from J1C14), human LDHA forms tetramers with hamster ldhb to give multiple isozymes which migrate both anodally and cathodally as shown in the Results chapter, "Radiation hybrids derived from a chromosome 11-only parent", Figure 3.1.1.
Ch. 2 Materials and Methods 81 Adenosine deaminase
Ada in hybrid cells (derived from the hamster parents) was assayed by horizontal starch gell electrophoresis (Harris et al, 1976). Cells remaining in 25 cm2 or 75 cm2 culture flasks after trypsinisation were washed in PBS, pelleted and frozen. Lysates were electrophoresed at 3.5 V/cm through 15 by 22 cm 11% starch/0.01 M phosphate pH 6.5 gels in 0.1 M phosphate pH 6.5 bridge buffer for 17 hours. Gels were sliced horizontally in the direction of electrophoresis and the half gel overlaid with the staining system (which leads to the formation of formazan dye). The reaction mixture was composed of
(1) 0.05 M phosphate buffer, pH 7.5, 25 ml, (2) Adenosine, 15 mg (1.1 M final concentration), (3) Nucleoside phosphorylase (25 units/ml): 25 pi, (4) MTT: 5 mg in 1 ml water, (5) PMS: 2.5 mg in 0.5 ml water, (6) Agar 2%: 25 ml.
LDH is a monomer. Isozymes migrate anodally and electrophoretic patterns vary according to tissue of origin. The simplest pattern is found in red cell lysates. "Tissue-specific" isozymes are probably secondary isozymes derived by post-translational modification.
Mammalian cell culture
Cell lines were cultured from stocks frozen in liquid nitrogen. The cells were thawed rapidly at 37 °C and then transferred to a 80 cm^ flask containing 15 ml pre-warmed medium (Appendix 3). Stocks removed from liquid nitrogen were replenished as soon as possible. Cells were pelleted in a bench-top centrifuge at 1000 rpm for 5 minutes and then resuspended in 1 - 2 ml glycerol medium (Appendix 3) depending on the size of the pellet, and stored in 1 ml aliquots, initially for one hour at 4 °C, then overnight in the vapour phase of a liquid nitrogen storage tank, and finally in liquid nitrogen until required.
Ch. 2 Materials and Methods 82 The cell lines J1C14, Wg3h and the radiation-reduced cell lines were grown as attached monolayers in supplemented Eagle's medium, FCS/E (Appendix 3). Radiation-reduced cell lines were cultured in FCS/E-HMT: FCS/E to which the purine metabolite hypoxanthine, the dihydrofolate reductase inhibitor methotrexate and thymidine had been added (Appendix 3).
Confluent cultures were washed in Hank's balanced salt solution (Appendix 3) and released from the surface of the flask by means of a brief incubation with a small volume of trypsin-versene solution (Appendix 3). The trypsin was 'neutralised' by the addition of some fresh medium and the culture was divided or transferred to a larger flask.
Radiation reduced hybrids
Radiation hybrids were generated according to the protocol of Benham et al., (Benham et al., 1989). Cell fusion was performed essentially as described (Goodfellow et al, 1988). 5 x 10^ of the HPRT+ parental donor cell line J1C14 were harvested from a sub-confluent culture by trypsinisation and resuspended in 20 ml FCS/E in a plastic universal (Appendix 3). The cell suspension was exposed to 40,000 rad at room temperature using an industrial X-ray unit (Pantak HF320 SR) at 1000 rad/minute in two fractions over 120 minutes (to allow the unit to cool between sessions). After this lethal irradiation the cells were combined with 5 x 10^ of the HPRT" recipient hamster cell line Wg3h and washed once in phosphate-buffered saline, PBS (Appendix 3). The cells were pelleted by centrifugation at 1500 rpm in a bench top centrifuge of standard rota radius for 5 minutes at room temperature. The supernatant was decanted and the pellet loosened by gently tapping the centrifuge tube. One ml of 55% w /v PEG (dissolved in PBS) prewarmed to 37 °C was added drop-by-drop, gently disturbing the pellet with the pipette tip. The cell suspension was incubated at 37 °C for one minute. 10 ml of PBS was added slowly over two minutes with gentle stirring using a gilson tip, followed by the addition of 10 ml of FCS/E over two minutes, again with constant
Ch. 2 Materials and Methods 83 stirring. The cells were pelleted by centrifugation at 1500 rpm for 5 minutes. The cells were gently resuspended in FCS/E and plated at a density of 8 x 10^ cells in 25 cm^ tissue culture flasks. After 24 hours incubation at 37 °C, selective growth medium, FCS/E with HMT
(Appendix 3) was substituted (to eradicate the unfused HPRT“ Wg3h). Colonies appeared after 20 days, medium gently aspirated and colonies picked with minimal disturbance to the residual medium to reduce the likelihood of contamination of other colonies in the same flask. Cell lines were numbered 1 to 50 depending on the flask from which they were picked together with the suffix ', " or to refer to independent colonies from the same flask. Over 100 colonies were picked and cultured: at least two colonies from each of the fifty 25 cm^ flasks. These were grown to 80 cm^ stage and an aliquot frozen in liquid nitrogen as live cells after ADA and LDFIA analysis, which was performed on 107 cell lines. One clone from each of the 25 cm^ flasks was chosen at random to be cultured further to three 175 cm^ flasks from which DNA was prepared. Over 50 cultures were selected, but a few became contaminated and were discarded: DNA was made from 47 cell lines. No analysis (other than LDHA and ADA assay) was performed on the remaining clones which were cryopreserved after expansion to the single 80 cm^ flask stage only.
Single cell cloning procedure
The radiation-reduced hybrid Jol2 was subcloned after resuscitation from liquid nitrogen. Cells were plated at limiting dilutions in multiwell plates and solitary colonies picked for expansion, DNA preparation and storage in liquid nitrogen.
A 80 cm2 flask containing confluent Jol2 hybrid cells was trypsinised and the cells disaggregated by repeated pipetting. An aliquot of the mixed suspension was counted in a haematocytometer to determine the cell number (20,000 cells per ml). Four separate dilutions were made from this stock suspension:
Ch. 2 Materials and Methods 84 1/1000 (a), containing 20 cells/ml, i.e. approximately 1 cell per 50 pi added to each well. 1/1000 (b), (a second dilution from the stock). 1/2000, containing 10 cells/ml, less than one cell per 50 pi 1/500, containing 40 cells/ml, approximately two cells per 50 pi
50 pi of each dilution was used to seed each well of one Nunclone 24-well plate. Each well was then topped up with 2-3 ml cell-free FCS/E medium (containing HMT, ciprofloxacin and GPS), placed in an ethanol-cleaned plastic box together with moistened tissue paper. Wells were examined on the following day (day 1), day 12 and day 15. Colonies were first seen on day 15. The plates were left undisturbed for a further six days. On day 21 the supernatant medium was carefully removed and replaced with fresh FCS/E. Nine clones were identified in a total of 144 wells. Six were in the tray which had been seeded with the 1/500 dilution suspension, two in the 1/1000 (a) plate and one in the 1/1000 (b). Six of these were in six separate wells; the remaining two clones were in a single well (J12.1A & J12.1B).
The following day the well containing the two clones was drained and each colony lifted into a separate 25 cm^ flask using a pasteur pipette or a blunted hypodermic needle. Each single colony was trypsinised conventionally within the native well. The following day (day 23) yeast infection was found in all the trypsinised wells and the 25 cm^ flasks (which probably arose from the trypsin-versene solution). The six wells and two flasks were aspirated and freshly-made medium added. The anti- fungal amphotericin B was added to the medium in addition to the GPS and ciprofloxacin antibiotics. The supernatant medium was replaced on day 24 and medium containing amphotericin used until day 29. No further fungal colonies were seen after day 26. On day 37, two more colonies were identified and treated as above. One of these subsequently failed to grow and was discarded.
Colonies underwent one further trypsinisation within the Nunc plate well and were then expanded via 25 cm^ and 80 cm^ flasks to three 175 cm^
Ch. 2 Materials and Methods 85 flasks from which DNA was made. Metaphase chromosome spreads of the subcloned hybrids were prepared for FISH to test for the presence or absence of human fragments present in the Jol2 parent. Cells from 80 cm^ flasks were harvested and frozen as live cells in glycerol freezing medium.
Fluorescent In Situ Hybridisation
Chromosome analysis was performed by Dr Margaret Fox (MRC Human Biochemical Genetics) and Mrs Lynne West. Karen J. Woodward hybridised the biotinylated RXRB cosmid to human metaphase spreads.
Metaphase preparations of lymphocytes
Metaphase chromosomes were prepared from phytohaemagglutinin (PHA)-stimulated normal lymphocyte cultures and FISH performed according to standard procedures (Lichter et ah, 1990). 1 ml of heparinised blood from a male donor was added to a 25 ml culture flask containing 17 ml Iscoves modified DMEM (Imperial), 1 % GPS (glutamine, penicillin, streptomycin, Appendix 3), 2 ml foetal calf serum (FCS) and 200 jllI phytohaemagglutinin (PHA, ICN Labs). The culture was incubated for 72 hours and was shaken gently once daily; all incubations took place at 37 °C except where stated. Thymidine (Sigma) was then added to give a final concentration of 0.3 mg/ml and the culture was incubated for a further 18 hours. 2-deoxycytidine (Sigma) was added to a final concentration of 2.3 |Lig/ml and incubated for 3 hours 55 minutes. Colcemid (Gibco) was then added and incubated for a further 20 minutes. The culture was transferred to two sterile 10 ml tubes and centrifuged in a standard bench-top centrifuge at 1000 rpm for 5 minutes. The supernatant was removed and the pellet resuspended dropwise in 5 ml pre-warmed 0.075 M KC1 (hypotonic) solution. The tubes were left at room temperature for 20 minutes and then centrifuged at 1000 rpm for 5 minutes. The supernatant was again removed and the pellet resuspended dropwise in 10 ml 3:1 absolute methyl alcohol: glacial acetic acid fixative. This process of centrifugation and resuspension in fixative was repeated at least twice
Ch. 2 Materials and Methods 86 until the pellet was a clean white colour. The final suspension was stored at -20 °C until required.
Metaphase preparations of hybrid cell lines
Sub-confluent flasks of hybrid cultures containing a reasonable number of dividing cells were selected for harvesting. Colcemid was added to each flask to give a final concentration of 0.025 pg/ ml and incubated at 37 °C for one hour. The medium was aspirated, centrifuged at 1000 rpm for 5 minutes and the supernatant decanted and discarded. Cells were washed from the floor of the flask with 5 ml KC1/EDTA hypotonic solution (Appendix 3) and added to the centrifuged cell pellet in a tube. A further 5 ml of hypotonic solution was added to the flask. Both flask and tube were incubated for 20 minutes at 37 °C after which the contents of the flask were added to the tube and centrifuged at 1000 rpm for 5 minutes. The cells were fixed as described above and suspensions stored at -20 °C until required.
Slide preparation and pre-hybridisation treatment.
Slides were cleaned and cooled by soaking in methanol containing 0.5 % v/v concentrated hydrochloric acid. Immediately prior to use the slide was wiped dry with a lint-free cloth, breathed upon and 1-2 drops of chromosome suspension added from a 1 ml plastic pipette. The slide was allowed to air dry slightly before the addition of two drops of fresh fixative. The slide was shaken so that it was almost dry and then flooded with 70 % glacial acetic acid. It was left for approximately 1 minute and then completely air dried by shaking. Slides were examined under phase- contrast microscopy and slides with a reasonable number of metaphase spreads not surrounded by cytoplasm were selected for use.
During the following procedures the slides were treated in 50 ml or 100 ml glass Coplin jars unless otherwise stated. Incubations were performed at 37 °C, and those under coverslips (22 x 50 mm) were in a volume of 100 pi, unless specified otherwise.
Ch. 2 Materials and Methods 87 The slides were treated with RNase A (100 pg/ml in 2 x SSC pH 7) for one hour in a moist chamber. After two 5 minute washes in 2 x SSC at room temperature and one 5 minute wash in proteinase K buffer (Appendix 3) at 37 °C, the slides were treated with proteinase K (50 ng/m l in proteinase K buffer) for 7 minutes. A rinse in formaldehyde buffer (Appendix 3) was followed by a 10 minute post-fixation step in 1% formaldehyde in formaldehyde buffer. The slides were washed in PBS for 5 minutes, dehydrated through an ethanol series and air dried.
Bio tin-labelling of probe by nick-translation
The concentration of DNA derived from the cosmid minipreps was measured by DNA fiuourimetry using a TKO-100 minifluorimeter(Hoefer). 0.5-1.0 pg DNA was labelled with biotin-14- dATP by nick translation using a BioNick™ labelling system (Gibco BRL). The reaction was performed according to the manufacturers' instructions; it is designed to generate small biotin-labelled DNA probes between 50-500 bp. After incubation at 16 °C for one hour the reaction was stopped with 5 pi 300 mM EDTA pH 8 and unincorporated nucleotides removed using a Nick™ Sephadex G-50 column (Pharmacia). The column was prepared with 400 pi 1 x TNE buffer (Appendix 3), the reaction added and the probe eluted with a further 400 pi 1 x TNE. The biotinylated probes were stored at -20 °C until required.
Preparation of labelled probe: cosmid probes
Cosmid probes were prepared with an excess of unlabelled C0t-1 DNA to suppress repetitive sequences (Lichter et al, 1990). For each hybridisation reaction the following components were added to a 1.5 ml eppendorf tube: 200 ng purified biotinylated probe, 10 pg sonicated salmon sperm DNA, 10 pg yeast tRNA, 10 pg Cot-1 DNA (Gibco BRL), 0.1 x volume 3 M sodium acetate and 2.5 x volume ice-cold absolute ethanol (99.7 - 100 %). After precipitation at -70 °C for one hour the tubes were centrifuged in a cold- room microfuge for 15 minutes. The supernatant was discarded, the pellet freeze-dried for 10 minutes, resuspended in 10 pi hybridisation mix
Ch. 2 Materials and Methods 88 (Appendix 3) and incubated at 37 °C for 10 minutes to ensure that the DNA was dissolved. The probe mixture was denatured at 70 °C for 5 minutes and the repeat sequences were allowed to pre-anneal to the Cot-1 DNA at 37 °C for about 90 minutes before hybridisation to the chromosome preparation.
Preparation of labelled probe: total human or hybrid DNA probes
Probe preparation was performed as described above but repeat sequences were not suppressed: unlabelled Cot-1 DNA was not added and the pre annealing step was omitted.
Hybridisation: cosmid probe
Chromosomal DNA on the slide was denatured under a coverslip with 100 pi 70 % v/v deionised formamide / 2 x SSC at 80 °C for 5 minutes. Immediate quenching in ice-cold 70 % ethanol was followed by dehydration through an ethanol series and the slide was then air dried. The denatured and pre-annealed probe was applied under a 22 mm diameter coverslip and the edges sealed with cow gum. Hybridisation took place at 37 °C in a moist chamber overnight.
Hybridisation: total human DNA probe
The 10 pi probe mixture was applied under a 22 mm coverslip and sealed. The probe and chromosomal DNA were denatured simultaneously in an 80 °C oven for 5 minutes. Hybridisation proceeded as above.
Post-hybridisation washes:
Coverslips were removed and the slides were washed three times for 5 minutes in 50 % formamide / 2 x SSC at 45 °C. This was followed by three 5 minute washes in 0.1 x SSC at 60 °C and a single wash of 5 minutes in 4 x SSC / 0.05 % Tween 20 at room temperature.
Detection of hybridisation
Biotinylated DNA was detected using the procedures described by Pinkel et al., (Pinkel et al, 1986). The slides were first equilibrated in 4 x SSC / 5 %
Ch. 2 Materials and Methods 89 non-fat milk (4xSSC/NFM, Marvel) for 20 minutes. The succeeding incubations were carried out at room temperature for 20 minutes in a volume of 100 jul under a 22 x 50 mm coverslip. The initial incubation was with avidin-fluorescein isothiocyanate (avidin-FITC, Vector Laboratories) at a concentration of 5 pg/ml in 4xSSC/NFM and thereafter the slides were protected from exposure to light. The slides were then washed three times for 5 minutes in 4 x SSC / 0.05 % Tween 20 and the hybridisation signal was amplified by incubation with 5 pg/ml biotinylated goat anti-avidin (Vector Laboratories) in 4xSSC/NFM, followed by washes as before and a second incubation with 5 pg/ml avidin-FITC. After a 5 minute wash in 4 x SSC / 0.05 % Tween 20 and two 5 minute washes in PBS, the slides were drained and mounted in 20 |il Vectorshield medium (Vector Laboratories) containing p-phenyldiamine dihydrochloride (anti fade), 10 pg/ml 4,6-diaminophenylindole (DAPI) and 2 pg/ml propidium iodide (PI) as counterstains for both Q- and R- banding.
Microscopy
The preparations were analysed by epifluorescence confocal microscopy using a laser scanning confocal microscope (BioRad MRC600). The laser beam was used to excite the fluorochromes which then emit light of a different wavelength. The emissions were collected separately by two photomultiplier tubes using the appropriate filter sets and then the chromosomal and signal images were merged. Photographs were produced using a Mitsubishi colour video copy processor.
Sequencing
The chain termination method of sequencing (Sanger et al, 1977) was carried out by using the Sequenase Version 1 sequencing system (USB).
D11S144
A subclone of this plasmid probe MCT128.9 (prepared by Dr Cathy Abbott) was used as the template for sequencing and the universal primers were used to prime the reaction. Plasmid miniprep DNA was made single
Ch. 2 Materials and Methods 90 stranded by alkaline denaturation using the protocol provided with the DNA sequencing kit (Sequenase, USB). 3-5 pg plasmid DNA and 0.1 x volume 2M NaOH / 2 mM EDTA were combined in an eppendorf tube. After an incubation at 37 °C for 30 minutes, 0.1 x volume 3M sodium acetate pH 5.3 was added to neutralise the mixture. The DNA was precipitated following addition of 2 x volume ethanol and freezing (-70 °C for 15 minutes). The DNA was pelleted by centrifugation, washed in 70 % ethanol and resuspended in 7 pi distilled water.
The annealing reaction consisted of: plasmid DNA in a volume of 7 pi, 1 pi of the appropriate primer (10 pM/pi) and 2 pi reaction buffer. The tube was incubated at 65 °C for two minutes, allowed to cool to ambient temperature then placed on ice. The labelling reagents consisted of 1 pi DTT (0.1 M), 2 pi labelling mix (Sequenase labelling mix diluted 1 in 5 with distilled water), 0.5 pi oc^S dATP (-1400 Ci/mmol) and 2 pi diluted Sequenase enzyme (diluted 1 in 4 with Sequenase enzyme dilution buffer). These were premixed, added to the annealing reaction and incubated at ambient temperature. After 5 minutes, 3.5 pi of the reaction was added to 2.5 pi of each of the four termination mixes ddG, ddA, ddT and ddC, pre warmed to 37 °C. After 5 minutes at 37 °C the reactions were stopped with 4 pi stop solution (formamide, see Appendix 3).
Polyacrylamide gel electrophoresis
The sequencing apparatus used was 21 x 50 cm and was supplied by Bio rad. The plates were cleaned with distilled water and ethanol before use and the top plate was treated with approximately two ml of silane to minimise adherence of the gel. The 0.4 mm thick gel was prepared with 60 ml acrylamide mix (7 M urea, 6 % acrylamide (Severn Biotech Ltd) and 1 x TBE), 74 pi TEMED (Bio-rad) and 74 pi freshly prepared 25 % w /v ammonium persulphate (Bio-rad). A well was formed in the upper edge by insertion of the comb upside down. After the gel had been allowed to set for one hour the comb was removed and re-inserted in the correct orientation to produce wells into which the samples were loaded.
Ch. 2 Materials and Methods 91 The gel was pre-run at 45 watts for 20 minutes in pre-warmed 1 x TBE to achieve an optimum running temperature of 50 °C. The samples were then denatured at 75 °C for 3 minutes and 3 pi was loaded into a 24 well sharks tooth comb or 2 pi into a 48 well comb. The length of time the samples were allowed to run was determined from the positions of the bromophenol blue and xylene cyanol dyes (included in the formamide stop solution).
Signal detection
After disassembling the sequencing apparatus the gel was transferred from the bottom glass plate to 3MM filter paper, covered with Cling-film™ and dried for two hours at 80 °C using a Bio-rad gel drier. Signals were detected by radioautography using Beta-max Hyperfilm™ (Amersham) for 12-72 hours at room temperature.
Ch. 2 Materials and Methods 92 C h a p t e r 3.1: Radiation hybrids derived from a chromosome 11-only parent
Introduction
The aim of the experiments described in this chapter was to generate numerous somatic cell hybrids containing small fragments of human chromosome 11. We anticipated that some of these would contain human chromosome llq23, the region of the chromosome containing loci to which linkage with TSC had been obtained (in 1989, the inception of the project). The human DNA content would be characterised using a variety of analytical techniques (enzymological, Southern hybridisation, PCR and FISH). Hybrids containing a suitable region or regions of the human chromosome could then be 1) exploited as a resource for the generation of novel probes from these regions, 2) used as a source of DNA for cloning the TSC gene (and other genes from the characterised region) and 3) could also be used for mapping candidate genes and independently-generated polymorphic markers.
Since there was no dominant selectable marker in the llq23 region, the technique of chromosome-mediated gene transfer could not be used to generate somatic cell hybrids. In addition, CMGT has some considerable disadvantages, if the resulting hybrids are to be used for mapping purposes. It has been the experience of several groups that the donor chromosomal material is rearranged and that interstitial deletions are frequent. In addition, centromeric alphoid sequences are over-represented (Porteous, 1987; Pritchard et ah, 1987; Goodfellow et ah, 1988). We chose to exploit the technique of radiation-fusion gene transfer, IFGT, randomly to incorporate small fragments of chromosome 11 into the hybrids.
The size and number of the chromosomal fragments produced is determined by the dose of irradiation applied to the donor hybrid
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 93 (Benham et al., 1989; Cox et ah, 1990). These initial experiments used radiation doses of between 6000 and 8000 rads. We decided to use a much higher dose, 40,000 rads, in the expectation of producing hybrids which would contain much smaller fragments of chromosome 11, more suitable for subsequent cloning experiments.
One aim of the irradiation-fusion experiment was to generate a panel of daughter hybrids containing fragments from all regions of chromosome 11 to serve as mapping and cloning resources for projects concerned with genes elsewhere on the chromosome, outside our region of primary interest, for example the region of llql3 to which the locus for Multiple Endocrine Neoplasia type 1, MEN1 had been mapped (Larsson et al., 1988). Hence, we did not attempt to identify those clones containing llq23 fragments at an early stage by PCR of crude extracts of the clones while they were being grown up. This more focussed strategy has been used by some workers in order to derive a panel of hybrids containing fragments containing a given region of a human chromosome (Glaser et al., 1990; Doucette-Stamm et al., 1991; Brook et al., 1992; Jackson et al., 1992). Others have not been successful in amplification of such cell lysates, and have demonstrated that there is an inhibitor to amplification in some growing cultures which it is difficult to eliminate (Thomas, 1991).
R esults
Markers tested on the hybrid panel
A total of 107 hybrids were generated, of which DNA was made from 47, as described in Methods (Chapter 2). The presence of fragments of human chromosome 11 in the hybrids was assessed by enzymology, by Southern hybridisation, PCR and, in selected hybrids, FISH. A total of 37 markers were tested on the panel of 47 hybrids (Table 3.1.1). Retention of 8 of these togther with a further 10 markers was studied in a smaller "subset" panel of 11 hybrids found to contain one or more markers in the llql3 region Chapter 3.3, "Isolation of hybrids containing human chromosome llql3".
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 94 Enzymatic Analysis: LDHA and ADA
LDHA (human lactate dehydrogenase-A) and ADA (rodent adenosine deaminase) were assayed in all 107 of the irradiation hybrids at an early stage of expansion of each clone by horizontal starch gel electrophoresis of lysed, pelleted cells (Harris et al., 1976), Figures 3.1.1 & 2. Eleven of the 107 hybrids expressed human LDHA (retention 10 %, vide infra). The parental cell lines J1C14 (the "donor") and Wg3H (the "recipient") were found to have different ADA isoenzymes. This permitted the identification of daughter hybrids retaining the ADA originating from J1C14 in addition to the Wg3H ADA. Fifteen (14 %) of the 107 hybrids tested expressed both J1C14 and Wg3H isoenzymes (2', 6', 8’, 11, 15’, 17, 24, 24’, 29’, 33', 36’, 38, 44, 46, 46'). 46 and 46' had identical results on enzyme analysis (LDHA negative, ADA weak positive) and it is presumed that they originate from the same clone, (both were picked from the same flask, 46). The ADA result confirms that the cell lines are triply hybrid (hybrids of Wg3H hamster, CHO hamster and human). It also suggests that the frequency of random retention of rodent and human chromosome fragments is similar (13 % c.f. 10 %, respectively).
Southern Hybridisation Analysis
Fifteen loci were tested by Southern hybridisation (Ms Deborah Hunt). The results together with the probes used are listed in Table 3.1.1. The average retention of a marker was 5.4 % (estimated from the 10 markers for which the complete panel of hybrids were scored and excluding D11Z1). The centromeric sequence D11Z1, probe pLCllA, was present in 20 of the 47 hybrids, 43%.
PCR Analysis
Twenty two loci were tested by PCR . The primer sequences are listed in Methods (Appendix 3). Primers for amplification at the D11S144 locus were obtained by DNA sequencing of a subclone of the probe MCT128.1
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 95 (see Methods). The average retention of a marker, scored by PCR, was 5.6 % . This is almost identical to the average retention assayed by Southern hybridisation, SH. This is a suprising result since SH is not as sensitive as PCR. It may indicate that after expansion of each culture into three 175 cm2 flasks the cutures were largely homogeneous. However, hybrid Jo 12' was not uniform, as discussed in Chapter 3.2, "Isolation of a hybrid containing llq23 only, J12.1A".
TYR and TYRL
Retention of TYR was analysed using two PCR amplifications, TYR4 and TYR1 (see Methods). The primers designated TYR4 (Spritz et al., 1990) amplify from adjacent intron sequence across exon 4 of the human tyrosinase gene, llql4.3 (Van Heyningen et al., 1995). This region of the gene (part of intron 3, exon 4, intron 4, exon 5 and the 3' untranslated sequence) is duplicated in the tyrosinase-related sequence TYRL located on the short arm at llpll.2. Figure 3.1.3 illustrates the use of the TYR4 primers to score for the presence of each of these loci. The TYR4 primers amplify both TYR and TYRL and the product from each is 271 bp. However, the sequence of the product differs at position 93 where there is a G in the TYRL sequence instead of the A found in TYR, introducing an Mspl restriction site, (Giebel et al., 1991). There is a additional MspI site at position 122 (in both products). Hence, Mspl digest of the TYR product results in two fragments, 150 and 121 bp, whereas digest of the TYRL product produces three, 150, 92 and 29. Hybrids containing either TYR or TYRL, both or neither, can easily be differentiated by electrophoresis of the digested product. In this experiment the digested products were separated in 5 %/12 % "stacking" acrylamide gels (Figure 3.1.3). The 19 bp fragment was usually not apparent or was electrophoresed off the gel. Results of TYR4 PCR and Mspl digestion of mapping panel somatic cell hybrids and Jo irradiation hybrids are listed in Table 3.1.2, below.
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 96 --HvbridJ Chr. content Size of fraements TYRL TYR TYR1
genomic llpter-qter 150,121, 92 + + nt J1C14 llpter-qter 150,121, 92 + + At EJNAC llpter-cen 150, 92 + - At A3RS llpter-q23.3 150,121, 92 + + At A3RW llpter-q24 150,121, 92 + + At M11X llpter-q22.3 150,121, 92 + + A t 39.8 150,121, 92 + + At CJ37 llql3.3-qter 150,121 - + nt CJ52 llq!3.3-qter 150,121 - + At
Jo 4 150, 92 + -- Jo 6' 150,121, 92 + + + Jo 12 150,121, 92 + + + Jo 14 150,121 - + + Jo 31 150,121, 92 + + + Jo 50 150, 92 + --
Table 3.1.2: Scoring of hybrids for TYR and TYRL using the TYR4 PCR plus MspI digestion (columns 3-5); and the TYR1 PCR (column 6) nt, not tested
The scoring of hybrids for TYR and TYRL was confirmed using primers specific for the TYR gene, designated TYR1 (Table 3.1.2, above). Amplification of sequence from the part of the tyrosinase gene 5' to intron 3 is specific for TYR; TYRL is not amplified. Primer sequences specific for human TYR were designed (by comparison with available mouse tyr sequence) to amplify a 478 bp fragment of exon 1 - intron 1. This includes the Mbol TCT/TAT polymorphism in codon 192, identified by Giebel and Spritz (Giebel et ah, 1990), and so the amplification and Mbol digestion of product can be used in linkage studies at the TYR locus (Povey et ah, 1994).
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 97 Alu-PCR analysis
Alu-PCR provided a further estimation of human DNA content. Inter- Alu PCR product was seen in 21 of 29 hybrids tested (72%). Those hybrids which scored negative with all markers or where only centromeric sequences were found to be present, in general had a simpler pattern of Alu-PCR product than hybrids found to contain one or more (other) marker/s, Figure 3.1.4. For instance, no product was seen in inter -Alu amplifications of hybrids 1', 5', 7', 10', 19, 20', and 22' which were negative for all markers including D11Z1 (centromere). However, hybrids 13' and 41' were Alu-PCR negative but were positive for one marker (NCAM and D11S97 respectively, each scored by Southern hybridisation). Conversely, hybrids 2', 4, 8', 16, 21' and 49'" each had multiple inter -Alu products but were positive for relatively few conventional markers. This is not suprising since the panel of markers used focussed on two regions of the chromosome (llql3 and llq23) and large regions of the chromosome were ignored.
Attempts were made to isolate individual inter -Alu products by picking discrete bands from the primary Alu amplification and re-amplifying the toothpick tip, Figure 3.1.4. Multiple products were seen in the secondary amplifications (mostly of the same or faster mobility) which did not invariably include the band picked. This strategy was not pusued as a means of isolating individual products to use as probes.
Summary
Of the 37 markers, one third map at intervals along the whole chromosome outside the main region of interest. The rest lie in llql4- llq23.
Twenty eight hybrids contained one or more human markers: 2', 3', 4, 6', 8', 9, 12, 13', 14, 15', 16, 17, 18, 21', 23, 24', 28', 30, 31, 32, 33, 34, 41', 46, 47'", 48', 49"', 50'. In four of these (9, 21', 28', 30), only centromeric sequences were detected. Nineteen hybrids were found to be negative with all
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 98 markers tested: 1', 5’, 7', 10', 11', 12', 19, 20', 22’, 26’, 27, 35’, 36, 37, 38’, 39, 40', 42", 44'". These have been omitted from Table 3.1.1. The percentage of hybrids with retention of any human material was 60% (28 of 47).
Combined marker analysis
Fourteen hybrids contained markers from llql4-23. These are Jo 6', 8', 12, 13', 14, 16, 17, 23, 31, 32, 33, 48', 49'", 50'. Of these fourteen, seven retained markers in the llql4-23 region and were negative for most other regions tested (8', 12,13', 14, 16, 23, 49"'). Hybrid Jo 12 seemed the best hybrid for our purposes and so was analysed in more detail.
Jo 12
Jo 12 was positive for all nineteen llq markers tested (including FUT4) extending from TYR (ql4-q21) to PBGD (q23.3), a region of about 40cM in length. It hybridised weakly to the centromeric probe D11Z1. The only lip marker detected was the tyrosinase-related sequence. Evidence from the relative intensities of the hybridisation signals on Southern filters indicated that the llq markers do not lie on a single human fragment: the more proximal llq probes gave mostly strong signals relative to control J1C14 tracks, whereas the more distal markers gave weaker signals, suggesting that retention of these sequences in the hybrid is on at least two separate fragments (Dr Frances Benham, data not shown).
This impression of the human chromosomal content in Jo 12 was confirmed by fluorescent in situ hybridisation. Using total human DNA as probe on Jo 12 metaphases, FISH showed that many of the cells contained at least two fragments of human DNA: one integrated into a hamster chromosome and one or two very small separate fragments, Figures 3.1.5(a, b, d). A few of the hybrid cells were found to contain only one of these two or three components which led us to attempt the single cell cloning experiment described in the next chapter.
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 99 Alu-PCR products from Jo 12 generated using Alu primers and painted onto normal human metaphase spreads hybridised to two distinct regions: a larger at Ilq21-q23, and a smaller one in proximal lip , Figure 3.1.5(c). These in situ mapping results are consistent with the marker analysis and indicate that the most, if not all, of the human DNA retained in Jo 12 derives from the two regions of chromosome 11 detected by the markers.
Discussion
Marker retention
The average retention of any given DNA marker (as assessed by Southern hybridisation and PCR) was 5.5 %. Similar values have been found in other high-dose hybrid series, such as that of Siden et al. where the marker retention frequency was 3% at an irradiation dose of 25 krad. This is less than the retention of the enzyme marker, LDHA, which was 10 %. This difference may be due to the stage of culture at which the hybrids were analysed. LDHA was assayed from cells retained in one of the early flasks remaining after trypsinisation of the clone when confluent, whereas the DNA markers were tested on DNA made from the final stage of culture (the three confluent 175 cm2 flasks). Culture conditions may influence retention: Siden indicated that culture in microtitre plates led to a two- to three- fold reduction in retention frequency compared with petri dishes but this phenomenon has not been seen by others (Siden et ah, 1992; W alter et ah, 1995). Prolonged culture or repeated freezing and thawing may lead to loss of chromosome fragments from the daughter hybrid, (referenced in (Walter et ah, 1995)). This may not be a disadvantage if the final hybrid is characterised when stable and may be regrown from frozen aliquots without loss of human material. It may hamper exploitation of a particular hybrid, however, if circumstances require that it be regrown from an aliquot of cells frozen at an early stage of culture. The frozen aliquot may contain additional fragments which were subsequently lost during the original culture and these would necessitate careful re-
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 100 characterisation. Previously unrecognised fragments present in the regrown hybrid could be eliminated by a subcloning procedure as performed with hybrid Jo 12 in this study, v.i.. We attempted to avoid these potential problems by prolonged initial culture (three large flasks) and making one large batch of DNA from each hybrid at this stage.
Retention of other llpl5 loci was greater than the average: HBB 10.6 %, MUC2 8.5%. An alternative hypothesis for the discrepancy between LDHA retention and the 5.5 % average retention of any given DNA marker could be that there are regional differences in retention. Conceivably llpl5 might contain one or more loci which have growth promoting properties and would provide a selective advantage to the hybrid if retained.
Regional discrepancies in retention frequency for markers on the same chromosome have been seen in other studies (Walter et ah, 1994; Gyapay et ah, 1996). Variation of twofold is common and on some chromosomes may be over threefold. In radiation hybrid series using low-dose irradiation, intended for mapping new markers or genes, it may be necessary to exclude from the analysis markers which show large differences in retention frequency compared with adjacent markers. This precaution is not relevant for high-dose series which cannot be used for pan-chromosomal mapping unless large numbers of hybrids are generated.
Part of the variation in retention frequency observed in this and other studies is due to the preferential retention of markers near the centromere (Benham et ah, 1989; Goodfellow et ah, 1990; Lawrence et ah, 1991; Sefton et ah, 1992). Increased retention of markers near the telomere has also been seen in some studies (Burmeister et ah, 1991) but not in others (Altherr et ah, 1992; Ceccherini et ah, 1992; Walter et ah, 1994). Two explanations have been suggested to account for these variations (Lawrence et ah, 1991). Stable retention of human fragments in radiation hybrid cell lines must require that they are either integrated within the chromosomes of the recipient cell line or that they acquire centromere- and telomere-
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 101 containing fragments from the irradiated donor. Chromosomal regions which are adjacent to these chromosomal elements necessary for successful segregation at mitosis may have a selective advantage. An alternative explanation is that the peri-centromeric and peri-telomeric regions have fewer expressed genes and that these fragments may be better tolerated by the recipient rodent cells.
Discussion of the numbers of fragments retained per hybrid is included in the Results chapter, "Isolation of a hybrid containing llq23 only, J12.1A".
Percentage of hybrids containing at least one human marker
60% of the hybrids characterised in this experiment contain at least one human marker. The range of frequencies reported in radiation hybrids derived using a similar irradiation dose (>20 krad) is wide: 28% (Sinke et al., 1992), 75% (Goodfellow et al., 1990), 76% (Fitzgibbon, 1993), 83% (Florian et al., 1991), 88% (Moore, JK, Walter, MA and Ponder BAJ, quoted in (Walter et al., 1995)) and 100% (Benham et al., 1989; Siden et al, 1992). This variability may partly be due to differences in initial culture conditions or in compatibility between donor and recipient cell lines, but it is difficult to address these factors from the radiation hybrid literature. Sinke et al. suggested that there may be a relationship between frequency of retention and the amount of human chromosomal material present in the donor hybrid parent. Walter and Goodfellow (op. cit) point out that the size of the donor region should not affect frequency of retention of markers within that region. The variation may simply reflect the difficulty of detection of hybrids containing fragments of a small target using insensitive methods. Sinke et al. screened their 60 hybrids by FISH using biotinylated total human DNA as probe, which may have failed to identify hybrids containing small regions of the donor human chromosome. Siden demonstrated the superior sensitivity of Alu-PCR (89% positive) compared with dot-blot analysis using total human DNA (55%) or alphoid DNA (69%) as probe. 21 of 29 (72%) of our series of hybrids tested showed some inter -Alu product, a higher frequency than
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 102 detected with enzymic, Southern hybridisation or conventional PCR (60%). Two of the 29 were positive for a conventional marker but negative by Alu-PCR. False-negatives with Alu-PCR have been noted by others (Warrington et al, 1991): even sensitive screening methods such as interspersed repetitive sequence PCR will not detect integration of regions of chromosome where the IRS is infrequent.
Screening of hybrids using A/w-PCR has been used in the initial characterisation of radiation hybrid mapping panels before extensive marker analysis to eliminate those hybrids containing little or no human DNA (Bouzyk et ah, 1996; Gyapay et ah, 1996). Minimising the number of unproductive amplifications is a practical necessity when working with the 100-200 hybrid DNA samples required for single chromosome or whole genome mapping panels. Alu repeats are distributed throughout the genome except for the centromeric heterochromatin regions where there is fifty-fold under-representation (Moysis et ah, 1989). Discarding those hybrids which do not show Alu-PCR product may remove those hybrids containing DNA originating from the donor centromeres. These hybrids may have breaks in the peri-centromeric region or regions which may be critical for the continuity of the map in those areas. The whole- genome radiation hybrid panel from which the Genbridge4 resource has been derived has several gaps around the centromeres where the adjacent framework markers have pairwise LOD scores of <9 (Gyapay et ah, 1996). 220 hybrids were originally derived but 21 of these were discarded because Alu-PCR amplification of their DNA produced few products (or because they did not contain sequences corresponding to human TK, the locus used for selection).
Exploitation of Jo 12,Alu -PCR cloning and mapping
Sequence tagged site markers were generated by inter -Alu PCR, cloning and sequencing from 3 of the 6 radiation hybrids containing fragments of chromosome 11 including the llq22-23 region (Jo 12, 13', 48’). This study demonstrated the value of well-characterised somatic cell hybrids for the
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 103 derivation of DNA fragments (in this case, by Alu-PCR) as markers from defined chromosomal regions. These markers were then used to identify YACs from which additional linked markers were obtained. Polymorphisms identified by the primary and secondary sequence tagged sites were combined to provide an informative haplotype which led to the reduction of the ataxia telangiectasia candidate region from 10.3 cM to 4.1 cM, and what subsequently proved to be the correct placing of the AT locus within the new candidate interval (by location score estimates). This work was performed by Drs Bird and McConville, Birmingham and is included as Appendix 2 and in Gillett et al, 1993.
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 104 T in ' ■ + . . + + . + + . . i , , , 05 4 9 " , , , , , , , i i + i i M-
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Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 105 Human B4A0
Fig. 3.1.1: Lactate dehydrogenase (LDH) starch gel electrophoresis
Anode top, cathode bottom.
From left to right, (hybrid score): *lysed human red cells,^Wg3h (-), V (-) , ^0 1 (-) , $47 1 1 • ( + ),t351 (-),>26 (-),*5- (-) ,*11 (-) .
A single isoform is present in each hybrid originating from Wg3h (hamster). Electrophoresis of hybrid 47''' demonstrates additional cathodal bands from the human chromosome 11 component including LDHA present in this hybrid. Five bands are seen, three of which are likely to be due to multimers formed with the hamster isozyme. The human red cell "control" serves to indicate the position of the human isozyme multimers only, which differ in mobility from the human- hamster multimers. **** expected position of the human B0A4 isozyme. Hb, haemoglobin.
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 106 Fig. 3.1.2: Adenosine deaminase (ADA) starch gel electrophoresis
Anode top, cathode bottom.
From left to right, (hybrid score): *11 (+),1 5 l (-),*26 (-),**35' (-), S4 7 i i i (-) , ^ 3 0 1 (-) ,'*9 ' (-) ,*Wg3 h -, ^lysed human red cells .
Electrophoresis of hybrid 11 demonstrates an additional cathodal band originating from the hamster parent of J1C14 (human ADA lies on chromosome 20 and is not present in J1C14).
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 107 Fig. 3.1.3: Use of TYR4 amplification and MspI digestion of product to score radiation hybrids for TYR and TYRL
5%/12% non-denaturing acrylamide gel, cathode top, anode bottom. From left to right, 'Kb marker, VJ1C14,^undigested TYR4 product,^Jo4, 5 Jo6 ' , *Jol2, *Jol4, *Jo31,*Jo50, '*marker, mEJNAC .
Both TYR (150, 121 bp products) and TYRL (150, 92 and 29 bp) are amplified in J1C14, Jo6 ', Jol2 and Jo31. Jo4, Jo50 and EJKTAC are positive for TYRL but not TYR. Jol4 includes TYR but not TYRL. See {Table (text). The 29 bp fragment is not visible: it is run off the gel. Some illegitimate or partial digestion has occurred resulting in faint bands of approximately 130 and 121 bp in many tracks. The hamster cell line Wg3h does not amplify (data not shown).
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 108 Fig. 3.1.4: Alu-PCR of selected irradiation hybrids
6%/3.5% non-denaturing acrylamide gel, cathode top, anode bottom. From left to right: (1) Jo6 1, (2) Jol2, (3) Jol3' (no amplification), (4) Jol4, (5) Jol7, (6 ) Jo23 (no amplification), (7) Jo23 (weak amplification), (8 ) Jo31, (9) Jo33, (10) Jo48, (11) marker jx/Haelll, (12) Jol4-l, (13) Jol4-l, (14) no DNA (marker sizes), (15) no DNA, (16) Jol4-2, (17) hamster Wg3h (no amplification), (18) no DNA, (19) Jol4-3, (20) Jol4-4.
The Jol4 product has five predominant components. Each of the four higher MW bands were toothpicked and the toothpick tip re-amplified. 14-1 was picked from the band of lowest mobility but the re-amplified products are all of faster mobility (lower MW). 14-2, 14-3 and 14-4 each contain major products corresponding to the band picked, together with lower MW species. The gel has suffered some edge distortion ("smile").
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 109 Fig. 3.1.5(a, b, d) : Fluorescent in situ hybridisation of hybrid Jol2 using total human DNA as probe
Metaphase chromosome preparations of Jol2 showing the presence of human DNA fragments. One is integrated within a hamster chromosome (largest signal); a smaller fragment appears to be separate. Two representative cells are shown: the number and type of human fragments was heterogeneous in the culture. 5(d), lower right, is the background video image of the hybrid metaphase spread 5 (b) (stained with propidium iodide) on which the probe image (total human DNA labelled with FITC) is superimposed in 5 (a, b, c).
Fig. 3.1.5(c): Fluorescent in situ hybridisation of human metaphase chromosomes using Alu-PCR products from hybrid Jol2 as probe
Lower left image. The larger signal delineates llq23, the smaller is from proximal lip.
Ch. 3.1 Radiation hybrids derived from a chromosome 11-only parent 110 C h a p t e r 3.2: Isolation of a hybrid containing llq 2 3 only, J12.1A
Introduction
Irradiation hybrid Jo 12 contained human DNA representing two regions of chromosome 11, llpll.2 and Ilql4-llq23.3. FISH analysis had indicated that some of the hybrid cells contained the llq fragment, solely. The aim of this experiment was to isolate that fragment of chromosome 11 containing the region Ilql4-llq23.3 (TYR-PBGD) in a subclone, w ithout the fragment containing the llpll.2 (TYRL) region. This was attempted by single cell cloning: the use of limiting dilutions of Jo 12 suspensions to approximate to one cell per well of a multiwell culture plate (see Methods).
R esults
Analysis of subcloned hybrids
The nine subclones (designated J12.1A & J12.1B, J12.2, J12.3, J12.4, J12.5, J12.6, J12.7 and J12.9) were analysed by marker analysis using Southern hybridisation and/or PCR, and by FISH of labelled human DNA onto hybrid metaphase spreads. J12.1A was also analysed by FISH of inter -Alu PCR product onto human metaphase chromosome spreads.
Analysis by Southern hybridisation and PCR
The markers were those used for the characterisation of the initial irradiation hybrid panel ( v.s. Table 3.1.1). Results of the marker analysis are shown in Table 3.2.1. One clone, J12.1, was positive for all l lq markers, but not for the lip locus, TYRL. Two clones were weakly positive for the
Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 111 centromere marker D11Z1 only, J12.1B and J12.4. Three clones were positive for one llq marker only, J12.2, J12.7 and J12.9 and three clones were negative for all markers examined, J12.3, J12.5 and J12.6. One aliquot of J12.5 DNA was positive for all lip and l lq markers tested (aliquot #5, see Figure 3.2.1, results not included in Table 3.2.1). Alu-PCR of the subclones were largely in agreement with these findings: no product was seen in J12.1B, J12.3, J12.4, J12.5 (apart from aliquot #5), J12.6 and J12.9 (Figure 3.2.1).
Analysis by FISH
Preparations of each subclone (except 12.1A, see below Table 3.2.3) were hybridised to total human DNA. For each subclone, metaphases were chosen at random and the human chromosome content noted. The results are summarised in Table 3.2.2 below. Subclones J12.2, J12.3 and J12.6 did not appear to contain any hum an material. Subclones J12.4 and J12.7 each contained one region of hybridisation of the same size in each of the ten metaphases examined. Subclones J12.1B, J12.5 and J12.9 were heterogeneous in fragment number per cell and fragment type.
J12.1B: a small signal was apparent in 63% of 30 interphase nuclei examined, Figure 3.2.3(c,d).
J12.4: all metaphase spreads contained a very small fragment (possibly of pericentromeric human chromosome 11, the only region shown to be present in the marker analysis), Figure 3.2.4(a,b).
J12.5: single interphase signals were seen in 48 % of 29 nuclei, multiple signals in 10 % and signal was not seen in 42 %, e.g. Figure 3.2.4(c,d).
Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 112 Sub- Metaphases Number of cells with clone examined different fragments none one two >two
IB 30 8 19 3 2 10 10 3 10 10 4 10 10 5 29 12 14 3 6 10 10 7 10 10 9 10 3 4 2
Table 3.2.2: Hum an chromosome content of Jo 12 subclones, J12.1B-J12.9.
In a separate experiment, Dr Margaret Fox studied 40 metaphase spreads from J12.1A probed with labelled total human DNA, Table 3.2.3. All but one had a large region of human chromosome integrated within a hamster chromosome. 80% had one or more small, apparently non integrated fragments, in addition, Figures 3.2.2(a-d), 3.2.3(a,b).
Subclone J12.1A Number of metaphases %
One integrated region, plus No "non-integrated" fragments 5 12.5 One "non-integrated" fragment 20 50.0 Two "non-integrated" fragments 11 27.5 Three "non-integrated" fragments 1 2.5
O ther 3 7.5
Table 3.2.3: Human chromosome content of the Jo 12 hybrid subclone, J12.1A.
Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 113 Isolation of cosmids from regions of human chromosome 11 contained in selected hybrids
J12.1A was also used by us to identify cosmids from a gridded human chromosome 11 genomic cosmid library (reference number 107, L4/FS11) which map to the region of chromosome 11 contained in the J12.1A subclone hybrid (Zehetner et al., 1994). This technique is described in Chapter 2, Methods and in Chapter 3.4, "The identification of genomic cosmids from the MEN1 critical region, using hybrid Jo2' ". In brief, bulk Alu-PCR products from amplification of subclone DNA were labelled and hybridised (with human Cot-1 DNA competition) to filters bearing the gridded clones. In a separate experiment, Alu-PCR product from irradiation hybrid Jo48' was hybridised to the same filters. Jo48' is positive for markers from llql3 (e.g. PYGM) and llq22-23.1 (NCAM and DRD2) but lacks markers from the intervening region of chromosome 11 present in J12.1A (see Figure 3.3.1(b), Tables 3.1.1 and 3.3.1). Cosmids which gave positive signals with both probes were predicted to lie in regions of chromosome 11 common to both hybrids. The predicted localisation was assessed by mapping of the individual cosmids inserts by FISH. Metaphase spreads were prepared both from a normal human 46 XY and from a cell line carrying a balanced constitutional 11:22 translocation (breakpoint in llq23.2).
Eleven cosmids were selected for analysis, five of which hybridised to J12.1A Alu-PCR products; the remaining six were positive with Jo48' products. Cosmid stocks provided by the Reference Library DataBase (Zehetner et al., 1994) were checked for contamination by re-hybridisation with labelled Alu-PCR product from the appropriate hybrid (column 2 of Table 3.2.4 below). Cosmids were also screened for (CA)n simple sequence repeats by hybridisation with labelled (GT)io probe (by Dr Frances Benham ).
Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 114 Cosmid Signal with Alu- Predicted FISH CA Repeat PCR Products Localisation Localisation Screen J12.1A Jo48'
D1016 - + ql2 - ql3 ql2 - ql3 -
F10149 - + ql2 - ql3 ql2 - ql3 -
C11179 - + ql2 - ql3 qi2 +
D1041 - + ql2 - ql3 qi2 - B027 - + ql2 - ql3 ql2 - ql3 - D08126 - + ql2 - ql3 ql2 - ql3 -
B11151 + + llq22-23.1 ql4 - q23.2 - C0572 + ? + llq22-23.1 ql4 - q23.2 -
E01115 + - ql4 or q23.2 q23.2 - 23.3 + E08171 + - ql4 or q23.2 q23.2 - 23.3 +
F0662 + - q!4 or q23.2 q23.2 - 23.3 -
J12.1A contains Ilql4.3-q23.2. Jo48’ contains parts of llql2-13 and q22-23.1.
Table 3.2.4: Localisation by FISH of ICRF cl07 cosmids which hybridise to Alu-PCR products from J12.1A a n d /o r Jo48'.
Cosmids E01115, E08171 and F0662 were shown by FISH to hybridise to the derivative chromosome 22, and must lie distal to the constitutional 11:22 breakpoint in llq23.2. B11151 and C0572 hybridised to the derivative chromosome 11.
Of the eleven cosmids in which the location has been confirmed by FISH, six map to Ilql2-ql3, two to Ilql4-llq23.2 and three to llq23.2-23.3. The flow-sorted chromosomes from which the chromosome 11 cosmid library was made contained a deletion in the llq23.1 region. If the deletion extends into llql4 this may explain the failure to isolate any cosmids in the second group (J12.1A +, Jo48' -) which map to the llq !4 region.
Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 115 Discussion
Numbers of fragments retained per hybrid
Fragment retention in this limited analysis is consistent with that found in other irradiation hybrid studies which have demonstrated using FISH that human fragments are retained as translocations or insertions into the "recipient" hamster chromosomes or as fragments apparently consisting entirely of human DNA (Cox et al, 1990). The use of FISH may underestimate the numbers of human fragments per hybrid since some FISH signals may arise from more than one fragment which are inserted too close together in the hamster chromosome to be easily differentiated (by the in situ techniques available at the time) or are fused together with human or mouse centromeric material to form an independent chromosomal fragment (Glaser et al, 1990).
If chromosome 11 comprises approximately 4.516 % of the human genome (Morton, 1991), that is 145 Mb (assuming the genome size is 3200 Mb), then given an average retention of 5.5 %, each hybrid should contain approximately 8.0 Mb. Pulsed field gel mapping using DNA made from another set of high-dose irradiation hybrids (chromosome 5, (Thomas, 1991)) has indicated that the average size of retained fragments was approximately 0.8 Mb and this figure is consistent with the estimates of Cox et al. which indicate that an irradiation dose of 40,000 rad (as used in our experiment) might be expected to generate fragments of about 1.5 Mb (Cox et al, 1990). If applicable to the Jo series, this would suggest that the average number of retained fragments per hybrid would be between 5 and 10, approximately. This analysis using FISH indicates that the number of visible fragments is very variable even within subclones from one hybrid. This variation in number between hybrids has been demonstrated in a study by Siden et al in which between one and five human fragments per cell were found using FISH in 7 hybrids randomly selected for analysis from 3 series generated with radiation doses of 5, 12.5 or 25 krad. Fragment size varied inversely with irradiation dose but there was no relationship
Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 116 between dose and number (Siden et al., 1992). A panel of 39 radiation hybrids was derived alongside the Jo-series hybrids from a human chromosome 9q-containing somatic cell hybrid using a similar radiation dose to our experiment (Fitzgibbon, 1993). More than three fragments of human origin were seen in 12 of 26 hybrids shown to retain human DNA by FISH. The other 14 hybrids contained 1-3 fragments per cell. Two hybrids were not tested by FISH and 9 of the remaining 11 were negative both on FISH and marker analysis.
Jol2 subclones
Three of the nine subclones did not appear to contain any human chromosomal material as judged by FISH. This probably reflects the proportion of cells in the parent culture of Jo 12 which did not contain any human chromosomal material. Of the subclones containing identifiable human material, the proportion of metaphase spreads which were negative was 0 % (J12.1A, J12.4 and J12.7), 10 % (J12.1B), 30 % (J12.9) and 42 % (J12.5), average 14.5 %. Scoring of interphase nuclei was less sensitive (small human fragments may have been difficult to distinguish from background): the proportion of nuclei which were negative was 35% (J12.1A), 41% (J12.5). The other subclones were not scored.
There was concurrence between the marker and the FISH analysis for subclones J12.2 (weak signal for STMY only), J12.3 and J12.6. However, J12.5 contained human material as judged by FISH but not by the marker analysis. This may have been due to loss of these fragments in the subsequent expansion of the clone from the 80 cm2 flasks (used for the FISH study) to the three 175 cm2 flasks from which DNA was prepared. Positive results in the marker analysis and Alu-PCR product obtained from one aliquot of J12.5 DNA but not from the others provides some evidence to support this hypothesis.
The pericentromeric region is not thought to harbour many Alu repeat sequences (Smit, 1996), which may account for the discrepancy between the
Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 117 FISH and the Alu-PCR results for J12.4. Amplification of one aliquot of J12.5 did not produce any Alu-PCR product, whereas another aliquot showed several bands, v.s. ). No product was seen with J12.9, although D11S35 was amplified in the same aliquot of DNA, and 70% of subclone metaphases contained one or more human chromosomal fragments.
144 Nunclone wells (four 24-well plates) were seeded with diluted stock culture. Ten subclones were obtained, one of which failed to expand. Of the remaining nine, only one was consistently found to contain any significant amount of human chromosomal material. It was therefore fortunate that this subclone lacked the region of lip which we had hoped to exclude, and contained the whole of the Ilql4-llq23.3 region which we intended to retain. This region included all 18 markers between (and including) TYR proximally and PBGD distally (Table 3.2.1).
This hybrid was subsequently exploited by Yosef Shiloh's research group in Tel Aviv to map several novel microsatellite markers obtainied from various sources (Genethon, Utah Department of Human Genetics and Iowa Cooperative Human Linkage Center) to llq22-23, the candidate region for the locus or loci mutated in Ataxia Telangiectasia. The markers found to be present in the J12.1A hybrid were confirmed and their localisation further refined by assaying them in a series of YAC clones spanning the region (Rotman et al., 1994; Vanagaite et al., 1994; Vanagaite et ah, 1995). Of the eight microsatellite markers mapped in this way, four subsequently proved to be within about 2 Mb of ATM, and one of these, D11S1294, within 250 kb of the gene. It is gratifying that two of the closest polymorphic markers to ATM were either derived from or mapped with Jo 12 or its derivative (Ambrose et al., 1994).
This strategy of sequential hybridisation of a gridded chromosome 11 cosmid library with Alu-PCR products from hybrids provides a rapid and efficient means to identify cosmids from regions of interest. It is dependent upon careful characterisation of the hybrid used to generate the probe. Cosmids which map by FISH outside the expected region may be
Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 118 due to unrecognised fragments contained within the hybrid. An ideal hybrid for this purpose would be one on which multiple markers throughout the length of the chromosome have been assayed. The most densely characterised chromosome 11 hybrid series is the Richard panel (Richard et al, 1991). Alu-PCR products from this panel of low-dose irradiation hybrids could be used in a similar manner as the Jo examples to "bin" the cosmids into physical intervals on the chromosome related to the centiRay data obtained from the marker studies. The human chromosomal content of the low-dose hybrids is much greater than the Jo series, and large numbers of cosmids would be expected to hybridise to the complex Alu-PCR products. However, as more of these hybrids are processed, the location of a given cosmid would become increasingly precisely defined, limited not by the resolution of the framework marker studies on the hybrids but by the number and position of breaks in the hybrid set. It is unlikely that every cosmid in the library would contain Alu repeat sequences oriented so as to be amplified by the chosen primers, and this would limit the assignment of all cosmids to a centiRay bin. However, the strategy should enable a useful preliminary assignment of cosmids prior to a more conventional contig building excercise (as is demonstrated by the cosmids from the MEN1 region, discussed in Chapter 3.4). It would be equally applicable to single chromosome or whole genome PAC and BAC libraries.
This tactic for obtaining genomic cosmid clones from a region of a chromosome of interest may be compared with an alternative method of constructing a cosmid library from the hybrid itself, and then screening the library with labelled human DNA or total human genomic Alu-PCR product to identify cosmids from that region. This is a much more laborious technique which requires that the hybrid be regrown to obtain high molecular weight DNA. DNA from the recultured hybrid must then be re-characterised to ensure that there has not been any loss of human chromosomal material during freezing, storage, thawing and subsequent culture. This approach was attempted by Jude Fitzgibbon (Fitzgibbon, 1993) using similar high dose irradiation hybrids made from a parent containing
Ch. 3.2 Isolation of a lhybrid containing llq23 only, J12.1A 119 human chromosome 9q as its sole human component. Two libraries were generated, both of which were small (approximately 20,000 clones per library). A total of only eight clones hybridised (four from each library) when probed with total human DNA (with Cot-1 DNA competition). Six of the eight mapped to the region of interest, and two to the centromere. This compares with over 26 cosmids identified in the first round screening with Alu-PCR product from Jo2' (see Chapter 3.4) all of which were subsequently shown by FISH to map to the expected region, llql3. The Alu-PCR screening procedure took 72 hours whereas the library construction approach took two weeks.
Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 120 SUBCLONES: Locus Probe Mode Jol2 12. 1A 12. IB 12.2 12.3 12.4 12.5 12.6 12.7 12. a I • I I • I 1 I 1 I I 1 • I I • I I ' I I • I I I I I 1*I 1I 1 1 1 1 * 1 1 * 1 1 1 1 1 1 I I rH H> r H rH rH rH >H rH rH rH * ft p u p ES] — CO a 1 h . slto fahbi otiiglq3ol,J21 121 J12.1A only, llq23 containing ahybrid of Isolation 3.2Ch. —1 ,— E P - I - - I - - 1 - - I - + + + + + + + + + + + + + + + + + + + + + • + + + + + + + + + + a a D I • I 1*11111*11*111111 h h
P VO CN l> rH CO *— N C H r Q ni mm m in in w a a a 0 + i i i • i i • i i i i + • 1 1 t H ^— rH CO CO in P P P IT) oo
1 P VO in in E PL, S h S <- P rH Q P u P rH CN CO LO co in U 0 h s U K H r-~ in l r CN CN CN i> rH sjf g o•o •o m s P P a a a I • I I I I •+ I I •
N 2 CN s Q P CN CM ^ P D o CO rH in CN LO rH CO I u U1 p — CO 1 *—I K <— P p — a 1 1 t H rH rH r- N ^ CN \ rH 1 S o P CO PL, O U P CO P P rH P CO rH rH rH .. VO rH P m — . P 0 P a PQ 0 pq p CO 1 rH CN CN CO ,—ICN A TS CN I \— CM I \— CM \—I CN CN t rH LO PU s < PQ to - ( u o o O 1 ) 1 ■rl ■H ■H ■rl -9 >3 1 ’d A l r d « CN rH m H CN S b E 4 w © id d (D id 2 % agarose, cathode top, anode bottom. From left to right:'Kb marker,lhamster Wg3h (no amplification),5 Jol', **Jol2, *12 . 1A, *12 . IB (no amplification) , ^12 . 2 (faint amplification), *12.3 (no amplification),^12.4 (no amplification),'M2.5 aliquot #5, l,12 . 5 aliquot #20 (no amplification) , '*12 . 6 (no amplification) , *Jl2 . 7 (faint amplification) ,*Vl2 . 9 (no amplification) ,**Kb marker (bands: smear, 1636, 1018, 517/506, 396, 344, 298, 220). Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 122 •s Fig. 3.2.2(a-d): Fluorescent in situ hybridisation of hybrid subclones J12.1A using biotinylated total human DNA as probe Metaphase chromosome preparations of hybrid subclone J12.1A showing the presence of human DNA fragments. Representative cells are shown. 2 (b) and 2 (d) are the background video images of the hybrid metaphase spreads (stained with propidium iodide) on which the probe image (total human DNA labelled with FITC) is superimposed in 2(a) and 2 (c) . 2 (a) top left, 2 (b) top right, 2 (c) lower left, 2 (d) lower right. Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 123 Fig. 3.2.3(a-d): Fluorescent in situ hybridisation of hybrid subclones J12.1A and J12.1B using biotinylated total human DNA as probe Metaphase chromosome preparations of subclones J12.1A, 3 (a, b) and Jol2.1B, 3(c, d) showing the presence of human DNA fragments. 3(b) and 3 (d) are the background video images of the hybrid metaphase spreads (stained with propidium iodide) on which the probe image (total human DNA labelled with FITC) is superimposed in 3(a) and 3(c) . 3(a) top left, 3(b) top right, 3(c) lower left, 3(d) lower right. Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 124 Fig. 3.2.4(a-d): Fluorescent in situ hybridisation of hybrid subclones J12.4 and J12.5 using biotinylated total human DNA as probe Metaphase chromosome preparations of subclones J12.4, 4 (a, b) and J12.5, 4(c, d) showing the presence of human DNA fragments. 4(b) and 4(d) are the background video images of the hybrid metaphase spreads (stained with propidium iodide) on which the probe image (total human DNA labelled with FITC) is superimposed in 4(a) and 4(c). 4(a) top left, 4(b) top right, 4(c) lower left, 4(d) lower right. Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 125 Fig. 3.2.5(a-d): Fluorescent in situ hybridisation of hybrid subclones J12.7 and J12.9 using biotinylated total human DNA as probe Metaphase chromosome preparations of subclones J12.7, 5 (a, b) and J12.9, 5(c, d) showing the presence of human DNA fragments. 5(b) and 5(d) are the background video images of the hybrid metaphase spreads (stained with propidium iodide) on which the probe image (total human DNA labelled with FITC) is superimposed in 5(a) and 5(c). 5(a) top left, 5(b) top right, 5(c) lower left, 5(d) lower right. Ch. 3.2 Isolation of a hybrid containing llq23 only, J12.1A 126 Chapter 3.3: Isolation of hybrids containing human chromosome llql3 Introduction MEN1, Multiple Endocrine Neoplasia, type 1, is a familial cancer syndrome of endocrine glandular tumours or adenomas involving predominantly the pituitary, parathyroid and pancreas. The tumours are generally histopathologically benign but are endocrinologically active and produce symptoms and signs which are dependent upon the hormone elaborated. Hormone synthesis and release is dissociated from the usual feedback endocrine regulation and the hormones are present in the bloodstream in excess. The common presentations are of hyperparathyroidism (hypercalcaemia), pituitary tumours (e.g. prolactinomas, or ACTH secreting pituitary adenoma producing hypercortisolism and Cushing's disease) and pancreatic islet cell or duodenal adenoma (e.g insulinoma). Many other tumours have been described in MEN1 kindreds. The related endocrinopathy MEN2 (medullary thyroid carcinoma, hyperparathyroidism and phaeochromocytoma) is inherited in an autosomal dominant fashion (as is MEN1), and is due to activating mutations in the RET oncogene (Mulligan et ah, 1993). The condition has high penetrance but variable expressivity (Wermer, 1954; Zollinger et ah, 1955). MEN1 was linked to markers on chromosome llq l3 in 1987 and 1988 by two groups (Bale et ah, 1987; Larsson et ah, 1988). Peak lod scores were initially obtained with markers for FGF3 (INT2, llql3.3) and PYGM (llql3.1). Shortly after linkage was established and loss of RFLP allele heterozygosity in insulinomas demonstrated in one family by Larsson, two further groups demonstrated loss of heterozygosity in parathyroid tumours from patients in MEN1 kindreds (Bale et ah, 1989; Thakker et ah, 1989). Subsequent linkage data and loss of heterozygosity studies narrowed the candidate region at the time of this study to Ch. 3.3 Isolation of hybrids containing human chromosome llq l3 127 approximately 3 cM just distal to PYGM, flanked by PYGM and D11S460/D11S807 (telomeric) (Larsson et ah, 1992a). It has been inferred from the loss of heterozygosity (LOH) data that the MEN1 gene has a tumour-suppressor function, the action of which is recessive at the cellular level. Homozygous loss of this function leads to neoplasia. Other evidence is more compatible with oncogene activation with a dominant gain of function at the cellular level (Brandi et ah, 1986; Scappaticci et ah, 1991). The two theories could be reconciled if the primary genetic defect was oncogene activation and loss of the normal allele was secondary. However, pre-adenomatous hyperplasia from tissue adjacent to MEN1 adenomas has also been shown to have the same loss of heterozygosity as the adenoma itself (Larsson et ah, 1988). This might imply that LOH is an early event in the process of MEN1 tumour development (in the pancreatic islet at least). Benign adrenocortical adenomas are a common associated tumour in MEN1, found to affect one in three patients at post mortem. In these tumours both parental marker alleles are preserved in the PYGM region: there is no loss of heterozygosity. The evidence is contradictory and does not provide conclusive support for either mechanism of carcinogenesis in MEN1. In 1992, few markers had been mapped to the PYGM candidate region. Sixteen markers had been mapped to the llql2-13 region in the Richard- Cox Chromosome 11 mapping panel and five of these had been placed (but not precisely ordered) between PYGM and FGF3 (Richard et ah, 1991). Only 33 loci were listed on the radiation hybrid map in this interval at the Fourth International Workshop on the Mapping of Human Chromosome 11 held in Oxford in September 1994 (Van Heyningen et ah, 1995). Several groups involved in MEN1 mapping began to construct YAC and cosmid contigs across this region in order to refine the map and to generate additional polymorphic markers to position the MEN1 locus more precisely. Ch. 3.3 Isolation of hybrids containing human chromosome llq !3 128 The aim of this experiment was to identify one or more of the Jo series irradiation hybrids containing the candidate region for MEN1 and little or no other human chromosomal material. Results The initial characterisation of the Jo irradiation hybrid series identified seven hybrids which contained PYGM, D11S97 or FGF3: 2', 15', 17, 31, 33, 41' and 48'. These hybrids formed a llql3 subset panel which was characterised further using additional markers in the llql3 region. At the time, the location of TYR was in doubt, and hybrids 6, 12 and 14' were included in the analysis. The markers used are in a PCR format and are listed in Methods, Appendix 3: CD5 antigen CD5, Pepsinogen PGA, Ferritin heavy chain 1 FTH1, Cytochrome C oxidase subunit VIII COX8, Phosphorylase glycogen muscle PYGM, the human homologue of avian retrovirus proviral tyrosine kinase SEA, keratin ultrahigh sulphur 1 KRN1, fibroblast growth factor 3 FGF3 (previously INT2), Glutathione S transferase P 1 GSTP1 (previously GST3), tyrosinase TYR, cofilin non muscle type CFL1 (see Chapter 3.6), fucosyltransferase 4 FUT4, and STS or microsatellite markers D11S97, D11S146, D11S527, D11S528, D11S533 and D11S534. Results of the further characterisation of the llq l3 subset panel are shown in Table 3.3.1. The marker characterisation of the llql3 subset panel indicated that five of the eleven hybrids contained the MEN1 candidate region telomeric to PYGM: Jo2', 15', 31, 48' and 50'. The preliminary characterisation of the full Jo series panel had shown that four of these five contained one or more regions of human chromosome 11 in addition to llql3. Only one hybrid, Jo2', appeared to contain the llql3 region and no other. This was confirmed by FISH of labelled Jo2' Alu-PCR product onto human metaphase chromosome spreads Figure 3.3.1. A single band in the llql3 region was the only visible region of hybridisation. The alternative hybrids each contained one or more additional fragments. Jo2' was therefore chosen as the hybrid with which to identify genomic cosmids from the MEN1 region (see the next chapter, 3.4). Ch. 3.3 Isolation of hybrids containing human chromosome llq l3 129 Jo48' was also used in separate but similar experiments to identify cosmids in the same llql3 region and in the llq22-23.1 region also contained in Jo48' (see Figure 3.3.1(b) and the previous chapter, 3.2). Discussion The marker loci in Table 3.3.1 are ordered so as to to minimise the number of apparent breaks in the subset hybrid panel (uppermost centromeric, lower telomeric). This most parsimonious order is PYGM-(SEA, CFL1)- GSTP1-D11S97, which conserves the continuity of the regions in 2' and 15'. This was initially inconsistent with the consensus order. D11S97 (pMS51) had not been positioned using the Richard hybrids which formed the basis of the fourth Chromosome 11 workshop's ordering of loci on llq. The approximate order (centromeric-telomeric) given in that report was PYGM-D11S97-(SEA, CFL1)-GSTP1 (Van Heyningen et ah, 1995). This would imply that there was a break in each of two large, otherwise continuous regions of retained human chromosomal material in hybrids 2' and 15', and an additional discontinuity in 48'. Frequent breaks are to be expected in high-dose irradiation hybrids, and the llql3 subset data provides evidence of this: the apparent small break in hybrid 48' identified by COX8, and breaks in the llql3.3-13.5 region in hybrid 31. However, Richard acknowledged in the report that the radiation hybrid data might support an inversion of loci in the SEA-GSTP1 region, which if it were to include D11S97, would have produced the order PYGM-GSTP1-(SEA, CFL1)-D11S97. The order PYGM-SEA-D11S97 is consistent with that determined by the Larsson and Nordenskjold group from the analysis of their MEN1 kindreds and from mapping of markers on the Prof. Carol Jones series of her own radiation reduced hybrids (Larsson et ah, 1992a; Larsson et ah, 1992b). The European MEN1 Consortium study of loci in llql3 region supports the order COX8-PYGM-(SEA-CFLl)-DllS97 (Courseaux et ah, 1996). The most telomeric marker in this analysis is D11S97; GSTP1 was not considered. The localisation of GSTP1 was also confused by the early Richard data until other evidence placed it distal to PYGM and proximal to FGF3 (Smith et ah, 1995). Our study is consistent Ch. 3.3 Isolation of hybrids containing human chromosome llq !3 130 with the most recent consensus report from the fifth Chromosome 11 workshop which orders these loci cen-PYGM-SEA-GSTPl-DllS97-FGF3- tel, (Shows et ah, 1996). The marker D11S146 was not included in either the fourth or the fifth workshop maps. D11S146 and D11S534 are known to be closely linked (maximum LOD score 11.88 at theta = zero, (Hauge et al., 1991)): the loci are shown adjacent in Table 3.3.1. D11S146 is less than 100 kb telomeric to D11S97 (Szepetowski et al., 1992). The results of the more detailed marker studies in this experiment suggest that high-dose irradiation hybrids may contain very small fragments of the parental hybrid human chromosome. The only positive markers in the llq l3 region in Jo50' are SEA and CFL1, which are shown in the Chapter 3.6 to be within 55 kb, at most. Further marker studies in this region would help to define the size of the fragment in Jo50' (which is potentially very small) and elucidate the order of the markers between PYGM and FGF3. Jo48' contains both FTH1 and PYGM, loci separated by 100 cRay^oo in the Richard series hybrids, equivalent to approximately 5 Mb (Richard et al., 1991). This interval was shown by PFGE to be 3.1 Mb by the European MEN1 Consortium. COX8 lies between these two loci, (distal to FTH1), 2 Mb cetromeric to FTH1 and 1.1 Mb telomeric to PYGM (Courseaux et ah, 1996). COX8 is absent in Jo48', suggesting that the apparently continuous fragment of llql3 contained in this hybrid must be composed of at least two components, separated by up to 3.1 Mb. This lacuna is not visible when labelled Alu-PCR product from Jo48' is hybridised to metaphase spreads, Figure 3.3.1(b). This indicates that the number of fragments seen by FISH may be an underestimate of the true number, given the resolution available at the time and the use of probes derived from Alu-PCR product. Recent developments of the FISH technique such as DNA fibre-FISH or Dynamic Molecular Combing (see Introduction) might now enable these gaps to be visualised (Michalet et ah, 1997). Ch. 3.3 Isolation of hybrids containing human chromosome llq !3 131 CM 3 ^ I> oo oo t" H OO ^ CN ro CN _ H c r i \ m h in in uo HOO § HftWrOHWWWW ^ CO LD S ^1 0 CN CN o d 1 LD I I I I I ++ 1 rH oo d 1 in oo rH 00 H CN a d1 + + + I + + + + I I I i I 00 CJ I CN 01 O d 1 i i i + i i i i •0 •H ■H 4-1 U ■H TS£ ro (d oo i i i i i i i i i + i i i i i i i + I n £ o ■H CN OO ■rl rH CN CN 4-> \—I D1 C1 1+ + + + + + +1 i + i i+ + fl) ■H13 CO u CN OO u CN CN O1 t? + + + + i i i i i i i i «h 4H 0 O W G LD iH O t—I + + + + + + + +I i i i i i i i i i ® -H si CD Ch. 3.3 Isolation of hybrids containing human chromosome llq !3 132 Fig. 3.3.1 (a, b) : Fluorescent in situ hybridisation of human metaphase preparations probed with biotinylated Alu-PCR product from radiation hybrids (a) Jo2', and (b) Jo4 8 1 (a), Jo2' appears to contain human chromosome llql3 in isolation, (upper). (b) A lu-PCR product from Jo481 hybridises with a similar region, and llq22-23.1 visible in some preparations, (lower, not shown). Marker studies indicate that there must be a gap in the llql3 region contained in Jo48' of up to 3.5 Mb which is not shown by FISH (see text). Ch. 3.3 Isolation of hybrids containing human chromosome llq !3 133 Chapter 3.4: The identification of genomic cosmids from the MEN1 critical region, using hybrid Jo2f Introduction In 1991, Monaco et al. indicated that cosmid and YAC libraries could be screened by hybridisation of Alu-PCR product, eliminating the purification, cloning and analysis of each product prior to screening (Monaco et al, 1991). They exploited this technique rapidly to isolate cosmids from an X-chromosome gridded cosmid library in the juxtacentromeric region of Xq (Muscatelli et al, 1992). In the same year we used a similar technique to isolate genomic cosmids from the llql3 region for a preliminary contig being built up by Dr Joanna Pang and Prof. Raj Thakker to assist the cloning of the MEN1 locus (Pang et al, 1996). The cosmid resource used was the ICRF RLDB chromosome 11 gridded genomic library, supplied on one high-density replica filter (reference library number 107, L4/FS11, Zehetner et al, 1994). Two identical filters were provided to permit both to be probed concurrently to facilitate the identification of true positive clones; only one was used. These Hybond N+ filters are approximately 22 by 22 cm onto which 20,736 cosmids are arrayed in 576 (24 by 24) blocks of 36 (6 by 6). Each cosmid on the filter can be uniquely identified by an x-y coordinate from a designated "origin". The library was constructed from cells cultured from an individual with an llq23.1 deletion (the FC11 cell line, provided by Dr Finbarr Cotter) by RLDB - ICRF staff. This enabled the smaller, deleted chromosome 11 to be separated from other chromosomes (9, 10 and 12) of the same fluorescent intensity by flow-sorting of chromosomes stained with the fluorescent dyes Hoechst 33256 and chromomycin A3. The procedure is similar to that described in the construction of gridded cosmid libraries for chromosomes X and 21, (Nizetic et al, 1991; Nizetic et al, 1994). Assuming that a normal, Ch. 3.4 The identification of genomic cosmids from the MEN1 critical region 134 undeleted chromosome 11 is 145 Mb (Morton, 1991), and the average insert size of each cosmid is 37 kb, then a chromosome equivalent is contained in approximately 4,000 cosmids. The parent library comprised about 12,000 clones which represents three chromosome equivalents. At ICRF, the entire library was gridded roboticly onto each filter together with a further 8,000 clones, so about two-thirds of the library was duplicated. Methods The screening of these filters is described in Methods, Chapter 2; the outline is recapitulated here. DNA from the Jo2' radiation hybrid was amplified using the Cotter Alu IV primers (Cotter et al, 1990). The product was cleaned up by passing through Promega PCR mini-columns. The concentration of the purified Alu-PCR product was estimated by fluorimetry, and 25 ng labelled with 32P using random-priming. In previous experiments using labelled Alu-PCR product from Jo series hybrids to probe this cosmid library, Frances Benham had used (unlabelled) Cot-1 DNA as competitor, to reduce background hybridisation. Large amounts of the Cot-DNA were required, considerably increasing the expense of the procedure. Dean Nizetic suggested that I should try unlabelled Alu-PCR product as an economical alternative, in a ratio of 50:1 unlabelledilabelled product. I used 640 ng of "cold" product from the parent chromosome 11-containing hybrid J1C14 incubated at 72 C for 80 minutes with an estimated 12.5 ng labelled Jo2' Alu-PCR product (half of the labelling reaction). A single Library 107 filter was prehybridised for 105 minutes (in 0.5 M sodium phosphate pH 7.2, 7 % SDS, 1 mM EDTA and 1 % albumin) and hybridised overnight at 65 C (in the same solution). The filter was rinsed at room temperature and washed in 100 mM sodium phospate, 0.1 % SDS at 65 C for 45 minutes and at 73 C for a further 50 minutes, down to 20 decays/sec. at 10 cm from the filter. The filter was autoradiographed with intensifying screens at -70 C for two hours. To obtain maximum definition the filter was also autoradiographed at room temperature overnight. Ch. 3.4 The identification of genomic cosmids from the MEN1 critical region 135 Results The labelled Jo2' Alu-PCR product hybridised with multiple clones: approximately 80 could be discerned, Figure 3.4.1. Each of the five (plus four duplicate) cosmids which hybridised to labelled CFL1 PCR product (Chapter 3.6) also hybridised to the Alu-PCR product, as expected (Jo2' was known to be positive with the CFL1 primers). The 22 most strongly hybridising, duplicated clones were selected, and obtained as 21 stabs from the ICRF Reference Library DataBase. One clone was present on the filter in quadruplicate. These were grown up in liquid culture, spread and single colonies plated in ordered arrays onto agar culture plates (containing kanamycin). Lifts were taken onto Hybond filters, which were then probed with the hybridisation solution saved from the initial experiment (approximately 10 days later). All of the 22 clones hybridised as expected (Table 3.4.1, column 2), though it was later recognised that clone ICRFcl07H0295 was contaminated with another colony of different morphology, which did not hybridise (which may account for the unexpected FISFI mapping result with DNA thought to be from this cosmid). ICRFcl07 identifications of these clones and the chromosome 11 FISH localisations are given in Table 3.4.1, below. Of the eleven clones mapped by FISH (by Drs Margaret Fox and Lynne West) all except H0295 were found to lie in the expected region of chromosome 11. There is no evidence from the marker studies, or from FISH of Alu-PCR product that the parent hybrid Jo2' contains any part of llq24-25. However, these high-dose irradiation fragment hybrids may contain very small regions of the human chromosome which may only become apparent with a much higher density marker analysis, as shown in the study of the MEN1 region hybrids, Chapter 3.4. H0295 is probably a contaminated clone, but it is possible that Jo2' does contain a previously unidentified part of the distal long arm of chromosome 11. Ch. 3.4 The identification of genomic cosmids from the MEN1 critical region 136 cosmid Jo2' Alu-PCR Localisation Local (Galton) ICRFcl07 qll-ql3.31 X bv* FISH Reference A0138 + + Jo2'-18 A062 + + Jo2'-17 A1277 + + ql3 CFL1 B0853 + + Jo2'-16 B0885 + + Jo2'-26 D01105 + + Jo2'-23 D05103 + + Jo2'-15 D0625 + + Jo2'-13 D0950 + + Jo2'-12 E0622 + + ql3 CFL1 E072 + + ql2 Jo2’-ll E1046 + + ql3 CFL1 E1288 + + ql3 CFL1 F0769 + + Jo2'-22 F0780 + + Jo2'-8 F0928 + + qi2 Jo2'-10 F1094 + + Jo2'-9 G028 + + Jo2'-5 G04104 + + ql2 Jo2'-4 G07102 + + ql3 CFL1 G077 + + Jo2'-7 G0799 + + Jo2'-21 G0870 + + Jo2'-6 H025 + + ql2 Jo2'-3 H0295 + + q24-25 Jo2'-2 H03104 + + q!2 Jo2'-l Table 3.4.1: ICRFcl07 cosmids which hybridise to labelled Alu-PCR product from Jo2'. Ch. 3.4 The identification of genomic cosmids from the MEN1 critical region 137 These cosmids were given to the Prof. Raj Thakker's group at the Royal Postgraduate Medical School, and their origin from the ql2-13 region was confirmed by hybridisation of cosmid insert to a panel of chromosome 11 mapping hybrids (Pang et ah, 1996). Jo48' was used in separate but similar experiments by Dr Frances Benham to identify cosmids in the same llql3 region and in the llq23.1 region also contained in Jo48' (Chapter 3.2). The localisation of eight cosmids was confirmed by FISH: six mapped to the llql2-13 region which is the predominant human component of the hybrid, one to a more centromeric region (llcen-llqll) and one to llql3 and also to 3q, 8p and to the short arm of either 4p or 5p. None of these few cosmids were found to originate from the small llq23.1 component of Jo48'. Nor were any of the cosmids mapped by FISH to llql2-13 the same as those isolated using labelled Jo2' Alu-PCR product. This is not surprising since relatively few cosmids were isolated and localised from this large region of chromosome 11 in either experiment. Discussion The results of this experiment provide further evidence of the value of a well-characterised hybrid for identifying cosmid clones from a selected region of a chromosome, despite the complexity of the probe material used (Muscatelli et ah, 1992). The (then) conventional approach was to make a cosmid library from the hybrid of interest. We felt that this strategy was not efficient, and elected to screen a gridded human chromosome 11 cosmid library. The screening method was novel in two respects: the probe used was 32P-labelled inter -Alu sequence obtained by PCR, and "cold" Alu-PCR product from the parent J1C14 hybrid was used for competition, rather than Cotl DNA. Cold Alu-PCR product sufficed, giving very clear discrimination between true positive clones and background hybridisation. The positives could be identified by the presence of a duplicate in another, predictable region of the filter. The intensity of signal on the autoradiograph from each of the pair of spots was very similar. Ch. 3.4 The identification of genomic cosmids from the MEN1 critical region 138 This strategy also has considerable advantages over the technique of generating cloned Alu-PCR products as performed by the Prof. Malcolm Taylor's research group in Birmingham using Jol2 (described in Appendix 2 and in Gillett et ah, 1993), or that of "tooth-picking" individual large inter -Alu products used elsewhere. The precision of the two methods is similar: six of the eight Birmingham sequences derived from Jol2 mapped back to the expected region of origin, compared with ten of the eleven cosmids identified with Jo2' product (and confirmed by FISH). But the latter is rapid, simple, robust and relatively cheap. In addition, the final resource obtained, cosmids, are more suitable for further mapping excercises, such as contig-building, than the short-insert pBluescript clones containing inter -Alu sequence. If these are required, for sequence tagged site generation for example, they may be obtained easily by cloning individual Alu-PCR product bands from amplification of the isolated cosmids. The choice of the J1C14 parent as the source of DNA from which the "cold" Alu-PCR product was generated is contentious. Inevitably, the parent hybrid contains the region of interest in Jo2', and this has the effect of merely "diluting-out" the specific 32P-labelled Alu-PCR product. A more logical approach might be to use cold product from Jo series irradiation hybrids known to contain regions of chromosome 11 other than that in Jo2 (qll-ql3.3), such as J12.1 (Ilql4.3-q23). This would have the advantage of obscuring the hybridisation of labelled product from any unrecognised fragment of Ilql4.3-q23 in Jo2'. The converse would also be true, however: cold products from unrecognised fragments of qll-ql3.3 in Jol2.1 would compete with hybridisation of labelled products from Jo2'. A simple alternative would be to use cold Alu-PCR product from a human cell line or a somatic cell hybrid containing multiple human chromosomes other than chromosome 11, such as MOG2E5 (Wong et al., 1987). The technique recommended by the RLDB for ensuring correct identification of positive clones was to hybridise the filters (prior to the Ch. 3.4 The identification of genomic cosmids from the MEN1 critical region 139 main experiment) with 33S-labelled Lawrist 8 vector. This highlights the backround pattern of the cosmid grid (and also draws attention to abnormal combinations of vector and insert, "vector monsters"). Unfortunately, this pre-treatment inevitably makes it more difficult to identify clones which hybridise weakly with the 32P-labelled Alu-PCR product. Clones with short inter -Alu sequence (of the correct orientation) may only produce weak signals when hybridised but may still come from the region of interest. The absence of the 35S-labelled vector background requires more careful attention to correct "registration" of the filter during exposure of the autoradiograph and calculation of the coordinates of positive spots, but ensures that these weakly-hybridising clones are not missed. The chromosome 11 gridded genomic cosmid library ICRFcl07 was constructed from flow-sorted chromosomes with an interstitial deletion in llq23.1. This was necessary in order to separate the chromosome from others which sort together with, and are inseparable from, normal chromosome 11. The presence of the deletion, and the consequent absence of clones from the llq23.1 region is of no consequence in this attempt to isolate cosmids from the llql3 region. However, it would now be possible to isolate normal chromosome 11 by the intermediate step of creation of a human-muntjac somatic cell hybrid. The Indian muntjac deer have the fewest diploid chromosomes of all mammals: six in the female (1, 2 and X) and seven in the male (1, 2, X and Y). Nos 1 and 2 are "giant" chromosomes. The difference in size between muntjac and human chromosomes is so great that human chromosomes in a hybrid may be separated from the muntjac by flow sorting or by preparative centrifugation. Generation of a hybrid containing human chromosome 11 as its only member of the 9-12 group would facilitate the FAC sorting of normal chromosome 11, from which a complete chromosome 11 cosmid library could be generated (Lee et al, 1994). Initial linkage studies in families with Multiple Endocrine Neoplasia, MEN1 identified the llql3 region to which we mapped CFL1 (Chapter 3.6) Ch. 3.4 The identification of genomic cosmids from the MEN1 critical region 140 as that within which the candidate gene for MEN1 must lie (Larsson et ah, 1988). The Larsson and Nordenskjold group narrowed the MEN1 candidate region to a region of about 3 Mb flanked by D11S427 (telomeric to PYGM) and D11S807 by a combination of deletion mapping and the identification of recombinants in MEN1 families (Larsson et ah, 1992a; Larsson et ah, 1992b). Marker localisation in the Carol Jones series of radiation-reduced hybrids placed SEA distal to D11S807 (Larsson et ah, 1992b). This indicated that neither SEA nor CFL1 could have been a candidate gene. However, the order derived by the Larrson group from identification of recombination in MEN1 families for the more centromeric markers (CD20-D11S480-PGA-PYGM) differed from that obtained from other data, such as irradiation hybrids (CD20-PGA-D11S480) (C. W. Richard in Van Heyningen et ah, 1995; Sandford et ah, 1995). The map of a 5 Mb region of llql3 was refined using a number of methods to produce a consensus order which placed MEN1 within a 2 Mb interval extending from D11S1883 to D11S449-CAPN1 approximately (Courseaux et ah, 1996). This map placed SEA and CFL1 telomeric to CAPN1 and outside the candidate region. Prof. Raj Thakker's group were attempting to derive additional cosmid clones from the irradiation hybrid Jo2' (which includes the candidate region) when the cloning of the MEN1 gene was announced. The MEN1 gene was identified in 1997 by groups at the National Institutes of Health and the University of Oklahoma (Chandrasekharappa et ah, 1997). Thirty-three candidate genes were identified in a 2.8 Mb contig of YAC, BAC, PAC and PI clones. This interval was further refined by loss of heterozygosity mapping in microdissected tumour tissue from affected individuals. Two BACs and two cosmids spanning appoximately 300 Kb were sequenced within which 8 transcripts were identified and studied. One of these was found to harbour mutations in individuals with MEN1. ESTs from this gene had already been localised to the critical region on the human transcript map (Schuler et ah, 1996). The gene spans 9 kb, contains 10 exons (the first of which is untranslated) and encodes an ubiquitously- expressed 2.8 Kb transcript of 610 translated aminoacids, Menin. There are few clues to its function from the aminoacid sequence which does not Ch. 3.4 The identification of genomic cosmids from the MEN1 critical region 141 show homology with other known proteins. Mutations were detected in 14 out of 15 unrelated affected individuals, the majority being frameshift or nonsense mutations likely to lead to loss of function and consistent with the hypothesis that the protein product has a tumour suppressor function. The European Consortium also located the gene by meiotic mapping to a 900 Kb interval bounded by VRF and D11S1783 (Lemmens et al, 1997). It was one of 22 candidates found in a 1.2 Mb contig (which included some of the Jo2' cosmids), within the central group of five expressed sequences and about 55 kb telomeric to PYGM, one of the first markers to be linked with MEN1. Ch. 3.4 The identification of genomic cosmids from the MEN1 critical region 142 I Fig. 3.4.1: Autoradiograph of genomic cosmid filter ICRF cl07 (set 12) probed with 32P-oligo-labeiled Alu PCR product from hybrid Jo2' Ch. 3.4 The identification of genomic cosmids from the MEN1 critical region 143 C h a p t e r 3.5: Localisation of a simple sequence repeat polymorphism, D11S614 Introduction The initial indication of the location of a putative second locus for TSC came from the investigation of a child with typical features of the syndrome who had an unbalanced translocation leading to trisomy llq23- llqter and trisomy 22pter-22qll. The mother had a balanced constitutional translocation 11;22 and did not have any phenotypic features of TSC, nor did other members of the family who were balanced translocation carriers. At the time, 1989, there was a paucity of polymorphic markers in the region of the constitutional breakpoint. Following the publication of Weber and May's description of polymorphic dinucleotide repeats (Weber et al., 1989), Dr Moyra Smith's group at the University of California, Irvine attempted to isolate similar markers in the llq23 region, with which to test TSC families not apparently linked to the TSC1 locus at 9q34. A highly informative (CA)n repeat was identified from a cosmid isolated from the chromosome llql3-qter cosmid library of Glen Evans (Sugiyama et al, 1991). It was known to originate from the Uq23 region, but its precise location was obscured by errors in identification and nomenclature which took some time to clarify. The Irvine group mapped a cosmid, named 8D11, to the llq23 region by FISH. Two further cosmids, 7H8 and 5G1, were isolated from the same library by screening with a fragment of 8D11 adjacent to the T3 promoter- end of the cosmid. All of these cosmids were shown by FISH to hybridise to the llq23 region in the normal chromosome 11 and to be translocated onto chromosome 22 in a constitutional 11;22 translocation, t(ll;22)(22pter-22qll::llq23-llqter). DNA sequence provided by Dr Moyra Smith, supposedly from 8D11, was used by Dr Finbarr Cotter to design pairs of primers from both the T3 and the T7 end of the cosmid insert Ch. 3.5 Localisation of a simple sequence repeat polymorphism, D11S614 144 (personal communication). He FACS flow sorted derivative chromosome 11 and chromosome 22 from the constitutional translocation ll;22(q23;qll.2) cell line GM6229 (NIGMS, 1994). Using the T3-end primers, the derivative 11 was negative and the der. 22 was positive. With the T7-end primers, the converse applied: the der. 11 was positive and the der. 22 negative. This indicated that the cosmid from which he had been given sequence actually spanned the constitutional breakpoint. This had not been apparent from the in-situ hybridisation of the cosmid onto a similar cell line by the Irvine group. This could be explained by the size of the component of the cosmid from the der. 11 region of llq23: if small, there may not have been sufficient signal from the der. 11 to be apparent by FISH. The initial expectation, therefore, was that the 8D11 cosmid contained DNA from the llq23, in the region of the constitutional breakpoint implicated in the unbalanced translocation TSC case. Any polymorphic dinucleotide repeat from that region would be ideal for addressing the issue of TSC linkage. Dr R. Sugiyama in Dr Smith's group identified a (CA)n dinucleotide repeat sequence, D11S614, in a cosmid, said to be 8D11, by screening with an (AC)io oligomer. Primers designed from sequence flanking the repeat amplified products from human DNA ranging in size from 160-180 bp. Deborah Hunt, Dr Frances Benham's research assistant and I used both the primer sequences given in R. Sugiyama's poster at the Paris Chromosome 11 mapping workshop and adjacent primers, designed by me to investigate the mapping of the polymorphism. Ch. 3.5 Localisation of a simple sequence repeat polymorphism, D11S614 145 Results The primers were used to score for the presence of D11S614 in DNA from a series of somatic cell hybrids containing human chromosomes with translocations in distal llq. The results of this experiment are shown in Table 3.5.1, together with the scoring of 14 additional markers from Ilq23.1-llq24. These showed that the sequence amplified by the primers lies distal to the t(ll;22) constitutional and the t(4;ll) leukaemia (MLL1) breakpoints and proximal to the t(ll;22) Ewing's sarcoma breakpoint. This localisation was supported by FISH analysis (Dr Margaret Fox) in which the 8D11 cosmid gave a positive signal on the der. 22 from the cell line GM6229, Figure 3.5.1. Analysis of the D11S614 PCR products in 34 Caucasian and one Afro- Carribean families (segregating for tuberous sclerosis, Povey et al., 1994) indicated ten different alleles which conformed to a codominant mode of inheritance. The observed heterozygosity at this locus in a population of 37 unrelated Caucasians was 85%. The allele frequency was: Al (160 bp) 0.16, A2 0.04, A3 0.04, A4 0.07, A5 0.25, A6 0.17, A7 0.12, A8 0.08, A9 0.03, A10 (180 bp) 0.04. Segregation of the D11S614 polymorphism with four other llq23 markers was analysed in the 35 families. DRD2 contains a CA repeat and was scored using a PCR-based assay (Hauge et al., 1991). Probes for D11S144, D11S29 and PBGD which detect RFLPs were assayed on Southern hybridisations. Two point lod scores were calculated using the LIPED programme (Ott, 1976), and are shown in Table 3.5.2 below. Ch. 3.5 Localisation of a simple sequence repeat polymorphism, D11S614 146 In the 26 of the 32 TSC families (above) in which D11S614 was informative, linkage was excluded to this marker (Table 3.5.3, below). M arker LOD score (Z) at recombination fraction: D11S614 vs: 0 0.01 0.05 0.1 0.2 0.3 0.4 DRD2 males -inf. 7.2 9.4 9.3 7.2 4.3 1.6 females -inf. -8.9 0.35 3.2 4.2 3.1 1.4 D11S144 m ales 5.3 5.2 4.7 4.0 2.7 1.4 0.4 females 5.3 5.2 4.8 4.2 2.9 1.6 0.5 D11S29 m ales 2.2 2.1 1.9 1.6 1.1 0.6 0.2 females 2.8 2.7 2.4 2.1 1.4 0.8 0.3 PBGD m ales 5.8 5.7 5.2 4.5 3.2 1.8 0.6 females 8.3 8.2 7.4 6.5 4.7 2.8 1.1 Table 3.5.2: Pairwise LOD scores for D11S614 v.s. four llq23 markers, in 35 families. Marker LOD score (Z) at recombination fraction: Zero 0.01 0.05 0.1 0.2 0.3 0.4 D11S614 -infinity -40.67 -18.68 -10.00 -3.00 -0.47 0.18 Table 3.5.3: LOD scores for TSC v.s. D11S614, in 26 families. I performed many of the marker analyses in the somatic cell hybrids. The D11S614 amplifications and the family studies were performed by Deborah Hunt. Ch. 3.5 Localisation of a simple sequence repeat polymorphism, D11S614 147 Discussion The position of D11S614 shown by these experiments was not the anticipated result. If the sequence from which these primers had been selected was from a cosmid spanning the constitutional breakpoint, the expected result would be either that given by the D11S144 primers (for example) or by the APOC3 primers, depending upon which side of the breakpoint the sequence was from. It appears that two cosmids must have been isolated from the Evans library. One, 8D11 which contains T3- and T7-end sequence from which Dr Finbarr Cotter designed primers to demonstrate that the cosmid spans the constitutional breakpoint. Another, not 8D11, contains the dinucleotide repeat and maps to the interval between the leukaemia and Ewing's breakpoints. This latter cosmid has since come to be known as 6D11, and has been shown by mapping on the Richard irradiation hybrid panel to lie distal and very close to the MLL1 leukaemia gene (Van Heyningen et al., 1995). It is unclear which cosmid was originally shown by FISH in Dr Moyra Smith's laboratory to lie distal to the constitutional breakpoint. The genetic data (Table 3.5.2) indicate linkage of D11S614 to three of the four markers D11S144, D11S29, and PBGD, are consistent with the physical mapping and concur with the linkage data available at the time (Kramer et al., 1992). The relative order of the five markers is shown in Table 3.5.1: DRD2 is most centromeric and PBGD most telomeric (Van Heyningen et al., 1995). PBGD has been renamed HMBS. The PBGD nomenclature has been retained in this section, since these results were published using the older gene symbol (Benham et al., 1993). Assuming homogeneity, linkage was excluded between TSC and D11S614 in the 26 families in which the marker was informative. However, in eleven of these families lod scores with chromosome 11 markers (including D11S614) were at least as high as with markers from chromosome 9 (five families) or 16 (three families) (Povey et al., 1994). Three families had very small positive lod scores for all three regions. Ch. 3.5 Localisation of a simple sequence repeat polymorphism, D11S614 148 Prof. John Edwards' linkage and heterogeneity program ZZZ was used to analyse the data (Edwards, 1994). This tests linkage to a single marker at each location and considers the hypothesis that TSC may be caused by any one of three loci . At a recombination fraction of 0.05 the highest likelihood is obtained by 50% of families on chromosome 9, by 44% on chromosome 16 and by 6% on chromosome 11. From these data there is no definite support for a locus on chromosome 11 but the possibility of a locus mutated in a small minority of families cannot be formally excluded. Ch. 3.5 Localisation of a simple sequence repeat polymorphism, D11S614 149 Hybrid J1C14 A3EW3B A3RS12B P3.27A M11X A3RS17B MCH110.Ic4 Translocation t(ll;22) t(4;ll) t(ll;22) t(ll;X) t(4;ll) t(X;ll) TD rH i \— i i— —l —I \— l \— CM P H c—IrH H rH rH CM P \—I rH rH rH P CM -P U U CM 4-> QJ U o* a co 0) U 1 1 1 CM + + + + + + + + + + P P rH rH g P _ . . + + + + + + + + + + + + + + LD ^ ^ T—I H rH rH co rH CO w •H rH rH rH p P P p Id R CQ (d QJ O O § § O M ^ CMCO Q H H t f < O CO CO U + + + + + + + + + + + + + + P O P P T (Ti P •H •H * —V r~H iS H r H r '— P P id O R QJ id id QJ + + + + + + + + rH rH + + + + + + + + if) rH P CO + + + + + + + + + + + + + a pq 0 P H r P P H r i f f X i—1 [X I l I l a CO o CM < \— W p CO — 1 1 H r E CO h x H r H r P — •». -P 1 CO \—1 LD p CO G — 1 ■H ■H •rl ■H H H rH ■H •d ■rl 4-> ■rl P 4J % A rH rH 2 ^ ,2 CQ H Id ^ 40T3 •d f G if) P H &• (0 W & W P U1 G QJ Q o O id o g G E P id G 0) (0 u id G a) 0 G G 10 u id U ID U G id G U 0* 6 id id O 0) • • h . O W p qj Fig. 3.5.1: Fluorescent in situ hybridisation of metaphase chromosomes from cell line GM6229A using biotinylated 8D11 cosmid DNA as probe The cell line contains a balanced constitutional translocation t(ll;22). The full karyotype is 46, XX, t(ll;22) (llpter>llq23::22qll.2>22qter;22pter>22qll.2::llq23>llqter) (NIGMS, 1994). Two signals may be seen on the normal chromosome 11; a third signal is visible on the derivative chromosome 22. Ch. 3.5 Localisation of a simple sequence repeat polymorphism, D11S614 151 C h a p t e r 3.6: Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 Introduction Cofilin is a widely-distributed, intracellular actin-modulating protein which binds and depolymerises filamentous, F-actin and inhibits the polymerisation of monomeric, G-actin at a 1:1 molar ratio and in a pH- dependent manner (Nishida et ah, 1984; Muneyuki et ah, 1985; Yonezawa et ah, 1985; Nishida et ah, 1987; Abe et al, 1989). These functions are inhibited by phosphoinositides and phosphoinositol through an interaction with one of the actin-binding motifs of cofilin (Yonezawa et ah, 1990; Yonezawa et ah, 1991). In fibroblasts, heat shock or exposure to dimethyl sulfoxide induces binding of cofilin to actin and translocation of the cofilin-actin complex from the cytoplasm to the nucleus, (Ohta et ah, 1989). Cofilin has been found to be involved in pinocytosis in the thyroid cell, (Saito et ah, 1994) and in the platelet response to aggregating agents such as collagen and thrombin, (Davidson et ah, 1994). It is also an essential component of the accessory pathway of T-cell activation (Samstag et ah, 1994). Two cofilin isoforms have been identified in mouse, M-type ("muscle", cfl2) and NM-type ("non-muscle", cfll) (Ono et ah, 1994). In contrast, chicken appears to have only a single cofilin, the primary structure of which is most similar to the mouse M-type. The M-type cofilin is expressed in heart, skeletal muscle and testis, whereas the NM-type is found in a wide variety of tissues studied (including heart and testis). NM-type cofilin expression is minimal in mature mammalian skeletal muscle. Increased expression has been found following denervation and in dystrophic muscle in both degenerating and regenerating myofibrils, (Hayakawa et ah, 1993) Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 152 The cDNA sequence of a cofilin, cloned from a porcine brain cDNA library, was first reported in 1988 (Matsuzaki et al., 1988). It encodes a 166 amino acid protein which has a molecular mass of about 18.5 kDa. Cofilins from chicken (embryonic skeletal), mouse and human (placental) have also been characterised (Abe et ah, 1990; Moriyama et ah, 1990; Ogawa et ah, 1990). They are highly homologous and have over 80% amino acid identity. The mouse M-type cofilin (Ono et al, 1994) has greater homology with chicken cofilin (96% aminoacid identity) than with the previously published mouse cofilin amino acid sequence (81% identity). This latter clone was obtained from a mouse brain library (Moriyama et ah, 1990), and has been designated a NM-type cofilin by Ono et ah. A homologue of cofilin has also been identified in Saccharomyces cerevisiae with 35-41% identity to mammalian cofilin (Iida et ah, 1993; Moon et ah, 1993). The growing recognition of the roles played by cofilin as an intracellular messenger or effector in diverse cell types in mammals begs the question whether derangements of the cofilin-actin signalling system may be involved in disease processes. An increasingly common means of linking disease to causative mutation has been through the testing of appropriate genes mapped to the region to which a clinical condition has been located. None of the cofilin isoforms had been mapped, and because of the significance of the novel roles of cofilin (especially in the accessory pathway of T-cell activation) we decided to attempt to localise them. Cloning of a NM-type Cofilin, CFL1, from an HL60 library: The human cofilin cDNA from which sequence was derived for this mapping project was isolated serendipitously. An attempt was made in 1989 by Peter Rowe to clone the smaller (the alpha) subunit of cytochrome b-245, the terminal component of the neutrophil microbicidal oxidase. This protein had been purified and the aminoacid sequence of the N- terminal 39 aminoacids was available, from which oligonucleotide sequence was derived by back-translation. A region of least nucleic acid redundancy was identified and several 17-mer oligonucleotides were synthesised (as separate molecules). These were used individually to Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 153 probe Northern blots of RNA made from the human promyelocytic cell line, HL60, using stringent hybridisation and washing conditions. One of these oligomers hybridised to a mRNA of 1.2 Kb, similar to the expected size of the alpha subunit mRNA. This oligo was then used to screen a HL60 cDNA library. Approximately 20,000 plaques were screened and two distinct, positively hybridising clones were identified. The two clones were found to cross hybridise. DNA sequencing demonstrated that the clones were identical, except for some alterations of vector sequence in one. CFL1 gene structure: The DNA sequence of the DNA clones was 1053 bases in length, and the longest open reading frame was 166 amino acids. Computer searches of EMBL and GENBANK DNA sequence databases showed near-perfect aminoacid identity with porcine brain cofilin cDNA. Bases near the putative initiator ATG methionine at nucleotide position 52 correspond to the consensus sequence for initiation of transcription: a purine (most often A) three bases 5' to the AUG initiator codon, and a G residue immediately 3' to the AUG, (Kozak, 1987). The porcine initiator methionine is in the same relative position. A polyadenylation signal TATAAA lies 110 bases 5' to the start of the poly-A tail (Figure 3.6.1). The sequence of these clones display close homology to pig cofilin and mouse NM-type cofilin (99.4 and 98.8% aminoacid identity). The sole aminoacid discrepancy compared with the porcine cofilin sequence is a cystine (TGT, pig) to serine (TCT, human) substitution at aminoacid position 108. This is not conservative and raises the question of a cloning- dependent sequence change in either cDNA. The rat brain cofilin sequence has a serine at this position (Shirasawa et al., 1991), and is identical to the pig and human sequence (apart from a threonine for alanine substitution at position 69, in common with the other rodent cofilin sequences). The mouse muscle-type cofilin and the chicken cofilin also have a serine at position 108. It is also identical to the published DNA Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 154 sequence of a partial human placental clone, (Ogawa et al., 1990). The 3' sequence is also identical or very similar to 19 cDNA expressed sequence tags (v.i.). From these comparisons it seems likely that the sequence of the human clone is correct. cDNA Mapping by Southern hybridisation: Attempts were made to map this human cofilin gene in 1990 by Southern hybridisation of regions of the cofilin cDNA to digested, electrophoresed DNA from various somatic cell hybrids (Dr Colin Casimir, personal communication). Multiple bands of hybridisation were detected in both hybrids and rodent control DNA using the complete cDNA insert as probe. This indicated the existence of a family of cofilin genes in both the rodent and human genomes. 5' and 3' probes were generated by restriction endonuclease digestion of the cosmid, but each gave rise to multiple bands. The reason for this failure is apparent when the sequence of the two probes is compared with published cofilin sequences available on the sequence databases at the time. The 5' probe not only included the 51 nucleotides of the 5' untranslated sequence, but also first 190 bases of the coding sequence encoding the first 63 aminoacids. In this region there is 100% aminoacid homology between mouse (Genbank MUSCOF) and human NM-type cofilins. The nucleic acid identity may be illustrated graphically by comparing the two sequences and plotting a dot wherever there is nucleic acid identity between the two (GCG "Dotplot", or Staden "Quickscan"). Points are suppressed in regions in which there is no greater homology than expected by chance (Figure 3.6.2). The 5' untranslated regions are less homologous but the human sequence of 51 nucleotides (89 bases in the sequence HUMCOF in Genbank) is too short to construct a workable amplification and there are regions of significant homology between the human (H) and mouse (M) sequences: M l 11 21 31 41 GCCGGGAGAG GCCGCGTTCA GTCGGATCCC GGCAGCAGCT GCGGCGGCTC ***** ***** **** ****** *** ** *** *** ------AAGTTCA GTCGGGTCCC GGCAGCGGCT GCAGCG-CTC H I 11 21 31 41 Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 155 M 51 61 71 81 91 AGGTCTTCAG TTGCTCTTTC — TG--TCT CCCTTTCGTT TCCGGAAAC ****** * ***** * ** *** ** ******* ********* TCGTCTTCTG CGGCTCT--C GGTGCCCTCT CCTTTTCGTT TCCGGAAAC H 51 61 71 81 91 Conservation (indicated by *): 65% The multiple bands of hybridisation using the 3' probe were less expected. The probe used was a 409-base pair sequence comprising the terminal 80% of the 3'UTS: no coding sequence was included (in contrast to the 5' probe). However, when this probe sequence is compared with the mouse cofilin, significant homology is apparent (Figure 3.6.3). The 3'UT regions of all the mammalian NM-type cofilins are highly homologous (Figures 3.6.3-5, 3.6.7). This finding was suprising and questions the presumption that there is little selective pressure to conserve 3'UTS. There is similar conservation of 3' sequences in the M-type cofilins (Figure 3.6.9), but these 3' sequences differ completely from the NM-type 3'UTS (Figures 3.6.6, 3.6.8,3.6.10,3.6.11). Homologies of this degree preclude mapping by Southern hybridisation of somatic cell hybrid DNA, even with the stringent washing conditions used. However, such is the specificity of the polymerase chain reaction (PCR) technique, that with careful design of primer sequences selective amplification of only one of two or more closely related templates may be obtained. The design of suitable primers is described in detail below. Strategies to ensure specificity of PCR amplification: Four techniques were used in this study to minimise non-specific amplification of rodent NM-type cofilins and other related genes. (1) Amplification of the 3' UTS. (2) Selection of primer sequences to maximise discrepancy between the human target and other mammalian sequences. Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 156 (3) Choice of length and GC nucleotide content of the primers to obtain an melting temperature between 60 and 70 °C, and to (4) Ensure that the 3'-most base or bases of the oligomer form "strong" (high negative Delta G) complementary pairs (e.g. G or C in ultimate position). The Gibbs free energy of the annealing of G and C is greater (more highly negative) than that of A to T, principally due to there being three hydrogen bonds between the former, and two between the latter. CFL1 primer design: Selection of 3'UTS is often sufficient to permit specific amplification of a human target in somatic cell hybrids, due to the divergence of human and rodent sequences in most genes in which 3'UTS have been compared. Choice of primers at random within the 3'UTS of cofilin would probably not have been successful, because of the homology between the human and rodent sequences. In order to facilitate the selection of primers, the human, mouse and pig cofilin 3'UT sequences were aligned and the regions of greatest discrepancy noted. With sequences as similar as the cofilin 3'UTS this could be done "by hand" (using GCG "Lineup"), however, sequence analysis computer programs exist which facilitate this, several of which were used: GCG (GCG, 1994), Staden (Gleeson et al, 1991) and Clustal, (Higgins et al., 1992)). An alignment of the 3' UTS of the human, mouse, pig and rat NM-type cofilins is shown (Figures 3.6.12a, b). The rat sequence was not available when the primers were chosen but is included here for completeness. It is very similar to the mouse sequence and confirms the selection made by comparison of the other three sequences. A more graphical display (such as a GCG "Dotplot" or Staden "Proportional Algorithm" comparison, v.s.) may provide an initial indication of regions of discrepancy (gaps in any line of identity) where primers may best be placed (Figure 3.6.3). Inclusion in the alignment of sequences from mammals other than human and rodent, where available, serves two purposes. It draws attention to regions of the 3'UTS which are highly conserved, and so to be Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 157 avoided when choosing sites for primers (for example, bases 338-399). It also highlights significant discrepancies between the human sequence and all other mammalian sequences, which raise the possibility of a sequencing artefact. The adenine at base 389 is absent from the mouse, rat and pig sequences in a region of homology. It is also not present in a sequence of another clone from a human colon cDNA library submitted to the sequence databases after the promyelocyte clones had been sequenced (Genbank HUGMS04080). I subsequently reviewed the sequencing autoradiographs and confirmed that the additional adenine was a sequencing error. Similar comparisons indicated that the omission of the thymidine residue at base 494 was also due to an error in the sequence. In addition, it is helpful to have another non-primate mammal in the alignment to indicate a possible sequence for the hamster. There are many useful somatic cell hybrids which have been generated by fusion of hamster and human parent cell lines. Unfortunately, very few hamster sequences have been submitted to the sequence databases. Mouse and hamster may have diverged from the rodent phylogenetic tree long before rat and mouse, such that the hamster sequence may be significantly different from either the mouse or rat sequences. A suitable site of the 5' ("forward") primer is indicated by the five base-pair discrepancy between the mouse and human sequences at bases 323-327 inclusive, the gap in the centre of Figure 3.6.3. A primer with a 3'-end formed by those five bases not only lacks any homology with the mouse sequence in the critical 3' region of the oligo, but the 3'-most two bases are GG (see (4) above) and there is a G/T discrepancy with the pig sequence at the last base. It also has a high GC content (12/19 bases) and so a high melting temperature (71.3 °C). Selection of the 3' ("reverse") primer is more difficult. The obvious position for the 3' end of this primer (which is the reverse complement of the sequence shown in the alignment) would be bases 412-414 (the next, more 3', gap in Figure 3.6.3). A 20 base primer ending with the complement of these bases, together with the 5' primer above, would Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 158 amplify a product of approximately 120 bases. This is slightly shorter than the length which I find optimal for efficient amplification. The reverse complement of bases 434-451 would have three mismatches with the first five bases of the mouse sequence, but there would be a risk of mispriming on the mouse sequence, bases 432-435. The penultimate 3' gap in Figure 3.6.3 is in the region 444-473 (human TTCCT to CGGCT). A primer which is the reverse complement of those 29 bases would have 11 bases mismatched with the equivalent mouse sequence, including three mismatches within the first five bases . However, its melting temperature would be 82.4 °C, much higher than that of the 5' "forward" primer. A shorter primer, the reverse complement of 461-480 would have a more similar melting temperature (66.7 °C) and 7/20 mismatches, but there is a likelihood of primer-dimer formation between the 3'-end of this primer (the reverse complement of bases 461-465) and the 5' primer (bases 323- 327). This is avoided by the primer which was chosen for this project, bases 468-489. The sequence of the 16 central to 5' bases of the primer is identical to the mouse (and rat and pig) sequence, but there are 4 mismatches between human and mouse sequences in the remaining 3' 6 bases. This primer has an melting temperature of 70.4 °C, very similar to the 5' primer. The third strategy to promote specific amplification of the target sequence is to maximise the melting temperature of the primers, aiming for an annealing temperature between 60 and 70 °C. The annealing temperature is 2-3 °C below the melting temperature (when the latter is calculated with the base stacking term predominant). Each cycle of the polymerase chain reaction is a sequence of annealing (of the primers to the target or the amplified product), extension by the DNA polymerase from the 3' ends of each annealed primer, and denaturation of the resulting product from the target. The reaction is commonly heated to 95-97 °C for between 20" and one minute to denature (or separate) the target and primer DNA in the first cycle and also to denature the annealed product from template in subsequent cycles. This is followed by cooling to the annealing temperature determined largely by the length and GC content of the Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 159 primers. Extension of the annealed primers will occur as soon as the DNA polymerase attaches to the 3'-end of the primer but the rate of extension increases as the temperature of the reaction is increased. PCRs using conventional Thermus species polymerase incubate at 70 or 72 °C for 20" to 4 minutes to enable extension to take place, this temperature being a compromise between the rate of extension and the degradation of the enzyme that occurs at higher temperatures. Early PCR protocols suggested aiming for annealing temperatures around 50 °C. These temperatures are usually suitable for uncomplicated reactions (in the absence of homologous sequences), e.g. amplification from cosmid library pools where there is a single target. However, low annealing temperatures permit primer annealing to similar target sequences even if there is some mismatch between primer and false target. This will not usually be a problem unless both primers anneal to the same false target in the appropriate orientation to amplify a product within the time permitted by the extension interval. This is more likely to occur in amplifications of complex mixtures of DNA, such as in somatic cell hybrids, where there may be a rodent target of a similar sequence to the human. Mis-annealing may be avoided by ensuring that the annealing of primer to target is as stringent as possible, which requires the use of longer primers with a high GC-content. The Gibbs free energy released when a primer anneals to a secondary or non-specific target is smaller (less negative) than the Delta G of the perfect match between primer and the desired target. A high annealing temperature renders false priming less likely by "burning off" mismatched primers form the target DNA. If the target sequence is sufficiently GC-rich, primers of reasonable length (between 20 and 30 nucleotides) may be selected to melt at around 74 °C, and so anneal at 72 °C: the same as the extension temperature. This results in a two-step per cycle reaction as opposed to the conventional three-step per cycle PCR. The two step cycle has a further advantage of being very rapid, since the cooling of the reaction from the denaturation temperature to the annealing temperature takes an appreciable proportion of the length of Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 160 each three-step cycle: cooling from 95 °C to 50 °C may take a further 20" compared with 95 °C to 72 °C (depending upon the instrument). CFL2 primer design: The human M-type cofilin, CFL2, could be mapped using the strategy described above. As with the NM-type 3'UTS, the M-type 3'UTS shows sifprising inter-species sequence conservation (Figure 3.6.9). There is no similarity between the 3' non-coding sequences of the two different isoforms, however (Figures 3.6.6, 3.6.8, 3.6.10-11). The differences between the two types are not confined to the 3'UTS: the coding sequence of the two types is homologous but each has preferred aminoacids at certain positions which are conserved between species, for example, at 12 sites in the C-terminal 40 aminoacids (Figure 3.6.17). The characteristic aminoacid sequence of the M-type cofilin in this part of the coding region and the nucleotide sequence of the 3’UTS permits identification of expressed sequence tags submitted to the sequence databases which are likely to be derived from human M-type cofilin cDNA clones. The deduced aminoacid sequences of four of these, T35436, HSB35H081, T31361 and HSDHEB062 are shown in Figure 3.6.17. T35436 is entirely coding sequence; there appears to be a sequencing error in T35436 at nucleotide 290, and the frame of the last four aminoacids is altered. Similarly, the last five aminoacids of HSB35H081 are out of frame because of a possible error at base 177. T31361, HSDHEB062 and HSB35F1081 span the end of the coding sequence. The putative 3'UTS of these differ at only one nucleotide and are very similar to both the mouse M-type cofilin and the chicken cofilin (only one type has been identified in this species, Ono et al., 1994). The 3'UTS of the M-type cofilins from chicken and mouse are shown in Figure 3.6.18a-c, together with the aligned sequences of nine human clones, (the three above and T70364, HSDHABA23, HSBC5G082, T31490, T77057, and HSBC6A042). Primers were designed to maximise discrepancy between human and rodent sequence. A forward primer, 27 bases in length (bases 127-153 in the alignment, annealing temperature 62.6 C) and a reverse primer (also 27 bases, 403-373, 62.5 C) should amplify a product of Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 161 271 bases from human DNA. There is an interesting discrepancy between T31361 and HSDHABA23 at the 5-end of the latter. The 5' HSDHABA23 sequence CTCGTGCCGAA does not resemble any of the M-type cofilin sequences in this region. These primers would fail if HSDHABA23 is the correct sequence in this region. However, it is probable that the HSDHABA23 sequence is an artefact since these are the terminal 11 bases at the end of a sequencing gel "read" (and so possibly vector sequence or from a chimeric clone). The sequence chosen for the 5' primer is derived from the middle of clones T31361 and T70364, regions in which the sequence is likely to be secure. T31361 is, moreover, the sequence which spans the 3' end of the coding sequence and which has aminoacid identity to the chicken-, and mouse M-type, cofilins in this region. In summary, I designed oligonucleotide primers from 3' untranslated sequence of human NM-type (CFL1) and M-type (CFL2) cofilins to amplify the human genes in DNA in somatic cell hybrids. Methods CFL1 PCR of the somatic cell hybrid panel: DNA from rodent parent cell lines, somatic cell hybrids, and human controls was amplified by PCR. The human chromosomal content of the somatic cell hybrids has been characterised extensively by two or more of these techniques: karyotyping, isoenzyme analysis, Southern hybridisation, PCR and FISH. The oligonucleotide primer sequences were chosen from 3' untranslated region of the human CFL cDNA (Casimir, unpublished) forward, 5'-ATC CCC ATT CCC CAC CTG G-3'; reverse, 5'- TCC TGC TTC CAT GAG TAG CCG T-3' and amplify a 179 bp product. The sequence of these primers and the intervening sequence is identical to regions of the human sequences Genbank T34758 (Adams et ah, 1995), HUMGS0480 (Okubo et ah, 1993) and the reverse complement of T07190 (Adams et ah, 1993), T56052 and T54088 (Hillier et ah, 1996). It is very similar to T31419 and T34499 (reverse complement), with which there are Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 162 one or two single base discrepancies (unique to each of these clones). Partial sequence is contained in T34556, T31350, T34398, T30107, T30441, T31324, T33933, T30334, T35473 and the reverse complement of M78086, T35473 and T68285 (M78086 (Adams et al, 1992), T68285 (Hillier et al, 1996), all others (Adams et al, 1995)). All these sequences are shown in Figure 3.6.14a-d, aligned with CFL1. The libraries from which these clones were obtained were made from the following tissues: Clone Tissue T34758, T07190 fetal brain M78086 hippocampus HUMGS0480, T31350, T31324 colon T68285 liver T34499 lymph node T56052 fetal spleen T34556, T33933 "white blood cells" T34398, T35473 lung T30441, T30334, T30060 endothelium T30107 uterus T54088 placenta T31419 fetus (tissue unspecified) In the mouse, the NM-type cofilin is expressed in brain, lung, gut and spleen, organs in which the M-type cofilin is expressed poorly, if at all (Ono et al, 1994). This suggests that the clones sequenced from these tissues are more likely to be from the human homologue of NM-type CFL than of M-type CFL. The smooth muscle cell line A10 expresses both NM- and M-type CFL, and NM-type is preponderant, which may explain the cloning of a NM-type CFL from uterus. Amplifications were performed in 25 jil volumes in a Plybaid Thermal Reactor for thirty-five cycles (denaturation 93 °C for 20 s, annealing 60 °C for 20 s, extension 72 °C for 20 s). Each reaction contained 0.25 jag genomic Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 163 DNA, 0.5 pM each oligonucleotide primer, and 0.8 units of Advanced Biotechnologies Thermus species polymerase. The enzyme was added to each reaction after an initial 5 minute denaturation at 97 °C. Final reaction mixes contained, in addition, 210 |iM each dNTP, 1.5 mM MgCl2, 50 mM KC1, 10 mM Tris-HCl pH 9.0, 0.01% gelatin, 0.1% Triton X-100 and 10% glycerol. 5 jil of each reaction product was electrophoresed in 2% agarose gels in TBE buffer and the product visualised by ethidium bromide staining under UV illumination. CFL1 PCR of a chromosome 11 somatic cell hybrid panel: DNA from somatic cell hybrids containing characterised fragments of chromosome 11 (Hunt et al., 1994) was amplified using the CFL1 primers, as described above. CFL2 PCR of a somatic cell hybrid panel: DNA from a similar panel of hybrids to those used in the chromosomal localisation of CFL1 was amplified using primers selected from the 3'UTS of human EST sequences with homology to mouse M-type cofilin and chicken cofilin to maximise discrepancies between the consensus human CFL2 sequence and the mouse sequence (see above). The primer sequences were, forward, 5'-ACA ATG AAT GAA GGA AAT ATC ATT TAT-3', reverse, 5'-AAA TAA TAC TGA AAA AAG TTG ACC ATC-3'. The product of the amplification is 271 bp. Reaction conditions were identical to those used for CFL1 (except for an annealing temperature of 58 °C). Identification of genomic clones from the ICRF RLDB gridded chromosome 11 cosmid library: 32P-oligo-labelled 179 PCR bp product amplified from human DNA template was hybridised to a human chromosome 11-specific genomic cosmid library gridded onto nylon membrane. The filters were kindly Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 164 provided by Dr G. Zehetner, Imperial Cancer Research Fund Reference Library DataBase (RLDB, library number 107, set 12) (Nizetic et ah, 1991). The PCR product was electrophoresed through 1.2 % agarose in TBE buffer. The 179 bp band was excised from the gel and centrifuged through siliconised glass wool. The concentration of DNA in the eluate was estimated by fluorimetry and 50 ng of DNA was random-prime labelled with 32P (AmershamMegaprime kit). The whole labelling reaction was hybridised to the filters at 65 °C overnight in 0.5 M sodium phosphate buffer, pH 7.2/7 % SDS/1 mM EDTA. The filters were washed at 65 °C in 40 mM sodium phosphate/l % SDS for a total of 45 min. and were autoradiographed overnight at room temperature. Fluorescent In Situ Hybridisation: Fluorescent in situ hybridisation to metaphase chromosomes was performed as described previously with the exception that 5- bromodeoxyuridine (BrdU) was incorporated into the chromosomes (Gillett et ah, 1993). The five cosmid clones isolated from the ICRF RLDB were studied (by Dr Margaret Fox). Whole cosmids were biotinylated by nick translation. Labelled cosmid probe was annealed with excess Cot-1 DNA to compete out human repetitive sequences, and hybridised to human metaphase chromosome preparations overnight at 37°C. The signal was detected using avidin conjugated to fluorescein isothiocyanate, and amplified as described previously. The chromosomes were counterstained with propidium iodide and diaminophenolindole (DAPI) to obtain R-banding when visualised under UV illumination. The images were collated by means of confocal laser microscopy (Biorad MRC 600). SEA PCR of cosmid glycerol stocks: The cofilin cosmids isolated were tested for the presence of SEA sequences using published primers (Richard et ah, 1991), and the same amplification conditions as for CFL1. These primers amplify a 130-base pair product. 0.5 jil of a glycerol stock of cosmid-containing E. coli was added to the reaction mix in place of target DNA (v.s.). Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 165 Results Amplification of CFL1 and SEA: A single product of the expected size, 179 bp, was seen when human DNA was amplified using the CFL1 primers. Larger-sized products were also visible in some of the somatic cell hybrids but these non-specific products could easily be differentiated from the specific 179 bp band. This latter product was not observed when mouse, rat or hamster control DNA was amplified (Figure 3.6.15a). The results of the amplifications of the somatic cell hybrid panel are listed in Table 3.6.1. There is complete concordance between retention of chromosome 11 in the hybrids and amplification of the CFL1 sequence, and at least six examples of discordance with each other chromosome. PCR of somatic cell hybrids containing characterised fragments of human chromosome 11 permitted regional localisation to the interval llql2- llql3 .3 (Table 3.6.2, Figure 3.6.15c). Retention of CFL1 in a panel of 47 high dose irradiation hybrids was studied by PCR of hybrid DNA using the primers listed above (Gillett et al, 1993). CFL1 product was obtained in five hybrids: Jo2', Jol5', Jo31, Jo48', Jo50' (data not shown). These same hybrids also gave positive results with an amplification of part of the SEA oncogene (Richard et al., 1991). All the other 42 hybrids were negative for both CFL1 and SEA. Calculation of Lod score, recombination fraction and centiray 40,000 by the method described by Cox (Cox et al., 1990) indicated that CFL1 and SEA are closely linked: LOD 6.92, theta = zero, cRay40/0oo = zero. SEA has been mapped previously to llql3 by in situ hybridisation (Williams et al, 1988). Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 166 CFL1 genomic cosmids: Hybridisation with labelled cofilin PCR product identified five cosmids (Figure 3.6.15d) which were isolated from the ICRF human chromosome 11 gridded cosmid library, number 107 (L4/FS11): ICRFcl07E1288, ICRFcl07E1046, ICRFcl07E0622, ICRFcl07A1277 and ICRFcl07G07102. The 130 bp product was amplified in four of these using the SEA primers; ICRFcl07E1046 was negative (Figure 3.6.15b). CFL1 FISH: In situ hybridisation of biotinylated cosmid probe to human metaphase chromosomes localised the signal to chromosome llql3; there was little or no non-specific background (cl07E1288 shown in Figure 3.6.16). The signal was sufficiently strong to be easily visible in the majority of metaphase spreads. For each cosmid, at least ten spreads were examined and specific signals were seen on each chromatid of the chromosome 11 homologues. Amplification of CFL2: A single product of the expected size, 271 bp, was seen when human DNA was amplified using the CFL2 primers and was not observed when mouse, rat or hamster control DNA was amplified (Figure 3.6.19). The results of the amplifications of the somatic cell hybrid panel are listed in Table 3.6.3. There is complete concordance between retention of chromosome 14 in the hybrids and amplification of the CFL2 sequence, and at least six examples of discordance with each other chromosome, in each case including at least one example in which the chromosome was present and amplification of CFL2 was not seen. Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 167 Discussion Cofilin CFL1 mapping: We have mapped a gene for NM-type Cofilin CFL1, to chromosome llql3 by PCR of two panels of somatic cell hybrids, and by fluorescent in situ hybridisation of genomic cosmid cofilin clones. The human homologue of avian retrovirus proviral tyrosine kinase, SEA, has been mapped to the same band, llql3, by in situ hybridisation (Williams et al, 1988). We have demonstrated that cofilin is closely linked to SEA by demonstration of both cofilin and SEA sequences in each of four of the five cosmids isolated from the ICRF chromosome 11 gridded cosmid library. We have not yet estimated the size of these cosmids. However, the average size of the partial digest used to construct a very similar cosmid library was approximately 55 kb (Nizetic et al, 1991) so it is likely that CFL1 and SEA are separated by not more than this distance. Loci in the llql3 region have been ordered in an irradiation hybrid panel which places SEA between PYGM (centromeric) and D11S913 (telomeric), separated by a centiray distance of about 54 cRay9000 approximately 2.7 Mb (Richard et al, 1991; James et al, 1994). This analysis shows SEA to be most closely linked to D11S1957E (centromeric) and D11S951E, expressed sequence tagged sites lying between PYGM and D11S913. D11S1957E and D11S951E are estimated to be 14.8 cRay apart, equivalent to approximately 750 kb. We anticipate that CFL1 is close to these loci. Sequence comparison with other mammalian cofilins indicates that the cofilin which we have mapped is the widely-expressed non-muscle form. The expression data of Ono et al. also suggests that this cofilin is the NM- type. The DNA sequence in our study was derived from cDNA clones from a promyelocytic cDNA library. In the mouse, NM cofilin is strongly expressed in liver and spleen (in which myeloid and lymphoid series precursors would be found), whereas there is minimal or no expression of the M-type cofilin in these tissues (Ono et al., 1994). Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 168 We have also mapped the human M-type cofilin, CFL2, to chromosome 14, using EST sequence which closely resembles the sequence of the mouse M-type cofilin (Ono et al, 1994). This is the first report of the mapping of cofilin genes in the human, with the exception of an abstract published but not presented at the Annual Meeting of the American Society of Human Genetics, Montreal, 1994. This abstract indicated localisation to chromosome lq25 (Hung et al, 1994). A human genomic lambda phage library was probed with a cDNA fragment of a human cofilin (source not stated) and four clones isolated, DNA from which was hybridised to human metaphase spreads. These discrepant results could be reconciled if there is more than one human NM-type cofilin, or one or more cofilin pseudogenes. There is some evidence for the existence of two NM-types in the mouse: Southern blots of DNA extracted from mouse liver, digested with four different restriction enzymes and probed with the mouse NM-type cofilin show two bands, whereas there is only a single band per track in equivalent blots probed with M-type cofilin (Ono et al, 1994). This band is of different size to either band in the blots hybridised with NM-type cofilin. We have used 3' untranslated sequence from cDNA, very similar to cDNA sequence submitted to the sequence databases (Adams et al, 1993; Okubo et al, 1993; Hillier et al, 1996) to localise the NM-type cofilin to human chromosome llql3 and the M-type to chromosome 14, and it is unlikely that all of these clones are derived from expressed pseudogenes and none from the true locus (with a different chromosomal location). We propose the symbol CFL1 for the NM-type cofilin mapping to llql3, and CFL2 for the M-type cofilin on chromosome 14. Function of Cofilin: Novel roles for cofilin as an intracellular messenger in the thyroid gland, in T cells and in platelets have been elucidated with the recognition that cofilin is homologous to previously-identified phosphoproteins and exists in both phosphorylated and dephosphorylated forms in these cells: pl9 (Samstag et al, 1994), p21 (Saito et al, 1994), pl8 (Davidson et al, 1994). Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 169 Thyroid Stimulating Hormone, TSH induces rapid dephosphorylation of cofilin and may be involved in the reorganisation of cytoskeletal actin filaments in response to TSH, similar to changes seen on exposure to 10% DMSO (Saito et al., 1994). However, unlike DMSO, TSH does not induce the formation of intranuclear cofilin rods. Cofilin is also dephosphorylated in response to triggering of the accessory pathway of T- cell activation, but not following T-cell receptor/CD3 complex stimulation alone, (Samstag et al, 1994). The former stimulus is associated with translocation of the partially dephosphorylated cofilin into the nucleus. It is significant that nuclei in the transformed T-cell lymphoma cell line Jurkat contain substantial amounts of dephosphorylated cofilin, and the authors postulate that cofilin may be involved in preventing apoptosis in T-cells stimulated through the accessory pathway. Cofilin has been recognised to have diverse functions in the regulation of the cytoskeleton (platelet, thyrocyte) and in intracellular signalling (T- lymphocyte). The possible involvement of cofilin in apoptosis and in immortalisation of the Jurkat cell line is of particular interest. The chromosomal location of CFL1 on chromosome llql3 in relation to the MEN1 gene and other markers in the region is considered in Chapter 3.3, "Isolation of hybrids containing human chromosome llql3". The function of the 3f untranslated sequence The conservation of the cofilin M-type and NM-type 3'UTS is intriguing, and suggests that the sequences have some functional role which has been conserved since the divergence of the avian and mammalian phyla. Cross-species conservation of 3'UTS is seen in other gene families, such as the actins (see below), and may have implications for the mapping of ESTs and EST clusters on radiation hybrid panels: this aspect is considered further in Chapter 4, Discussion. Given the functional relationship between actin and cofilin, is there any similarity in nucleotide sequence between 3'UTS of the actin and cofilin gene families? Preliminary comparisons show some faint identity, e.g. Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 170 between mouse M-type cofilin and both rat cytoskeletal gamma actin and mouse cytoskeletal beta actin. It is of interest that the region of weak homology lies entirely within the 3'UTS of the mouse M-type cofilin, MUSCOFILIN, but extends across much of the rat gamma actin coding sequence and all of the RRGAMACT 3'UTS (Figure 3.6.20). This same extension of homology into the actin coding sequence is also apparent in the comparison of MUSCOFILIN and the mouse beta actin, MMACTBR (Figure 3.6.21). This may indicate shared motifs in both coding and 3'UT sequence which have been conserved in the same relative positions in both M-type cofilin and the cytoskeletal actin gene families, either because of functional constraints (actin-cofilin interaction) or due to a common evolutionary origin. What is known about the function of this region? One of the candidate functions proposed for the 3' untranslated sequence after its discovery in 1970 was in mRNA stabilisation. This has been confirmed by experiments which have indicated that the poly (A) tail and 3'UTS are not an obligatory requirement for translation initiation but appear to be modulators of translation efficiency, at least in vitro ; this is reviewed in (Jackson et ah, 1990). The poly(A) tail of all cytoplasmic mRNAs in most if not all eukaryotes is bound with high affinity by a poly(A) binding protein, PABP, which is conserved across species as diverged as human and xenopus. The stabilising effect of the poly(A) tail requires PABP and the tail becomes degraded once it loses its association with PABP. Upstream AU-rich motifs in the 3'UTS can bind PABP leaving the poly(A) tail "naked" and vulnerable to degradation. These motifs, with a consensus sequence UAUUUAU, are found in short-lived mRNAs of proto-oncogenes such as c-fos and c-myc and lymphokines (not in the cofilins). Proteins other than PABP may interact with separate 3'UTS motifs, as occurs in the stabilisation of transferrin mRNA in iron deficiency states. There are a variety of additional but subsidiary mechanisms which influence mRNA stability which are dependent upon sequences elsewhere in the gene (both coding and 5'-proximal). For these to influence the rate of poly(A) Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 171 shortening and 3'UTS degradation implies some kind of "cross-talk" or functional interaction between the upstream elements and the poly(A) tail. Trans-acting factors which positively or negatively regulate polyadenylation and deadenylation have also been identified. This interaction is not merely "one-way". Sequences in the 3' untranslated region, protein-3'UTS interactions or changes in the polyadenylation state may influence upstream events such as translation initiation or reinitiation, in addition to mRNA stability. Experiments in Xenopus oocytes in which 3'UTS have been exchanged between genes have shown that the AU-rich motifs in lymphokine mRNAs v.s ( .) also have an inhibitory effect on translation. Tandemly repeated, pyrimidine- rich sequences in the rabbit erythroid 15-lipoxygenase (LOX) gene are bound by a 48 kDa protein which together repress LOX (and reporter construct) translation (Ostareck-Lederer et al., 1994). The poly(A)-PABP complex may also enhance initiation or reinitiation of translation, as well as stabilising the mRNA. 3'UTS may also mediate differential regulation of very similar genes. Beta and gamma actin coding sequences differ by only four aminoacids (at the amino terminus). Despite this near identity, there is divergent expression of beta and gamma actin in HL60 promyelocytes following induction of differentiation with phorbol acetate or DMSO and amiloride (Chou et al., 1987). Like the cofilin M- and NM-type 3'UTS, the actin beta and gamma 3'UTS have no homology, and may be involved in the separate regulation of expression. Does naturally-occurring variation in the 3'UTR provide any information about the function of the region? Pathological mutations are predominantly due to large-scale gene rearrangements (translocations, insertions, duplications, inversions and deletions), nucleotide substitutions, deletions, insertions or trinucleotide expansions in coding sequence, introns (splice donor or splice acceptor sequences) or 5' promoter or locus control regions. A minority of mutations are known directly to affect RNA processing and translation: base changes at the cap site, the Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 172 polyadenylation and cleavage signal, or at initiation or termination codons. A very few disease states have been reported to be associated with sequence variations confined to the 3'UTS; a few are outlined below. One patient with aspartylglucosaminuria (AGU) has been found to be homozygous for a novel 876 base pair deletion (Ikonen et al., 1992). This spares the coding sequence but removes the 3'UTS. Transcription termination is not impaired, the truncated mRNA is polyadenylated and mRNA concentration is comparable to the non-AGU control. However, virtually no aspartylglucosaminidase (AGA) protein is detectable in cultured fibroblasts from the patient. This mutation suggests that sequences in this 3'UT region are essential for normal initiation or re initiation of translation, at least in the AGA gene. Several single base pair substitutions have been reported in the cleavage- polyadenylation signal sequences (consensus AAT AAA) of the haemoglobin genes, HBA2 and HBB. These lead to a reduction of HbA2 synthesis to 3-5 % of normal and result in a relatively mild form of thalassaemia. In the mutants which have been studied, there is minimal cleavage and polyadenylation at the normal site and alternative sites further 3' from the mutated AATAAA are used. The mutant transcripts are larger than the wild-type and are difficult to detect in vivo either because of instability or impaired transport from nucleus to cytoplasm (reviewed in Cooper et al., 1993). A beta thalassaemia has been reported in a Turkish patient associated with a 13 base pair deletion in the 3'UTS of HBB (Basak et al., 1993). This does not appear to involve the cleavage- polyadenylation signal sequence. One of the most intensively studied mutations in 3'UTS is the unstable CTG trinuclotide repeat expansion in the myotonin protein kinase (DMK) gene which leads to myotonic dystrophy, DM (Brook et ah, 1992). Clinical severity varies with the number of CTG repeats: normal individuals have between 5 and 30 copies of the triplet, mildly affected persons from 50 to 80, and the severely affected over 2000 repeats. The molecular effect of this expansion is unclear: studies using reverse transcription - polymerase Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 173 chain reaction (RTPCR) suggest that the expanded repeat impairs both the synthesis and normal processing of the primary transcript (Carango et al., 1993; Fu et al., 1993). This is confirmed by demonstration of reduced protein expression (using anti-MPK antibodies, Fu et al., 1993). However, the evidence is not unanimous, and others have shown increased levels of DMK mRNA in tissue from congenital DM (Sabourin et al., 1994). The same group have confirmed this finding in vitro using a model reporter constructs composed of the CMV promoter, chloramphenicol acetyl transferase coding sequence and DMK 3'UTS repeats. Constructs with 3'UTS containing repeats in the premutation (50 triplets) and mutation (90 triplets) range show more than ten-fold increase over control CAT activity (Ang et a l, 1994). They propose that increased expression of the mutant mRNA has a dominant negative effect. Both lines of evidence are open to the criticism that the altered expression is an in vitro artefact, either related to the use of RTPCR or of artificial constructs. There is increasing support for the role of a that second gene DM locus-associated homeodomain protein DMAHP, 3' to DMK, the function of which may be compromised by expansion of the CTG repeat (Boucher et al, 1995; Klesert et al, 1997; Thornton et al, 1997). The relevance of these finding to the regulation of translation of other mRNAs could be investigated using in vitro mutagenesis to generate contructs with mutated 3'UTS and in vitro translation of the mRNA, or injection of the contructs into Xenopus or Spisula oocytes, or more laboriously by 3'UTS knockout in mammalian embryonic stem cells. In summary, mutations in the 3'UTS, though rare, do occur and may be associated with both reduction or increased expression of the primary transcript. This provides additional evidence that sequences in the 3' untranslated region are involved in modulation of translation. Ch. 3.6 Use of resources generated: mapping of cofilin genes, CFL1 and CFL2 174 MAS gctctcgtcttctgcggctctcggtgccctctccttttcgtttccggaaacATGGCCTCC 60 4 GVAVSDGVIKVFNDMKVRKS GGTGTGGCTGTCTCTGATGGTGTCATCAAGGTGTTCAACGACATGAAGGTGCGTAAGTCT 120 24 STPEEVKKRKKAVLFCLSED TCAACGCCAGAGGAGGTGAAGAAGCGCAAGAAGGCGGTGCTCTTCTGCCTGAGTGAGGAC 180 44 KKNI ILEEGKEILVGDVGQT AAGAAGAACATCATCCTGGAGGAGGGCAAGGAGATCCTGGTGGGCGATGTGGGCCAGACT 240 64 VDDPYATFVKMLPDKDCRYA GTCGACGATCCCTACGCCACCTTTGTCAAGATGCTGCCAGATAAGGACTGCCGCTATGCC 300 84 LYDATYETKESKKEDLVFIF CTCTATGATGCAACCTATGAGACCAAGGAGAGCAAGAAGGAGGATCTGGTGTTTATCTTC 360 104 WAPESAPLKSKMIYASSKDA TGGGCCCCCGAGTCTGCGCCCCTTAAGAGCAAAATGATTTATGCCAGCTCCAAGGACGCC 420 124 IKKKLTGIKHELQANCYEEV ATCAAGAAGAAGCTGACAGGGATCAAGCATGAATTGCAAGCAAACTGCTACGAGGAGGTC 480 144 KDRCTLAEKLGGSAVI SLEG AAGGACCGCTGCACCCTGGCAGAGAAGCTGGGGGGCAGTGCGGTCATCTCCCTGGAGGGC 540 164 K P L * AAGCCTTTGTGAgccccttctggccccctgcctggagcatctggcagccccacacctgcc 600 cttgggggttgcaggctgcccccttcctgccagaccggaggggctggggggatcccagca 660 gggggaggcaatcccttgcaccccagttgccaaacagaccccccaccccctggattttcc 720 ttctccctccatcccttgacaggttctggccttcccaaactgcttttgtacttttgattc 780 ctcttgggctgaagcagaccaagttccccccaggcaccccagttgtgggggagcctgtat 840 tttttttaacaacatccccattccccacctggtcctcccccttcccatgctgccaacttc 900 taaccgcaatagtgactctgtgcttgtctgtttaagttctgtgtataaatggaatgttgt 960 ggagatgacccctccctgtgccggctggttcctctcccttttcccctggtcacggctact 1020 catggaagcaggaccagaagggaccttcgattaaaaaaaaa 1061 Fig. 3.6.1: Nucleotide and predicted aminoacid sequence of human promyelocyte (non-muscle) cofilin cDNA, CFL1 Nucleotides are numbered on the right; aminoacids (one letter codes) are numbered on the left. Nucleic acid sequence of the 5' and 3' untranslated regions is shown in lower case. Ch. 3.6 Use of resources generated: mapping of CFL1 and CFL2, figures & tables 175 Fig. 3.6.2: CFL1 5' probe, Staden proportional algorithm comparison x axis CFL1 cDNA nucleotides 1-241 (5' Sail fragment). The coding sequence is bases 52-552. y axis Genbank MQSCOF (mouse NM-type cofilin) , nucleotides 1-284, the equivalent fragment of murine clone. The coding sequence is bases 95-595. All the Staden proportional algorithm comparisons are shown in the same format. The scale differs for each axis and for each comparison, and unfortunately could not be displayed. Ch. 3.6 Use of resources generated: mapping of CFL1 and CFL2, figures & tables 176 Fig. 3. .3: CFLl 3' probe, Staden comparison x axis CFL1 cDNA nucleotides 651-1061 (3' BamHI fragment). The coding sequence is bases 52-552. y axis Genbank MQSCOF (mouse MM-type cofilin), nucleotides 695-1134, the equivalent fragment of the murine clone. The coding sequence is bases 95-595. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 177 Fig. 3.6.4: Cofilin NM-type, Staden comparison x axis Genbank MUSCOF (mouse NM-type cofilin) , nucleotides 1-1134. The coding sequence is bases 95-595. y axis Genbank PIGCOFIL (pig NM-type cofilin), nucleotides 1-1390 The coding sequence is bases 364-864. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 178 Fig. 3.6.5: Cofilin NM-type 3'UTS, Staden comparison x axis Genbank MUSCOF (mouse NM-type cofilin), nucleotides 596-1134. The coding sequence is bases 95-595. y axis Genbank PIGCOFIL (pig NM-type cofilin), nucleotides 865-1390 The coding sequence is bases 364-864. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 179 Fig. 3.6.6: Cofilin M-type & NM-type, Staden comparison. x axis Genbank MUSCOFILIN (mouse M-type cofilin) , nucleotides 1-2974. The coding sequence is bases 132-632. y axis Genbank PIGCOFIL (pig NM-type cofilin), nucleotides 1-1390. The coding sequence is bases 364-864. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 180 Fig. 3.6.7: Cofilin NM-type, Staden comparison x axis Genbank MUSCOF (mouse NM-type cofilin) , nucleotides 1-1134. The coding sequence is bases 95-595. y axis Genbank RNCOFIL (rat NM-type cofilin), nucleotides 1-1039. The coding sequence is bases 6-506. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 181 J / / / / * / }____ Fig. 3. i.8: Cofilin M-type & NM-type, Staden comparison x axis Genbank MQSCOFILIN (mouse M-type cofilin), nucleotides 1-2974. The coding sequence is bases 132-632. y axis Genbank RNCOFIL (rat NM-type cofilin), nucleotides 1-1039. The coding sequence is bases 6-506. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 182 Fig. 3.6.9: Cofilin M-type 3'UTS, Staden comparison x axis Genbank CHKCOF (chicken cofilin), nucleotides 543-1369. The coding sequence is bases 42-542. y axis Genbank MQSCOFILIN (mouse M-type cofilin), nucleotides 633-2974. The coding sequence is bases 132-632. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 183 Fig. 3. 6.10: Cofilin NM-type & M-type, Staden comparison x axis Genbank MUSCOF (mouse NM-type cofilin), nucleotides 1-1134. The coding sequence is bases 95-595. y axis Genbank MUSCOFILIN (mouse M-type cofilin), nucleotides 1-2974. The coding sequence is bases 132-632. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 184 Fig. 3.6.11: Cofilin NM-type & M-type 3'UTS, Staden comparison x axis Genbank MQSCOF (mouse NM-type cofilin), nucleotides 596-1134. The coding sequence is bases 95-595. y axis Genbank MQSCOFILIN (mouse M-type cofilin), nucleotides 633-2974. The coding sequence is bases 132-632. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 185 1 50 CFL1_3 1 GCCCCTTCTG GCCCCCTGCC TGGAGCATCT .GGCAGCCCC ACACCTGCCC muscof_3 GCCACCTCCA GCCCCCTGCC TGGAGCATCT TAACAGCCCC AGACCTGCTC pigcofil_3 GCCCCCTCCA GCCCCCTGCC TGGAGCATCT .GGCAGCCCC AGACCTGCCC rncofil_3 GCCACCTCCA GCCCCCTGCC TGGAGCATCT .AGCAGCCCC AGACCTGCTC NM_TYPE GCC.CcTCca GCCCCCTGCC TGGAGCATCT ..gCAGCCCC AgACCTGC.C 51 100 CFL1_3 ' TTGGGGGTTG CAGGCTGCCC CCTTCCTGCC AGACCGGAGG GGCTGGGGGG muscof_3 TTGGGTGTTG CAGGCTGCCC CTTTCCTGCC AGACCGGAGG GGCTGGGGGG pigcofil_3 ACGGGGGTTG CAGGCTGCCC CCTTCCTGCC AGACCGGAGG GGCTGGGGGG rncofil_3 TTGGGTGTTG CAGGCTGCCC TTTTCCTGCC AGACCGGAGG GGCTGGGGGG NM_TYPE ttGGG.GTTG CAGGCTGCCC c .TTCCTGCC AGACCGGAGG GGCTGGGGGG 101 150 CFL1_3 ' ATCCCAGCAG GGGGAGGCAA TCCCTTGCAC CCCAGTTGCC AAACAGACCC muscof_3 ATCCCAGCAG GGGGAGGGCT ATCCCTTCAC CCCAGTTGCC AAACATCCCT pigcofil_3 ATCCCAGCAG GGGGAGGGCA ATCCCTTCAC CCCACTTGCC AAACAGCCCC rncofil_3 GTTCCAGCAG GGGGAGGGTT TTCCCTTCAC CCCAGTTGCC AAACATCCCT NM_TYPE aTcCCAGCAG GGGGAGGgc. .tCCcTtCAC CCCAgTTGCC AAACA.cCC. 151 200 CFL1_3 ' CCC..ACCCC CTGGATTTTC CTTCTCCCTC CAT.CCCTTG ACAGGTTCTG muscof_3 CCC..ACCCC CTGGACCGTC CTTCTCCCTC CAT.CCCT.G AC.GGTTCTG pigcofil_3 CCCCAACCCC CTGGACCTTC CCCCTCCTCC CACCACCCTG AC.GGTTCTG rncofil_3 CCC..ACCCC CTGGACCGTC CTTTTCCCTC CAT..CCCTG AC.GGTTCTG NM_TYPE CCC..ACCCC CTGGAcc.TC CttcTCCctC CAt.cCC.tG AC.GGTTCTG 201 250 CFL1_3 ' GCCTTCCCAA ACTGCTTTTG TACTTTTGAT TCCTCTTGGG CTGAAGCAGA muscof_3 GCCTTCCCAA ACTGCTTTTG ATCTTCTGAT TCCTCTTGGG TTGACGCAGA pigcofil_3 GCCTTCCCAA ACCGCTTTTG ATCTTCTGAT TCCTCTTGGG TTGAAACAGA rncofil_3 GCCTTCCCAA ACTGCTTTTG ATCTTCTGAT TCCTCTTGGG TTGAAGCAGA NM_TYPE GCCTTCCCAA ACtGCTTTTG atCTTcTGAT TCCTCTTGGG tTGAagCAGA 251 300 CFL1_3 ' CCAAGTTCCC CCCAGGCACC CCAGTTGTGG GGGAGCCTGT ATTTTTTTT. muscof_3 CCAAGTCCCG TCCTAGGCAC CCAGTTTGGG GGGAGCCTGT ATTTTTTTTT pigcofil_3 CCAAGTTCCC CCCAGGCACC CCTGTTTGGG GGGGGCCTGT ATTTTTTTT. rncofil_3 CCAAGTCCCG TCCTAGGCAC CCAGTTTGGG GGGAGCCTGT ATTTTTTTTT NM_TYPE CCAAGT.CC. .CC..G...C CCaGTTtgGG GGGaGCCTGT ATTTTTTTT. Fig. 3.6.12a: Cofilin NM-type 3'UTS sequence alignment, GCG "Lineup" Nucleotides 1-300 (numbering from the first base of the 3'UTS) CFL1_3' Human NM-type cofilin cDNA, 3'UTS. muscof_3 Genbank MUSCOF (mouse NM-type cofilin) 3 'UTS. pigcofil_3 Genbank PIGCOFIL (pig NM-type cofilin) 3 'UTS. rncofil_3 Genbank RNCOFIL (rat NM-type cofilin), 3 'UTS. NM_TYPE Consensus sequence, upper case - unanimous agreement; lower case - majority agreement; "." - no consensus. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 186 301 350 CFL1_3 ' ..AACAACAT CCCCATTCCC CACCTGGTCC TCCCCCTTCC CATGCTGCCA muscof_3 TTAACGACAC CCCTACTCCG TA.... TCC CTCCCCATCC CATGCTGCCA pigcofil_3 ..AACGACAC CCCAGTTCCC GACCTGTTCC TTCCTTTTCC CATGCTGCCA rncofil_3 TTAACGACAC CCCTACTCCT GA.... TCT GTCCC.ATCC CATGCTGCCA NM_TYPE ..AACgACAc CCCta.TCCc gA.... TCc ttCCcc.TCC CATGCTGCCA 351 400 CFL1_3 1 ACTTCTAACC GCAATAGTGA CTCTGTGCTT GTCTGTTTAA GTTCTGTGTA muscof_3 ACTTCTAACC ACAATAGTGA CTCTGTGCTT GTCTGTTTA. GTTCTGTGTG pigcofil_3 ACTTCTAACC GCAATAGTGA CTCTGTGCTT GTCTGTTTA. GTTCTGTGTA rncofil_3 ACTTCTAACC ACAATAGTGA CTCTGTGCTT GTCTGTTTA. GTTCTGTGTG NM_TYPE ACTTCTAACC .CAATAGTGA CTCTGTGCTT GTCTGTTTA. GTTCTGTGT. 401 450 CFL1_3' TAAATGGAAT GTTGTGGAGA TGACCCCTCC CTGTGCCGGC TGGTTCCTCT muscof_3 TAAATGAAAT G...TGGAAA TGACCC.TCC CTGCCCCAGC TGGCTGCCCT pigcofil_3 TAAATGGAAT GATGTGGAGA TGACCCCTCC CTGCGCCACC TGGTCCCCCC rncofil_3 TAAATGAACT G...TGGAAA TGACCC.TCC CTGCACCAGC TGGTTGCCCT NM_TYPE TAAATG.AaT G...TGGA.A TGACCC.TCC CTGcgCCagC TGGtt.CcCt 451 500 CFL1_3 ' CCC.TTTTCC CCTGGTCACG GCTACTCATG GAAGCAGGAC CAG.AAGGGA muscof_3 CCCC.TTTCC TTTGATCTTG ACCACTCATG GAAGCAGGAC CAGTAAGGGA pigcofil_3 CCCCCTTTCC CCTGGTCACA GCCACTCATG GAAGCAGGAC CAGTAAGGGA rncofil_3 CCCCTTTCCC TTTGATCTTG GCCACTCATG GAAGCAGGAC CAGTAAGGGA NM_TYPE CCCctTTtCC . . TG.TC. . g gCcACTCATG GAAGCAGGAC CAGtAAGGGA 501 550 CFL1_3' CCTTCGATTA AAAAAAAA muscof_3 CCTTCAATTT AAAACAAAAC AAAACAAAAA AACAATAAAA AGGCTAATTA pigcofil_3 CCTTCAATTA AAAAAGAAAA AGACACAACA AT rncofil_3 CCTTCAATTT AAAAAAAAAA AAAAACACAA TAAAAAGGCT AATTAACAAA NM TYPE CCTTCaATT. AAAAaaAAAa AaAaacAaaA aa.AA A....A...A 551 muscof_3 ACAAC NM TYPE ACAAC Fig. 3.6.12b: Cofilin NM-type 3'UTS sequence alignment. Mammalian cDNAs, GCG "Lineup" Nucleotides 301-555 (numbering from the first base of 3'UTS) CFL1_31 Human NM-type cofilin cDNA, 3'UTS. muscof_3 Genbank MUSCOF (mouse NM-type cofilin) 3 'UTS. pigcofil_3 Genbank PIGCOFIL (pig NM-type cofilin) 3 'UTS. rncofil_3 Genbank RNCOFIL (rat NM-type cofilin), 3 'UTS. NM_TYPE Consensus sequence, upper case - unanimous agreement; lower case - majority agreement; "." - no consensus. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 187 Fig. 3.6.13: Cofilin NM-type 3'UTS, Staden comparison x axis CFLl cDNA nucleotides 553-1061. The coding sequence is bases 52-552. y axis Genbank MUSCOF (mouse NM-type cofilin), nucleotides 596-1134. The coding sequence is bases 95-595. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 188 1 50 CFL1_3 ' GCCCCTTCTG GCCCCCTGCC .TGGA.GCAT CTGGCAGCCC CACACCT. GC tll799r GCCCCTTCTG GCCCCCTGCC .TGGA.GCAT CTGGCAGCC t06272 GCCCCTTNTG GCCCCCTGCC .TGGN.GCAT CTGGCAGCCC CACACCT. GC t56086 GCCCCTTCTG GCCCCCTGCC . TGGGAGCAT CTGGCAGCCC CACACCTTGC t68348 GCCCCTTCTN GCCCCCTGCC .TGGGAGCAT CTNGCAGCCC CACACCT. GC t54447e GCCCCTTCTG GCCCCCTGCC .TGGA.GCAT CTGGCAGCCC CACACCT.GC t68285r NCCCCTTCTG GCCCCCTGCC CTGGA. NCAT CTGGCAGCCC CAAACCT.GC t54501r AGCCC CAAACCTTGC NM TYPE gCCCCTTcTg GCCCCCTGCC .TGGa.gCAT CTgGCAGCCC CAcACCT.GC 51 100 CFL1_3 ' CCTTGGGGGT T .GCAGGCTG CCCCCTTCCT .GCCAGACCG GAGGGGCTGG t06272 CCTTGGGGGT T t56086 CCTTGGGGGT TTGCAGGCTG CCCCCTTCCT TGCCAGACCG GAGGGCTTGG t68348 CCTTGGGGGT TTGCAGGCTG CCCCCTTCCT TGCCAGACCG GAGGGCTTGG t54447e CCTTGGGGGT NTGCAGGCTG CCCCCTTCCT .GCCAGACCG GAGGG.CTGG t68285r CCTTGGGGGT T.GCAGGCTG CCCCCTTCCT .GCCANACCG GAGGGGCTGG t54501r CCTTGGGGGT T .GCAGGCTG CCCCCTTCCT .GCCANACCG GAGGGGCTGG NM TYPE CCTTGGGGGT t .GCAGGCTG CCCCCTTCCT .GCCAgACCG GAGGGgcTGG 101 150 CFL1_3 ' GGGG..ATCC CAGCAGGGGG AGG.CAATCC CTTCACCCCA GTTGCCAAAC t56086 GNGGGGATCC CAGCAG t68348 GGNGG.ATCC CANAAG t54447e GNGGG.ATCC CANAGNGGGG AGGGCAATCC CTTCACCCCA GTTGCCAAAC t68285r GGGG..ANCC CAGCAGGGGG AGGGCAANCC CTTCACCCCA GTTGCCAAAN t54501r GGGG..ATCC CAGCAGGGGG AGGGNAANCC CTTTACCCCA GTTGCCAAAA t34556 CTTCACCCCA GTTGCCAAAC t35473 CACCCCA GTTGCCAAAC t31350 GCCAAAC t34398 CCAAAC t30107 AAAC NM TYPE GggG..AtCC CAgcagGGGG AGGgcAAtCC CTTcACCCCA GTTGCCAAAc 151 200 CFL1_3 ' AGACCCCCCA CCCCCT.GGA TTTT.CCTTC TCCCTCCAT. CCC.TTGAC. t54447e AGACCCCCCA CCCCCTGGGA TTTT.CCTTC TCCCTCCATT CCCTTTNAC. t68285r AGACCCCCCA CCCCCT.GGA TTTT.CCTTC TCCCTCCAT. CCC.TTGAN. t54501r AGANCCCCCA CCCCCT.GGA TTTT.CCTTC TCCCTCCAT. CCC.TTGAN. t34556 AGACCCCCCA CCCCCT.GGA TTTT.CCTTC TCCCTCCAT. CCC.TTGAC. t35473 AGACCCCCCA CCCCCT.GGA TTTT.CCTTC TCCCTCCAT. CCC.TTGAC. t31350 AGACCCCCCA CCCCCT.GGN TTTT.CCTTC TCCCTCCAT. CCC.TTGAC. t34398 AGACCCCCCA CCCCCT.GGA TTTT.CCTTC TCCCTCCAT. CCC.TTGAC. t30107 AGACCCCCCA CCCCCT.GGN TTTT.CCTTC TCCCTCCAT. CCC.TTGAC. t30060r CCCCCAA CCCCCCTGGA TTTTTCCTTC TCCCTNCAT. CCC.TTGAC. t56052r CCT.GGA TTTT.CCTTC TCCCTCCAN. CCC. TTGAC. t30441 TTTT.CCTTC TCCCTCCAT. CCC.TTGAC. t07190r CCCTCCAT. CCCCTTGAC. t54088r TCCAT. CCCCTTGANG NM TYPE AGAcCCCCcA CCCCCt.GGa TTTT.CCTTC TCCCTcCAt. CCC.TTgAc. Fig. 3.6.14a: Cofilin NM-type 3'UTS sequence alignment, Human cDNAs, GCG "Lineup" Nucleotides 1-200 (numbering from the first base of 3'UTS) See foot of Figure 3.6.14b for key. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 189 201 250 CFL1_3' GGTTCT.GG. CCTT.CCC.A AACTGCTTTT GATCTTTTGA TTCCTCTTGG t54447e GGTTCTTGGG .CTTTCCC.A AATTGTTTTT GATTTTTTN...... t68285r GGTTCT.GG. CCTT.CCC.A AACTGCTTTT GATCTTTTGA NTCCTCTTGG t54501r GGTTCT.GG. CCTT.CCC.A AACTGCTTTT GATCTTTTGA TTCCTCTTGG t34556 GGTTCT.GG. CCTT.CCC.A AACTGCTTTT NATCTTTTGA TTCCTCTTGG t35473 GGTTCT.GG. CCTT.CCC.A AACTGCTTTT GATCTTTTGA TTCCTCTTGG t31350 GGTTCT.GG. CCTT.CCC.A AACTGCTTTT NATCTTTTGA TTCCTCTTGG t34398 GGTTCT.GG. CCTT.CCC.A AACTGCTTTT GATCTTTTGA TTCCTCTTGG t30107 GGTTCT.GG. CCTT.CCC.A AACTGCTTTT GATCTTTTGA TTCCTCTTGG t30060r GGTTCT.GG. CCTT.CCC.A AACTGCTTTT GANCTTTTGA NTCCTCTTGG t56052r GGTTCT.GG. CCTT.CCC.A AACTGCTTTT GANCTTTTGA TTCCTCTTGG t30441 GGTTCT.GG. CCTT.CCC.A AACTGCTTTT GATCTTTTGA TTCCTCTTGG t07190r GGTTCT.GG. CCTT.CCC.A AACTGCTTTT GANCTTTTGA TTCCTCTTGG t54088r GGTTCT.GG. CCTT.CCCCA AACTGCTTTT GATCTTTTGA TTCCTCTTGG t31419 CA AACTGCTTTT NATCTTTTNA TTCCTCTTGG t31324 TGCTTTT AATCTTTTNA TTCCTCTTGG humgs04080 GATCTTTTGA TTCCNCTTGG t33933 TCTTTNGA TTCCTCTTGG NM_TYPE GGTTCT.GG. cCTT.CCC.A AAcTGcTTTT gAtcTTTtga ttcctcttgg 251 300 CFL1_3' GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t54447e t68285r G .TGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t54501r GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t34556 GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t35473 GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t31350 GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t34398 GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t30107 GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t30060r GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t56052r GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t30441 GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t07190r GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t54088r GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t31419 GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG t31324 GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG humgs04080 GCTGAAGCAG ACCAAGTNCC CCCCAGGCAC CCCANTTGTG GGGGAGCCTG t33933 GCTGAAGCAG ACCAAGTTCC CCCCAGGCAC CCCAGTTGTG GGGGAGCCTG NM_TYPE gctgaagcag accaagttcc ccccaggcac cccagttgtg ggggagcctG Fig. 3.6.14b: Cofilin NM-type 3'UTS sequence alignment. Human cDNAs, GCG "Lineup" Nucleotides 201-300 (numbering from the first base of 3'UTS) Genbank identification given at the beginning of each row. Suffix r reverse complement of sequence in Genbank; Suffix e sequence edited (usually to remove coding sequence). CFL1_31 human NM-type cDNA, 3'UTS. NM_TYPE consensus sequence, letter case as above (Fig. 3.6.12a). N any nucleotide. Sequencing errors in CFLNM1 identified by sequence comparison with NM- type cofilins from other mammals (Figure 3.6.12) are corrected. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 190 301 350 CFL1_3' TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t68285r TATTTTTTTT AANAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t54501r TATTTTTTTT AANAAAACC t34556 TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t35473 TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t31350 TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t34398 TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t30107 TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t30060r TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t56052r TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t30441 TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t07190r TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t54088r TATTTTTTTT AACAANATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t31419 TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t31324 TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC humgs04080 TATTTTTTTN AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t33933 TATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t34758 ATTTTTTTT AACAACATCC CCA.TTCCCC ACCTGGTCCT CCCCCTTCCC t34499r TTTTTTT AACAACTTCC CCAATTCCCC ACCNGGTCCT CCCCCTTCCC t30334 CTGGTCCT CCCCCTTCCC m78086r TCCT CCCCCTTCCC NM TYPE TATTTTTTTt AAcAAcatCC CCA.TTCCCC ACCtGGTCCT CCCCCTTCCC 351 400 CFL1_3 ' ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT .GTCTGTTTA t68285r ATGCTGCCAA CTTCTAACCG CAAATAGTGA CTCTGTGCTT .GTCTGTTTA t34556 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT .GTNTGTTTA t35473 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT .GT t31350 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTNTGCTT .GTTTGTTTA t34398 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT .GT t30107 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT .GTNTGTTTA t30060r ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT .GTCTGTTTA t56052r ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT .GTCTGTTTA t30441 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT TGTCTGTTTA t07190r ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT .GTCTGTTTA t54088r ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT .GTCTGTTTA t31419 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTNTGCTT .GTTTGTTTA t31324 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTNTGCTT .GTNTGTTTA humgs04080 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTNTGCTT .GTTTGTTTA t33933 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTGTGCTT .GTCTGTTTA t34758 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTNTGCTT .GTTTGTTTA t34499r ATGCTGCCAA CTTCTAACCG CAA.NAGTGA CTCTGTGCTT .GTCTGTTTA t30334 ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CTCTNTGCTT .GTNTGTTTA m78086r ATGCTGCCAA CTTCTAACCG CAA.TAGTGA CNCTGTGCTT .GTCTGTTTA NM TYPE ATGCTGCCAA CTTCTAACCG CAA.tAGTGA CtCTgTGCTT .GTcTGTTTA Fig. 3.6.14c: Cofilin NM-type 3'UTS sequence alignment. Human cDNAs, GCG "Lineup" Nucleotides 301-400 (numbering from the first base of 3'UTS) Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 191 401 450 CFL1_3' GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG t68285r GTTCTGTGTA TAAA.TGGAA TGTTGTGNAA ATGAAAAA t34556 GTTCTGTGTA TAAA.TGGAA T t31350 GTTCTGTGTA TAAA.TGGAA TNTTGTGGAG ATG t30107 GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG t30060r GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATG t56052r GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG t30441 GTTCTGTGTA TAAAATGGAA TGTT t07190r GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG t54088r GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG t31419 GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG t31324 GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGA humgs04080 GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG t33933 GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATG t34758 GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG t34499r GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG t30334 GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG m78086r GTTCTGTGTA TAAA.TGGAA TGTTGTGGAG ATGACCCCTC CCTGTGCCGG NM TYPE GTTCTGTGTA TAAA.TGGAA TgTTGTGgAg ATGAccccTC CCTGTGCCGG 451 500 CFL1_3’ CTGGTTCCTC TCCCTTTTCC CCTGGTCACG G .CTACTCAT GG.AAGCAGG t30107 CTGGTTCCTC T t56052r CTGGTTCCTC TCCCTTTTCC CCTGGTCACG G.CTACTCAT GG.AAGCAGG t07190r CTGGTTCCTC TCCCTTTTCC CCTGGTCACG G .CTACTCAT GG.AAGCAGG t54088r CTGGTTCCTC TCCCTTTTCC CCTGGTCACG G .CTACTCAT GG.AAGCAGG t31419 CTGGTTCCTC TCCCTTTTCC CCTGGTCACG GGCTACTCAT GGGAAGCAGG humgs04080 CTGGTTCCTC TCCCTTTNCC CCTGGTCACG G.CTACTCAT GG.AAGCAGG t34758 CTGGTTCCTC TCCCTTTTCC CCTGGTCACG G.CTACTCAT GG.AAGCAGG t34499r CTGGTTCCTC TCCCTTTTCC CCNGGTCACG G.CTACTCAT GG.AAGCAGG t30334 CTGGTTCCTC TCCCTTTTCC CCTGGTCACG G .CTACTCAT GG.AAGCAGG m78086r CTGGTTCCTC TCCCTTTTCC CCTGGTCACG G .CTACTCAT GG.AAGCAGG m77933re CTC TCCCTTTTCC CCTGGTCACG G .CTACTCAT GG.AAGCAGG NM TYPE CTGGTTCCTC TCCCTTTtCC CCtGGTCACG G.CTACTCAT GG.AAGCAGG 501 550 CFL1_3’ ACCAG.AAGG GACCTTCGAT TAAAAAAAAA t56052r ACCAGTAAGC CCCTTCGATT t07190r ACCAGCTGAG NTCTTTAGTA ACTTGATATG AC t54088r ACCAGTAAGG GACCTTCGAT t31419 ACCAGTAAGG GACCTTCGGA TT humgs04080 ACCAGTAAGG GNCCTTCGAT TAAA t34758 ACCAGTAAGG GACCTTCGAT T t34499r NCCAGTAAGG GNCCTTCGAT T t30334 ACCAGTAAGG GACCTTCGAT TAAAAAAAAA AAAGACAATA AT m78086r ACCAGTAAGG GACCTTCGNT TAAAAAAAAA AAAANACAAT AATAAAAAGG m77933re ACCAGTAAGG GACCTTCGNT TAAAAAAAAA AAAAAAAGAC ACATTAACTT NM_TYPE aCCAGtaagg gaCcTtcgat taaaaAaAaa AaAaaaaaa. A....AA... Fig. 3.6.14d: Cofilin NM-type 3'UTS sequence alignment. Human cDNAs, GCG "Lineup" Nucleotides 401-550 (numbering from the first base of 3 'UTS) Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 192 Fig. 3.6.15a: PCR amplification of CFLl in human, rodent and somatic cell hybrid DNA. Electrophoresis of PCR product in 2% agarose; stained with ethidium bromide; viewed under UV illumination. Origin at top (cathode). Key: (from left):*Kb marker, Genomic Controls:*Human (+),3RAG mouse (- ),HFAZA rat (-),*WG3H hamster (-), Hybrids:\jlCL4 (+),,HORP9.5 (+), *H0RL411B6 (+) , ^MOG2E5 (- ) , ,*TWIN19F9 (-),"EDAG3R (-),'vFST9/5 (-), '^FST9/10 (-) ,f,*CTP34B4 (-),** Kb marker. Fig. 3.6.15b: PCR amplification of CFLl and SEA in cosmid glycerol stocks from the ICRF human chromosome 11 library, number 107 (L4/FS11) Electrophoresis of PCR product as above. Key: (from left),','kb marker, CFLl primers: *»Human genomic control ( + ) , 3ICRFc 107E1288 (+), **ICRFcl07El046 (+),*ICRFcl07E0622 ( + ),fcICRFcl07A1277 (+),*ICRFcl07G07102 (+) and*ICRFcl07B0333 (negative control, -), SEA primers Human genomic control (+),'*ECEFcl07El288 (+),m ICRFc 107E1046 (- ) , »*-ICRFcl07E0622 (+) , '*ICRFcl07Al277 (+) , u*iCRFcl07G07102 ( + ) and '*ICRFcl07B0333 (negative control, -). Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 193 cn *—1 i> *—I5—1 CM p- 0 0 LD 0 0 cn Cn 00 cn cn 0 r- 0 r- CO Cn P- r- r- Cn cn CTi c-~ p- 5—1 5—1 l> p- CO 0 0 CO CD CO CT\ 5—1 00 0 0 0 0 5—1 5—1 5—1 0 0 0 0 0 0 0 0 CTi cn cn e' CTi x—1 O'! Cn CTi CTi cn cn cn 5—1 \—1 5— 1 en 5—1 5—1 C-1 5— 1 5— 1 5— 1 M 1— 1 t—1 1— 1 i— 1 M r~H r~H 0 0 • >~H 0 0 0 0 XP XP M M 0 M M M M Cn Cn 4J 4J r~4 r~H 0 0 0 0 1 1 0 -U 0 0 0 4J 4J 4J 0 CTi Cn 0 0 0 0 0 O 0 -U 0 0 0 0 5H 5-4 d 4-J 4J ■U 4J 0 4J 4J 4J •U -U g 0 4J 4J 4J 0 0 0 0 0 0 0 0 0 0 > 1 0 0 0 £ 0 0 U 0 0 0 d d V CO CO g 0 0 tn 0) Cn 01 0113 tn 1 ) 15 1-1 Cn tJ) 0 U CJ) tn 14-1 d a d 0 d u 0 d > 1 C u u 0 d s s d 1—1 u d d 0 0 0 0 0 0 d ■H 0 O 0 •H 0 H H 0 0d 0 0 Pi 5 s s x 3 pq P 5 PQ 3 ss. X s S £ 3 co < 5 3: rl p h + + + + + 1 1 1 1 1 1 1 1 1 1 1 1 1 u + + X M-l + + 1 + 1 1 + 1 M-l + + + + 1 1 + + 1 + 1 X CO X— 1 ["• 0 0 CM CM + + + 1 + 1 1 1 1 1 1 1 + + 1 1 + + 1 1 + 5—1 5—1 i n CM CM 5—1 5— 1 5—1 + 1 1 1 + 1 1 1 + 1 + + 1 1 1 1 + + 1 1 + CM m 0 CO CM CM 5—1 O O + 1 1 1 + 1 1 1 1 1 1 1 + + 1 1 1 1 + + m 5—1 m CM + CM CM 5—1 c n c n 1 1 1 1 + I 1 1 + 1 1 1 1 1 1 + 1 + + 1 1 *—1 CM rH t—1 'vt1 5—1 -cp 00 CO 5—I + + 1 1 + 1 1 + + 1 + + + + 1 1 + + 1 1 + *—1 m CM l> m r - + II I + I 1 + + 1 1 I 1 1 1 I + I 1 r - CM m 5—1 i n 5~1 + + 1—1 5—1 CD CO 1 1 1 1 + 1 1 + + 1 1 1 1 1 1 1 + D- 1 1 1 x—1 CM 5—1 *—1 5—1 c n 0 ID i n M-l + 1 1 + 1 1 1 + 1 + + + + 1 1 + O- 1 + 1 CM CM CO e' 8 *—1 x—1 + 1 + 1 + 1 1 + + + 1 1 + + 1 1 + + 1 CM en w 5—1 + + X—1 CO t" O on + + 1 1 1 1 1 + + 1 1 1 1 1 1 1 CO m 5—1 in 0S *— 1 + 1 1 + + x— 1 CM 5— 1 u + 1 + 1 1 1 1 + + 1 1 1 + + 1 1 + + 1 m 00 0 0 A + + CM 5— 1 \—1 u 5— + 4- + + + 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X LD 0 0 i —1 1 6 0 0 1 1 1 cn 5—1 + + 1 + 1 + 1 + 1 1 + + 1 1 + + + 1 *—1 CO CM i> 5—1 cn 1 1 1 1 + 1 1 1 1 1 1 1 + M-l + 1 + 1 1 1 + cn 5—1 5—1 0 0 1 1 1 1 + 1 1 + 1 1 + + + + 1 1 + 1 1 1 + 0 0 x— 1 cn i> 0 p- 1 1 1 1 1 1 + 1 1 1 + 1 1 1 D- 1 + 1 r- x— 1 + + + 5— 1 in CD 1 1 1 1 1 1 1 + 1 1 + 1 + + 1 1 + + 1 + + CD O in 0 0 CO 0 LD + l 1 1 in CM cn CD + 1 1 + + + 1 + + 1 1 1 + 5—1 — 1 5—1 1 1 1 + 1 1 1 1 1 ++ 1 + + 1 1 + 5 5— 1 in on + + 1 + + + 1 1 1 + + 1 + + 1 1 + CO m CM cn r- CM CM 1 1 1 1 <>■ 1 1 + 1 1 1 1 1 1 1 + + CM 0 in 5—1 m 0 + 1 1 5—1 CM cn in *—I 1 + 1 + + + M-l I I I I I + + 5-4 0 e 0 + 1 1 + CO 0 CM 0 O W CO r—1 CM CM CQ p P P CM cn § + + U 5—1in ^P u a H 0 c— 1 CM XP PQ in P I U ■H CO 5—l CM 0 PQ Pi cn CO 00 in \—I *—1 5—1 in 1 5 0 O \—I Q in CO EmP 0 O CO CO § & Q Q O H ■g X p Key: ? not tested or equivocal result; f hybrid contains a fragment of the chromosome; C concordant; D discordant. D concordant; C chromosome; the of a fragment contains hybrid f result; Key: equivocal or tested not ? a P u S 00 r - U W p p P P P 0 0 s a P CO Eh u UUP U Q Table panel mapping hybrid cell 3.6.1: a somatic CFLl in of Segregation Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 194 Fig. 3.6.15c: PCR amplification of CFLl in human, rodent and somatic cell hybrid DNA (chromosome 11 mapping panel). Electrophoresis of PCR product as Figure 3.6.15a. Key: (from left) : 'Kb marker, Genomic Controls : lHuman ( + ) ,-*WG3H hamster (-), Hybrids: *»A3EW3B ( + ),sA3RS12B ( + ) ,kMllX ( + ),*PG48 (+),*MAR1 ( + ) , *J1-11 (-) ,,aEJNAC (-) CJ52 (-),'VJ1CL4 ( + ),'JKb marker. Hybrid Chr. Component CFLl Reference B2 llpl3 llqter + Larizza et a l . 1983 1B5 llpter Ilq24-q25 + Katz e t a l . 1983 A3EW3B llpter llq24 + Guerts van Kessel 1985 A3RS12B llpter llq23.3 + Sacchi et a l . 1986 Mll-X llpter llq23.1 + Chiang et a l . 1984 PG48 llpter llq2 1 + Gillett et a l . 1993 MARI llpter llq2 1 + St Clair et a l . 1990 EJNAC llpter llcen - Porteous et a l . 1989 Jl-11 llpter llql2 - Glaser et a l. 1989 CJ52 llql3.3 - llqter - Koeffler et al. 1981 CJ37 llql3.3 - llqter - Mohandas et a l . 1980 PCLE3 llq23.1 - llqter — (Brown, unpublished) Table 3.6.2: Segregation of CFLl in a human chromosome 11 mapping panel. Key: + CFLl per product amplified, - CFLl per product not amplified. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 195 F i g . 3.6.15d: Autoradiograph of genomic cosmid filter ICRF cl07 (set 12) probed with 32P-oligo-labelled CFLl product Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 196 Fig. 3.6.16: Human metaphase chromosomes showing fluorescent in situ hybridisation of biotinylated cofilin cosmid probe localised to chromosome llql3 Clone ICRFcl07El288 shown; Upper: entire spread. Lower: partial spread, enlarged. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 197 60 t35436 R ...KQ..... CHKCOF ...... P ...... K ...... TR..... MUSCOF' N MASGVTVNDEVIKVFNDMKVRKSSTQEEIKKRKKAVLFCLSDDKRQIIVEEAKQILVGDI MUSCOF A.S.G...... P..V...... E. .KN. .L. .G.E.... V CFLl A.S.G...... P. .V...... E. .KN. .L. .G.E.... V PIGCOFIL A.S.G...... P. .V E. .KN. .L. .G.E.... V RNCOFIL A.S.G...... P. .V...... E. .KN. .L. .G.E.... V 120 hsb35h081 t35436 S CHKCOF ...... A ...... MUSCOF 1N GDTVEDPYTSFVKLLPLNDCRYALYDATYETKESKKEDLVFIFWAPESAPLKSKMIYASS MUSCOF .Q. .D. . . .T. . .M. .DK...... N ...... CFLl .Q. .D. . .AT. . .M. .DK...... S ...... PIGCOFIL .Q. .D. . .AT. . .M. .DK...... C ...... RNCOF IL .Q. .D. . . .T. . .M. .DK...... S ...... 166 hsdheb062 ...... N ...... L31361...... N ...... hsb35h081...... N .... t35436 ...... CHKCOF ...... N ...... mmb613...... MUSCOF' N KDAIKKKFTGIKHEWQVNGLDDIKDRSTLGEKLGGSVWSLEGKPL MUSCOF ...... L ..... L.A.CYEEV. . .C. .A.....A.I...... CFLl ...... L ..... L.A.CYEEV. . .C. .A.....A.I...... PIGCOFIL ...... L ..... L.A.CYEEV. . .C. .A.....A.I...... RNCOF IL ...... L ..... L.A.CYEEV. . .C. .A.....A.I...... Fig. 3.6.17: Alignment of predicted aminoacid sequences of M- and NM-type Cofilins. Dots " . " indicate aminoacid identity with the MUSCOFILIN sequence (shown in full). Spaces are used to denote aminoacids where translation was not possible because of nucleotide sequence ambiguity. Aminoacids (one letter codes) are numbered on the right. Genbank sequence identifiers used (except for CFLl). Hus M-type clones t35436, t31361 (Adams et al. 1995) clones hsb35h081, hsdheb062 (Houlgatte et al. 1995) Mus M-type clone mmb613 (Davies et al. 1994) CHKCOF Chicken M-type cofilin (Abe et al. 1990) MUSCOF'N MUSCOFILIN, Mouse M-type cofilin (Ono et al. 1994) MUSCOF Mouse NM-type cofilin (Moriyama et al. 1990) CFLl Human NM-type cofilin (Ogawa et al. 1990; Gillett et al. 1996) PIGCOFIL Pig NM-type cofilin (Matsuzaki et al. 1988) RNCOFIL Rat NM-type cofilin (Shirasawa et al. 1991) Note: there appears to be a sequencing error in T35436 at nucleotide 290, and the frame of the last four aminoacids is altered. Similarly, the last five aminoacids of hsb35h081 are out of frame because of a possible error at base 177. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 198 50 chkcof3 AAAGACAGAC AAGTGCCATC TGGATCT. AA GGAGCTTCCA TTTCT muscof'n_3 AATAATAGCC AAGTGCCATT TG.ATCTTAA GGGGCTTACA CGT.ATCTCT inrab613e AATAATAGCC AAGTGCCATT TG.TACTTAA GGGGCTTACA CGTTATCTCT hsb35h081e AATGACAGTC AAGTGCCATC TGGATCTTAA GGAGCTTCCA TTTCT hsdheb062e AATGACAGTC AAGTGCCATC TGGATCTTAA GGAGCTTCCA TTTCT t31361_3 AATGACAGTC AAGTGCCATC TGGATCTTAA GGAGCTTCCA TTTCT M TYPE AAtgAcAGtC AAGTGCCATC TGgatCTtAA GGaGCTTcCA TtTCT 51 100 chkcof3 GCAGCTCGTT CAATTGGAAT AGTATTAGTC TCCCTTTCT...... CCTTC muscof1n_3 CCAGCTCAGT CCACTGGAAT TGTATTAGGT TITGTTTTTT TTGTTTATT. mmb613e CCAGCTCAGT CCACTGG hsb35h081e TCAGCTCA hsdheb062e CCAGCTCAGT CCATTGGAAT AGTATTAGGT TTTGGTTTTT TG t31361_3 CCAGCTCAGT CCATTGGAAT AGTATTAGGT TTTGGTTTTT TGTTGTATTT t70364 GTATTT M TYPE cCAGCTCagT CcAtTGGAAT aGTATTAGgt Tttg.TTtTt tg.tgtaTTt 101 150 chkcof3 CCTCCTCAAA AATAAGCCCC TTCCCTTCTG CCCCTGAAGG AGATGTCATT muscof1n_3 ..CCCTTTTC ACTGGTCCCG TTCG..TGAA TGAGTGAA.. t31361_3 CCCCCTTTCC ACTGGGCCCT TCCAACACAA TGAATGAAGG AAATATCATT t70364 CCCCCTTTCC ACTGGGCCCT TCCAACACAA TGAATGAAGG AAATATCATT hsdhaba23 CTC GTGCCGAA.. hsbc5g082 CATCATT M TYPE cccCCTttcc AcTgggCCCt T .Caac.caa tga.tGAAgg a .atatcatt 151 200 chkcof3 ..GTTAAGCA GTCTACCAGT GATTGCCATT AGACTGTTTA ATCCTGGTAG muscof'n_3 ..TATAAGAA GCCTGTCAGT .ATTGCCATG AGACTGTTTC ATATGGTTAC t31361_3 TATTTAAGCA GCCTATCAGT GATTGCCATT AGACTGTTGA ATACTGTTAC t70364 TATTTAAGCA GCCTATCAGT GATTGCCATT AGACTGTTGA ATACTGTTAC hsdhaba23 ..TTTAAGCA GCCTATCAGT GATTGCCATT AGACTGTTGA ATACTGTTAC hsbc5g082 TATTTAAGCA GCCTATCAGT GATTGCCATT AGACTGTTGA ATACTGTTAC M TYPE ..ttTAAGcA GcCTatCAGT gATTGCCATt AGACTGTTga ATactGtTAc 201 250 chkcof3 TTTTATGTAG GATCCAAGGA ATGCTTTCAC GTCATACTCT TAGCCAAAAC muscof'n_3 TTTTCTGTAT T .CCCAAGGA ATGCCTTCCT GTCTTATT.T TAGCCAAAAC t31361_3 TTTTATATAG GACCC t70364 TTTTATATAG AACCCAAGGA ATGCCTTCCT GTCATATT.T TAGCCAAAAC hsdhaba23 TTTTATATAG AACCCAAGGA ATGCCTTCCT GTCATATT.T TAGCCAAAAC hsbc5g082 TTTTATATAG AACCCAAGGA ATGCCTTCCT GTCATATT.T NAGCCAAAAC M TYPE TTTTaTaTAg aacCCAAGGA ATGCcTTCct GTCaTAtT.T tAGCCAAAAC 251 300 chkcof3 TGACGC..TG CATGCATTCC TTGC...CAC ACGTACAATG AATGTGATAG muscof'n_3 AAACTGGTTC CATGCCTTCC TTGCAGTGAG CGTTACAATG GATGTGGTTG t70364 AA.CTGGTTA TATGCCTCCC TTGCAGCAAG CACTACAATG TGTGTGATCG hsdhaba23 AA.CTGGTTA TATGCCTCCC TTGCAGCAAG CACTACAATG TATGTGATCG hsbc5g082 AA.CTGGTTA TATGCCTCCC TTGCAGCAAG CACTACAATG TATGTNATCG M TYPE aa.CtggtTa tATGCcTcCC TTGCagcaAg cacTACAATG taTGTgaTcG Fig. 3.6.18a: Cofilin M-type 3'UTS sequence alignment, GCG "Lineup" Nucleotides 1-300 (numbering from the first bp of 3'UTS) For key see foot of Figure 3.6.18c. Ch. 3.6 Use of resources generated: mapping of CFLl and CFL2, figures & tables 199 301 350 chkcof_3 TTAATGTGAA TAGTCTAGCA GACAGCAAAG GGTAAGCTAA TTGAATGCCT muscof1n_3 TCAATGTGAA TAGCTTAGAG TACTACAAAG GGTAAGCTAA CTGAATGCCT t70364 TCAATGTGAA TAGCTTAGAA TACTGCAAAG GATAAGCTAA TTGAATGCCT hsdhaba23 TCAATGTGAA TAGCTTAGAA TACTGCAAAG GATAAGCTAA TTGAATGCCT hsbc5g082 TCAATGTGAA TAGCTTAGAA TACTGCAAAG GATAAGCTAA TTGAATGCCT t31490 ATGTGAA TAGCTTAGAA TACTGCAAAG GATAAGCTAA TTGATTGCCT M TYPE TCAATGTGAA TAGctTAGaa tACtgCAAAG GaTAAGCTAA tTGAaTGCCT 351 400 chkcof_3 TGAAAGTATT GT.CACTGGT GGG....ATG GTAGACTCTA TACAGTATTA muscof1n_3 TGAAAATATT ATCCACTGGT CGGTCATATG GGAGACTTGT TTCAGTATTA t70364 TGAAAGTATT ATCCACTGGT CAG ATG GTCAACTTTT TTCAGTATTA hsdhaba23 TGAAAGTATT ATCCACTGGT CAG ATG GTCAACTTTT TTCAGTATTA hsbc5g082 TGAAAGTATT ATCCACTGGT CAG ATG GTCAACTTTT TTCAGTATTA t31490 TGAAAGTATT ATCCACTGGT CAG....ATG GTCAACTTTT TTNAGTATTA M TYPE TGAAAgTATT aTcCACTGGT caG....ATG GtcaACTttt TtcAGTATTA 401 450 chkcof_3 CTTACAG.TT .GCACTTGAT T .GCAGTTCC ATGAGGCTCT TGTGCATT.C muscof'n_3 TTTATAG.TT .GCACTTGAT T .ACCGTTCT CTGAGGCACT GGAGCCTT.C t70364 TTTATAG.TT GGCACTTGAT TTGCAGTTCT GTGGAGG.CT TGAGCATTTC hsdhaba23 TTTATAG.TT GGCACTTGAT T .GCAGTTCT GTGAGG..CT TGAGCATT.C hsbc5g082 TTTATAGGTT GGCACTTTGA TTGCAGTTCT GTGNAGGCTT TGNGNCATTC t31490 TTTATAG.TT GGCACTTGAT T .GCAGTNCT GTNAGG..CT TGAGCATT.C t77057r AGT GGCNCTGGAT .GCNAGTCCT GNGAGG.CNT GGAGCACTCA hsbc6a042 CT GTGAG..CCT TGAGCAT.CC M TYPE tTTAtAG.tT gGCaCTtgat t .gcaGTtCt gtgagg..cT tGaGcatt.c 451 500 chkcof_3 ATACACCTCA CCTG.CC.TT GACAAGCCTA TTTTTG..TG ACATGGCAGC muscof'n_3 ATACACCTCA CCTG.CC.TT GGCAAGCCTA TTTTTG..TG ACCTGGCAGC t70364 ATACACCTCA CCNGGCCNTT GGNCAAGCCT ATTTTTAGTG GATATGGGCC hsdhaba23 ATACACCTCA CCTG.CC.TT GGAAG hsbc5g082 ATACACCTNA ACCTGNCCTT GGGAAGGCCT TTTTT t31490 ATACACCTCA CCTG.CC.TT GGCAAGCCTA TTTN..AGTG ATATGGCAGC t77057r AAANACCTCA CCTG.CC.TT GGNAAGCCTA NTTT.AAGGG ATAGGGCAGC hsbc6a042 ATACACCTCA CCTG.CC.TT GGCAAGCCTA TTTT..AGTG ATATGGCAGC M_TYPE AtAcACCTcA cCtg.cC.TT GgcaagcCta tTTtt.agtG atatgGcagC 501 550 chkcof_3 AC ACA ACACTATGCA TTTAAA. GCA CTTTTTTGTA ATATGTTTGA muscof'n_3 ACAGATT.TA ACACTATTCG TTAAAA.GCA CTTTTTTTTA ATGCGTTTAA t70364 AGCACG t31490 ACGGATA.TA NCACTATGCA TTAAAA.GCA C.TTTTTGTA ATAAGNTTAA t77057r ACGGATAATA ACNCTATGCA TTAAAAAGCA CTTTTTGGNA ATAAGTTTAA hsbc6a042 ACGGATA.TA ACACTATGCA TNAAAA.GCA C.TTTTTGTA ATAAGTTTAA M__TYPE Acggata.tA aCaCTATgCa TtaAAA.GCA CtTTTTtgtA ATaaGtTTaA Fig. 3.6.18b: Cofilin M-type 3'UTS sequence alignment, GCG "Lineup" Nucleotides 301-550 For key see foot of Figure 3.6.18c. Ch. 3.6 Use of resources generated: mapping of CFL1 and CFL2, figures & tables 200 551 600 chkcof_3 CCTGCCCCTC CCCCCCAAAG GAATGCCAA. TTAAGTGTGT AACTGTGT.. muscof'n_3 ...... T CCCTTATAAA GAATGCCAA. TTAAGTTTTA TTACCTGT.. t31490 TAT...... CCTAAAAG GAATGCCAAT TAAAGTTTTG t77057r ANAN...... CCTAAAAG GAATGCCAAN TAA.GTTTTG TTAACTGTGT hsbc6a042 TAN...... CCTAAAAG GAATGCCAAT TAA.GTTTTG TTAACTGTGT M_TYPE tat...... CctaaAAg GAATGCCAA. TaAaGTtTtg ttaacTGT.. 601 650 chkcof_3 C A. CAATTAT TG.TGGTACC TCAGT..TCA TTCCTGTTAC ATGCATA.TC muscof'n_3 CATCAATTTA TCCTAGTATC TCAGTGTTCA TTCTTCTTGC CTTCATATTT t77057r CATCAACTTA NCCTAGTACC TCAGTGTTCA NTCCTGTTAC CTGCATATCT hsbc6a042 CATCAACTTA TCCTAGTACC TCAGTGNTCA TTCCTGTTAC CTGCATATCT M_TYPE CAtCAA.Tta tccTaGTAcC TCAGTgtTCA tTCcTgTTaC cTgCATAt.t 651 700 chkcof_3 TTATAAATGA AGTAGCTGTT ACG.ATGCCT TTTGTTTT.C CATTGAATGT muscof'n_3 TTTTCAAAGA AACAGCTGTG CTA.ATGTCT TTGGTTTC.C CGATGAGTGT t77057r TCTTAAAAGA AATAGCTGTT ATTAATGCCT TTTTGTTTTC CATTGAGTGT hsbc6a042 TCTTAAAAGA AATAGCTGTT ATTAATGCCT TTTTGTTTTC CATTGAGTGT M_TYPE T .tTaAAaGA AatAGCTGTt att.ATGcCT TTt..TTt.C CatTGAgTGT 701 750 chkcof_3 ACACTACTGA ACAGGAGTAG AAGTTATCTG TTTACCATGT GAGTCTTGAA muscof'n_3 ACACTACTG...... T ATAATTTATG TTTACCATAT GAGTCTTGAA t77057r ACACTACTGA ATAAGTGTAG GAGTTTTATG TTTACCATGT GAGTCCTGCA hsbc6a042 ACACTACTGA ATAAGTGTAG GAGTTTTATG TTTACCATGT GAGTCCTGCA M_TYPE ACACTACTGa ataagtgtag .agtTtTaTG TTTACCATgT GAGTC.TG.A 751 800 chkcof_3 ACACTAAAGT TTTGTAAAAC ATCGGTCATG ATGGCAATTT CTGTATTAAA muscof'n_3 ACACTACAGA TATTTTGAAT ATCAGTCATG ATGGCAATTT CTGTATAAAA t77057r ACACTAAAGA TATTTTGAAT ATCAGTCATG ATGGCAATTT CTGTATAAAA hsbc6a042 ACACTAAAGA TATTTTGAAT ATCAGTCATT TAAGGCTG M_TYPE ACACTAaAGa TaTtTtgAAt ATCaGTCATg atgGcaatTT CTGTATaAAA 801 850 chkcof_3 AAGAGCCTTA AATGGAACAT TGTTTTTGAG ATCAAA...... muscof'n_3 ..GAGCCTTA AATGGAACAT TGTTTT.GAG ATCAAACTCC CTACCCTCAC t77057r ..GAGCCTTA AATGGAACAT TGTTTT.GAG ATCAAACTCC CCACCCTCAC M_TYPE ..GAGCCTTA AATGGAACAT TGTTTT.GAG ATCAAActcc c .accctcac 851 900 chkcof_3 ..AACTGGCC ACGTTGCAAT AAAACTTGTG GCTTATTACA G muscof'n_3 AAAAGTGGCC ACGTTGCAAT AAAAATTGTG GCAGATTACA GAATGTTGCC t77057r AAAAATGGCC NCGTTGCAAT AAAAATTGTG GCATATTACA AAAAAAAAA M TYPE aaAA.TGGCC aCGTTGCAAT AAAAaTTGTG GCatATTACA gAA..... C Fig. 3.6.18c: Cofilin M-type 3'UTS sequence alignment, GCG "Lineup" Nucleotides 551-900 chkcof_3 Genbank CHKCOF (chicken M-type cofilin), 3 'UTS muscof'n_3 Genbank MQSCOFILIN (mouse NM-type cofilin), 3 'UTS mmb613e Genbank mouse cDNA clone with homology to cofilin hsb35h081, hsdheb062, t31361, t70364, hsdhaba23, hsbc5g082, t31490, t77057, hsbc6a042 - Genbank human cDNA clones with CFL homology M_TYPE Consensus sequence (letter case, see Fig. 3.6.12a) Ch. 3.6 Use of resources generated: mapping of CFL1 and CFL2, figures & tables 201 I t 1 s * u * % ^ t» I V «J i s »fc Fig. 3.6.19: PCR amplification of CFL2 in human, rodent and somatic cell hybrid DNA. Electrophoresis of PCR product as Figure 3.6.15a Key: (from left): Genomic Controls:1 Human (+),lRAG mouse (-), ^ FAZA rat (-) ,**WG3H hamster (-) , Hybrids :^GM10479 ( + ) , ^C10B2BU ( + ) , ^ HOFP9 . 5 ( + ),*CON2 (-) , *>5647CL22 (-) , ,0FW4V6 - A (-) , n HORL411B6 (N4) (-), 762-8a (-) ,’*GM07299 (-),*GM10612 (-) ,'*GM10611 (-) ,fwKb marker. Ch. 3.6 Use of resources generated: mapping of CFL1 and CFL2, figures & tables 202 co 00 __. CO CTl x—1 -—- e' ^—. ^ ^ CD -—- .—^ —.^— . ,—* er* ^—, i> [-" e ' i> i> r - I> r - X—1 l> r - 4J 00 00 er* 00 00 00 00 00 00 00 00 CT* 1—1 CT X—1 CT| Ch cr* cr* cn cr* or* cr* x—1 id xH x—1 xH X—1 x—1 X—1X—1 X—1x—1 - r-H U 4J ** . ^ ^— id O CD . • ^ o r~H r~~) Id M M CTl r~H M M •U M co id CO Id (d id id on (d id Id CD (d id •H CD CD -U x—1 tJ 0 - u ■ H -U CD 4J • u 4J 4J 4J -U 4J 4J C CD P CD CD CD CD v CD CD CD 0 CD CD p a; A CO f t a p ft ft f t a> ft no ft S ftftfto ftft a) c s PP P >i a U C a fn I- 1 CC 4J CM .0 p 0 0 .0 0 0 H 0 o 0 o 0 0 CD S X S ft s m 5 5 S 5 5 W S S § X '—" — ■—■— -—■— -— —'-— — "—' — — - -—■ T! +i uo c o (M CM u A fa + + + + + + + + + + + + + + fa U U a) g a) CO « o X X id a ft o + I + + CM I U X I + + + O- + I X 00 -5+ A cm CM ft U CM I I I + I I + + + + I + CM i> o c •H cd + 1 I I + + I I I + + + I + r~ i> ft A ft 4-> id CM o O g 0 CM I I I l + l + l I + I+ + + CM Ml CT* cr* CM x—I I I I I I + I I I + I I I I X—I CM •rl a H a) I I + O- + + + I I + + + I + CO oo i n r- ■Si id + I + + + + I I I + + + I + cr* c n A +i X—I CM *—ICD | | + p.- I O- I CM I + + I I I co oo CO (D £ x—ii n + 1 I O- <4-1 D- + I I + + I + I x—Iuo -H 8 4->id w xH + + + + + + + + + + + + + + X—I a CO CO 0 a 5— I I I + I + + + I I l + l ID- X—I o u + + + I + + +I + I+ +I + CM A X—I ■H X—I O X—I + 1 I l + l I I + + I + I I +1 a 1 D* I I I+ + + I + + +I + I P- CD ■rl CM o CT* I I I I I I D- I + + I I + cr* CO x—I CM -P a i—I CX) I l + l I l + l + + + I + CD CD 00 fa 3 u co [> + + I + I + I I I + + + + I 00 CD aj i+i p CO I I + + I l + l I I + + + + co t"- !> o i—iid a u o o in 1 1 + 1 + + 1 1 1 + + 1 1 1 in i n cr* ■rl > A ■H ■cji 1 + 1 1 1 1 + 1 1 + + + 1 + CD 00 id ft (Ds CO 1 + + 1 + + + 1 1 + + + 1 + CO CT* in a) u P CM 1 1 + + 1 1 1 1 1 1 1 + + + CM in cr* ft O a> Tf X—1 1 1 + 1 1 + 1 1 1 + + 11 + x—1 in cr* 02 CD -P CO id CD CO ■P co1 O + I VO ■Po g - - c CM CT* CO 'd iD-sjiisc; ocnin in ft 'd A + + ■rl inpqpqcMcopcliHi> • cm in w ft ci ■H U i—I cm 'cJi x—I • co tn U W • in H to Ch. 3.6 Use of resources generated: mapping of CFL1 and CFL2, figures & tables 203 V£> CN 00 vo 00 ,—. c n 00 .—v cn CN .—. ^—i c n .—. i> .—. .—. ,—. ,—. X—1 00 .—. [> \ —i 00 X—1 X—1 X—1 m c n ---- CN 00 - 00 cn CTl cn CTl cn ,_. - x —1 __ O cn cn G - cn X—1 cn cn O cn o .__ l> 00 cn x —1 (1) t—i x—1 X—1 x —1 X—1 00 VO r~H 00 c n *—I i—l cn r- id c n V—1 - i—l Id - • --- X—1 cn x—1 (ti 0 M r 4 4J Id » M U 4J id id M M ^ __. ,_, ,_, s CD • id CD W id id fd id ^ ^ ^ • 4J * M iC 13 -u cn (N n I i i i i i i I i I i i A h Pm U U 4 4 I I I I I n oo 5 cn ® O o O a I I I I I rH O vo CN •H CTl I M-l I M-l I cn CN A 00 411 4 4 00 VO 00 j£] LT) Pm u l> + 1 I 1 0 - ["■ 00 VO rH M-l VO + I I M-l I VO t> i > J£J co o 0g LD 1 1 1 4 1 4 4 4 4 1 1 1 1 1 1 1 1 1 LD LD cn ^ LD ■H P ■vl* 1 1 1 4 4 1 1 1 1 1 1 4 1 1 1 1 1 4 VO CO ^ id di CO 1 1 1 1 o- I 1 1 1 1 4 1 1 1 4 1 1 1 ro OV ID ® CN d) p 1 1 1 1 1 1 1 1 lci cn ^ CO d> CN 1 1 4 1 4 4 1 1 1 1 CN d> T—1 1 1 1 1 M-l 1 1 1 1 4 1 1 1 1 1 1 1 1 X—1 in cn h 01 (D D 1 A cn § CO h ) W U VO Ch £ 0 4 1 1 vo CN CN CN CN VO co not tested or equivocal result; f hybrid contains a fragment of the chromosome; C concordant; D discordant. CN *—I CM CM CM PQ w •d CN G pLlWW^crinoo^-icN^H ■ — • •d rG 4 4- I -H id o J m 00000D I OlinOHHH P5 LDCN ■H U oo k u co u HHpqvOCNCN^VOVD-^^CQCNf^ u I ID h I cNOOo>r~-ooooiJiGcDF£]^. . CN A CT; CN O ^ O g1 x—I x—I x—I ^ O x—I x—I rH x—I pd O CM pLi A A A >1 >1 CO VO 0 0 VO N 1 O _ _ _ >i Pm id Ch. 3.6 Use of resources generated: mapping of CFL1 and CFL2, figures & tables 204 Fig. 3. 6.20: Cofilin M-type & Gamma-Actin, Staden comparison x axis Genbank MUSCOFILIN (mouse M-type cofilin), nucleotides 1-2974. The coding sequence is bases 132-632. y axis Genbank RRGAMACT (rat cytoplasmic-gamma actin) , nucleotides 1-1880. The coding sequence is bases 24-1151. Ch. 3.6 Use of resources generated: mapping of CFL1 and CFL2, figures & tables 205 / J i Fig. 3. 6.21: Cofilin M-type & Beta-Actin, Staden comparison x axis Genbank MUSCOFILIN (mouse M-type cofilin), nucleotides 1-2974. The coding sequence is bases 132-632. y axis Genbank MMACTBR (mouse cytoskeletal beta actin), nucleotides 1-1892. The coding sequence is bases 81-1208. Ch. 3.6 Use of resources generated: mapping of CFL1 and CFL2, figures & tables 206 C h a p t e r 3.7: Use of resources generated: mapping of FUT4 Introduction A human myeloid-associated surface antigen, CD15, was mapped to llql2- qter by expression studies in human-mouse myeloid somatic cell hybrids in 1984 (Geurts van Kessel et ah, 1984). This epitope was later shown to be synthesized by a myeloid-associated alpha-3-fucosyltransferase (Tetteroo et al., 1987) also localised to chromosome 11. Sequence which Dr Philip Johnson (Galton Laboratory, UCL) concluded was likely to be from this fucosyltransferase was published in 1990 (Goelz et ah, 1990). I designed oligonucleotide primers from the 3' untranslated sequence in order to refine the localisation using both somatic cell hybrids containing fragments of chromosome 11 and the irradiation-fusion hybrid panel. The fucosyltransferases are enzymes which catalyse the transfer of fucose from a nucleoside diphosphate fucose to an acceptor molecule, frequently another carbohydrate, a glycoprotein or a glycolipid molecule. Seven human alpha-L-fucosyltransferases have been described, named FUT1 to FUT7. FUT1 (Bombay) and FUT2 (Secretor) encode alpha(l,2)fucosyltransferases. The FUT1 gene from which the H- transferase is transcribed maps to chromosome 19ql3.3. FUT2 encodes the related Se-transferase. It has also been localised to 19ql3.3, only 35 kb from FUT1 and has been cloned. FUT3, 4, 5, 6 and 7 are alpha(l,3)fucosyltransferases. FUT3, 4, 5 and 6 share between 60-90% amino acid sequence identity, but show very little sequence similarity to FUT1. FUT3 (Lewis), FUT5 (plasma) and FUT6 (plasma) are closely linked on the short arm of chromosome 19 within an interval of less than 70 kb. FUT3 encodes the fucosyl transferase responsible for the last step of the synthesis of the Lewis red cell antigen epitope (although this step may also be catalysed by FUT1, FUT2 and FUT4 in vitro). FUT6 is the gene which encodes the principal plasma fucosyltransferase, and a homozygous Ch. 3.7 Use of resources generated: mapping of FUT4 207 missense mutation accounts for the plasma alpha(l,3)fucosyltransferase deficiency found in 9% of the population of Java (Mollicone et ah, 1994). FUT5 has 91% aminoacid sequence homology to FUT3 and was initially thought to be a plasma-type of alpha(l,3)fucosyltransferase (Weston et ah, 1992); its tissue expression has not been fully elucidated. None of the polymorphisms identified by Mollicone et ah in the coding sequence of FUT5 in the Javanese plasma enzyme-deficient individuals resulted in any reduction of expression of the enzyme when both FUT5 and FUT6 were transfected into COS cells so it is unlikely that FUT5 is involved in the Javanese plasma enzyme deficiency. FUT7 is a novel leucocyte alpha(l,3)fucosyltransferase transcribed in HL60 and YT cell lines (promyelocyte and natural killer-like, respectively) (Natsuka et ah, 1994; Sasaki et ah, 1994). It has 43% aminoacid identity to the chromosome 19- mapped alpha(l,3)fucosyltransferase group (FUT 3, 5 and 6, which share approximately 90% identity) and 47% identity with FUT4. FUT7 has been assigned to chromosome 9 by amplification of DNA from somatic cell hybrids (Natsuka et ah, 1994). FUT4 is described below. In 1991 Couillin et ah mapped an alpha(l,3)fucosyltransferase (expressed in polymorphonuclear leukocytes, monocytes and brain) to the long arm of chromosome 11 by quantitation of enzyme activity in somatic cell hybrids (Mollicone et ah, 1990; Couillin et ah, 1991). From the substrate specificities and tissue expression of this enzyme they concluded that they had assigned the myeloid type. At that time neither FUT5 nor FUT7 were recognised, but it was known that the Lewis antigen mapped to chromosome 19 and that the plasma alpha(l,3)fucosyltransferase was probably closely linked to Le. FUT4 was previously known as the ELAM ligand fucosyltransferase or ELFT. Endothelial cell-leucocyte adhesion molecule 1, ELAM-1 or E- selectin is one of the LEC-CAM or selectin family of adhesion molecules, which mediates leucocyte trafficking and recruitment to sites of inflammation, together with members of two other adhesion receptor families, the immunoglobulins and integrins. ELAM1 contains an N- Ch. 3.7 Use of resources generated: mapping of FUT4 208 terminal lectin domain which permits adhesion by binding carbohydrate ligands on opposing leucocyte and activated endothelial cells. Shortly after the Mollicone paper was submitted, the sequences of two ELFT cDNAs were published (Goelz et al., 1990). These were isolated by expression cloning using a monoclonal antibody which blocked the adhesion of HL60 promyelocytes to IL1-activated endothelial cells expressing cell-surface EL AMI. The larger of the two clones ELFT-L was 2861 bases long and included the entire sequence of the shorter ELFT clone (2159 bases) apart from the terminal four adenines. From Northern hybridisation with probes common to both clones and unique to ELFT-L, cells lines known to display an ELAM-1 ligand expressed three mRNAs, 2.3, 3 and 6 kb in size. Of these only the minor 3 kb transcript hybridised to the unique ELFT-L oligomer. However, COS cells transfected with either ELFT or ELFT-L had identical properties, in so far as each were bound by the antibody used to identify the cDNA clones. In either clone, translation from the only methionine with a reasonable translation initiation consensus sequence (8 out of 9 nucleotides) predicts a protein of 405 aminoacids with a calculated molecular weight of 46 kD. The ELFT gene product was shown to have alpha(l,3)fucosyltransferase activity. ELFT was also found to be expressed in cell types known to bind ELAM-1 and conferred ELAM-1 binding activity when transfected into cell lines lacking that ability. It is possible that ELFT-L results from variant splicing in the sequence encoding the 5' and 3' untranslated regions. However, the genomic structures of other fucosyltransferases which have been characterised indicate that each is encoded by a single exon (of about 1.2 kb). Ch. 3.7 Use of resources generated: mapping of FUT4 209 Methods Primer design Primers were selected from within the 3' untranslated sequence of ELFTL. As in the other mapping experiments described in this thesis, 3'UTS was chosen in order to ensure specific amplification of the human FUT4 gene, rather than any of the other human (hamster and rodent) alpha( 1,3)fucosyltransferases with which it shares aminoacid and nucleotide homology. Melting (or dissociation) temperatures were matched using the Primer program (Lincoln et ah, 1991), and a high melting temperature was selected to improve specificity and reduce the time taken by the assay. The 5' primer was bases 2215-2238 of ELFTL (bases 1724-1747 of ELFT) and the 3' primer was the reverse complement of bases 2773-2799 of ELFTL (not present in the ELFT cDNA). The dissociation temperatures were 69.8 °C and 70.5 °C respectively, and the annealing temperature chosen for the PCR, 68 °C. The predicted size of the product amplified by these primers is 585 bp. Amplifications were performed as described in Methods and 5 ul of the product electrophoresed through 1.8% agarose in 10% TBE buffer. The product was viewed by ultraviolet illumination after staining with ethidium bromide. Ch. 3.7 Use of resources generated: mapping of FUT4 210 Results Somatic cell hybrid mapping of FUT4 Amplification of the chromosome 11 translocation somatic cell hybrids (Table 3.7.1 below) indicates that FUT4 lies within the interval llq21-22.2, defined by hybrids MIS7.4 and PG48. MIS7.4 contains the derivative chromosome 1 of the translocation t(l;ll)(q42.2;q21), which defines the proximal limit of FUT4 (Fletcher et al., 1993). The breakpoint is in a 7.8 cM region bounded by tyrosinase proximally and D11S388 distally. Hybrid PG48 is positive for markers mapping between llpter and llq22.2. It contains D11S84 (llq22) but not D11S35 (llq22) (Gillett et al., 1993). Hence, from this study FUT4 has been mapped to a region of llq delimited by TYR and D11S35. Hvbrid Chr. 11 Component FUT4 Reference -- 7 — J1C14 11 + (Kao et al. 1976) H um an + Wg3h hamster - A3EW3B llp te r - Ilq24 + (Guerts van Kessel, 1985) A3RS12B llpter - Ilq23 + (Sacchi et al. 1986) M ll-X llp te r - Ilq23 + (Chiang et al. 1984) PG48 llp te r - Ilq22.2 + (Gillett et al. 1994) EJNAC llpter - llcen - (Porteous et al. 1989) MIS39.8 llp te r - Ilq21 - (Fletcher et al 1993) B2 llp l3 - llq ter + (Larizza et al. 1983) CJ52 llql3.2 - llqter + (Koeffler et al 1981) MIS7.4 llq21 - llq ter + (Fletcher et al 1993) PCLE3 llq23.l-.2- llqter - (Brown, unpublished) Table 3.7.1: Segregation of FUT4 in a human chromosome 11 somatic cell hybrid mapping panel. Ch. 3.7 Use of resources generated: mapping of FUT4 211 Irradiation hybrid mapping of FUT4 Results of amplification hybrids from the subset of the Jo irradiation fusion hybrid panel are shown in Tables 3.1.1 and 3.3.1. Hybrids 12, 14', and 31 are positive; 2', 6', 15', 17, 33, 41', 48', 49'" and 50' are negative. These data corroborate the mapping of FUT4 to a region of llq21 near tyrosinase: all three of the hybrids positive for FUT4 also contain TYR, and TYR and D11S84 are the only llql4.3 - q21 markers present in all three hybrids. 6' contains TYR, possibly on a small fragment of chromosome 11, but does not contain FUT4; D11S84 was not tested in this hybrid. Isolation of FUT4 genomic cosmids The 585 bp PCR product from the FUT4 3' untranslated region was random prime labelled and used to probe the ICRF RLDB chromosome 11 gridded genomic cosmid library (as described in Methods and Chapter 3.4, "The identification of genomic cosmids from the MEN1 critical region, using hybrid Jo2'"). Five positively-hybridising clones were identified, of which three were unique, ICRF cl07-G1297, -B0182 and -B0333. Given the representation of the library, the identification of three separate cosmids is consistent with hybridisation to a single copy sequence on chromosome 11 (see Results chapter, as above). Each of the five clones also hybridised to labelled Jol2 Alu-PCR product as expected from the amplification of FUT4 product from Jol2 DNA. PCR of fifteen cosmids isolated by hybridisation with Jol2 Alu-PCR product was attempted using the FUT4 primers: all were negative (ICRF cl07-A052, B0481, C0244, C0572, C11178, E01115, E01130, E08171, F01115, F0662, F09140, G09131, H0549, H11164, H1150). FISH mapping of FUT4 ICRFcl07-B0333 cosmid DNA was fluorescently labelled and hybridised by Dr Margaret Fox to human metaphase chromosome spreads. The result is illustrated in Figure 3.7.1. This shows localisation to Ilql4-q22. Ch. 3.7 Use of resources generated: mapping of FUT4 212 Discussion The assignment of the myeloid-associated alpha(l,3)fucosyltransferase, FUT4, to chromosome 11 was confirmed by PCR amplification of 3' untranslated sequence in somatic cell hybrids. Regional localisation to llq21-22.2 was achieved by amplification of DNA from a chromosome 11 mapping panel, and to llql4-22 by FISH of a genomic cosmid isolated from a chromosome 11 cosmid library. This study illustrates the utility of the combination of somatic cell hybrid mapping and cosmid isolation plus FISH. These results are consistent with published data. The group at Ann Arbor confirmed the mapping of the FUT4 to chromosome 11 by PCR amplification of FUT4 sequence in somatic cell hybrids (Weston et al., 1992). Subsequently, regional localisation to llq21, between D11S388 (cJ52.4, proximal) and D11S919 (afm203vg7, distal) was achieved using translocation and radiation hybrids (Reguigne et al., 1994). This was further refined in the analysis of the Richards irradiation hybrids, which placed FUT4 within an interval of 10 cRay9000 (approximately 0.5 Mb) between D11S706 (cClll-521proximal) and D11S1757 (afm218ygl, distal) (Van Heyningen et al, 1995). McCurley confirmed the assignment of FUT4 to llq21 by fluorescent in-situ hybridisation of cosmid containing FUT4 sequence (McCurley et al, 1995). The sequence published in the paper by Goelz et al. is virtually identical to those of the alpha(l,3)fucosyltransferases cloned by Lowe et al. and Kumar et al. a year later (Goelz et al, 1990; Kumar et al, 1991; Lowe et al, 1991). The identity of these enzymes was initially in doubt since transfection studies with the different clones gave different results. Goelz showed that Chinese hamster ovary (CHO) cells transfected with the ELFT cDNA expressed an enzyme with alpha(l,3)fucosyltransferase activity and were able to bind E-selectin. The Lowe and Kumar groups confirmed alpha(l,3)fucosyltransferase activity in CHO transfectants, but neither were able to demonstrate any E-selectin binding nor synthesis of the E-selectin ligand, sialylated Lewis x, SLex. These conflicting results were later shown Ch. 3.7 Use of resources generated: mapping of FUT4 213 to be due to differences in the CHO cell lines (Goelz et al., 1994). The ability of FUT4 to synthesize the SLex determinant appears to be dependent upon the glycosyltransferases expressed by the transfected cell. Transfected dihydrofolate reductase-negative CHO cells expressed SLex and bound E- selectin, whereas Pro-5 CHO transfectants did not. There was some evidence of this phenomenon when the alpha(l,3)fucosyltransferase was first mapped by functional activity. Expression of SLex on the cell surface of the somatic cell hybrids correlated with the presence of human chromosome 11 and with expression of an alpha(2,3)sialyltransferase, but not with activity of the alpha(l,3)fucosyltransferase known to be involved in the SLex synthesis. It was suggested by these workers that the alpha (2,3)sialyltransferase, involved in the sialylation of the O-linked Gal beta(l,3)GalNAc alpha-R core structure, also maps to chromosome 11 (de Heij et al, 1988). This observation has not been followed up. Ch. 3.7 Use of resources generated: mapping of FUT4 214 Fig. 3.7.1: Human metaphase chromosomes showing fluorescent in situ hybridisation of biotinylated FUT4 cosmid probe localised to chromosome Ilql4-q22 Clone ICRFcl07B0333 is shown. Ch. 3.7 Use of resources generated: mapping of FUT4 215 C h a p t e r 3.8: Resolution of chromosome 11 - related mapping issues: Mitochondrial NAD+ -dependent malic enzyme Introduction ME isoforms Malic enzyme (ME) catalyses the oxidative decarboxylation of L-malate (HOOC.CH(OH).CH2.COOH) to pyruvate (CH3.CO.COOH): malate + NAD(P)+ -> pyruvate + NAD(P)H + C02 Three different isoforms of malic enzyme have been recognised in m am m als: (1) cytosolic, NADP+ -dependent, gene symbol ME1, mapped to 6ql2 (Chen et al., 1973; Meera Khan et al., 1984). (2) mitochondrial, NADP+ -dependent, gene symbol ME2, mapped to 6pter-p24 (Kompf et al., 1985). (3) mitochondrial, NAD+ (or NADP+), unlocalised (Frenkel, 1975; Loeber et al., 1991). This mitochondrial isoform can use either coenzyme NAD+ or NADP+, but activity is much greater with NAD+; this enzyme is referred to below as ME3. It is activated by fumarate (Mandella et al., 1975). The activity of the cytosolic malic enzyme, ME1, is ubiquitous. It is a key enzyme in de novo fatty acid synthesis and the highest levels are found in liver and adipose tissue where this isoform generates the reduced NADP used in fatty acid synthesis by fatty-acid synthase and provides a precursor for oxaloacetate replacement in the mitochondria. The NADP+ - Ch. 3.8 Mitochondrial NAD+ -dependent malic enzyme 216 dependent mitochondrial ME2 also occurs in many tissues (e.g. brain, myocardium, skeletal muscle and adrenal). It is thought to be important as a means of cycling reduced NADP into the mitochondria for biosynthesis. NAD+ -dependent mitochondrial ME3 is found in tissues with high rates of cell division (small intestinal mucosa, spleen, thymus and malignant and transformed cell lines, including lymphocyte cell lines). It is also expressed in the rapid cleavage stages of early Xenopus development (Dworkin et al., 1990). The enzyme may be a component of a pathway facilitating the conversion of aminoacid carbon (from glutamine, for example) to pyruvate (Moreadith et al., 1984). ME mapping There has been a research interest in the mapping of the malic enzymes at the Galton Laboratory since the 1970's (Povey et al., 1975). We were aware of the suggestion made by Dr Phyllis McAlpine in 1981 that since a mouse mitochondrial malic enzyme Mod2 lay within the same linkage group as Ldha and Hbb, there could be a homologue in the syntenic region of the human genome, lip (personal communication to V. McKusick, McKusick, 1997). When a further hum an mitochondrial malic enzyme was cloned in 1991 (Loeber et al., 1991), we attempted to localise this by amplification of 3'UTS in somatic cell hybrid DNA. Loeber et al. purified a NAD+ -dependent mitochondrial malic enzyme from a transformed lymphocyte cell line by ion exchange- and affinity- chromatography. Isolated peptides obtained by trypsin digestion of the enzyme were aminoacid-sequenced and degenerate sense and antisense oligonucleotides designed by reverse translation of two of the longer peptides. These were used to amplify a 1100 bp fragment from a human fibrosarcoma library, which was found to have significant sequence homology with other known ME genes. This fragment was in turn used to rescreen the library to obtain a full-length clone of 1923 bp, which was sequenced. The longest open reading frame was 584 aminoacids, encoding a protein with predicted molecular weight of 65.4 KDa, similar to that of Ch. 3.8 Mitochondrial NAD+ -dependent malic enzyme 217 the purified enzyme. The first twenty aminoacids of the predicted protein had features of a mitochondrial leader sequence. An amplification product of the open reading frame without the leader sequence (aminoacids 19-584) was cloned into a bacterial expression vector. Bacteria carrying this plasmid were induced to express a novel protein of molecular weight 64 KDa, the expected size of the enzyme. The recombinant malic enzyme was separated from the bacterial enzyme by ion exchange chromatography. The recombinant ME was shown to have the same specific activity, Km, substrate and allosteric characteristics as the human enzyme from which the tryptic digests were made. These parameters were very similar to those recorded by other investigators for human mitochondrial NAD+ -dependent ME. Both the recombinant enzyme and the lymphocyte ME had little activity when NADP+ was substituted for NAD+ as electron acceptor. Methods Primer Design Oligonucleotide sequences were selected from the 3' region of the coding sequence and the 3'UTS, using the Primer program (Lincoln et ah, 1991) to obtain primers with melting temperatures above 60 °C. The 3'UTS of the ME3 clone obtained by Loeber et al. was only 79 bp in length (bases 1845- 1923 of the Loeber seqrjnce, excluding the stop codon and the polyA tail), which I felt to be too short a target sequence for a successful PCR amplification. The forward primer was chosen from within the coding sequence (bases 1804-1823, Tm 64.3 °C) and the reverse primer from the most 3' sequence giving a 20-mer with similar melting temperature and a GC-rich bases at the 3'-end of the primer (the reverse complement of bases 1873-1892, Tm 64.8 °C). The resulting product is 89 bp in length. Amplifications using these primers are referred to as ME3-3'89 or 3'89 in the Results. Ch. 3.8 Mitochondrial NAD+ -dependent malic enzyme 218 The initial amplifications of DNA from the somatic cell hybrid mapping panel indicated that these primers amplified two different loci (see Results), so additional primers were designed. A second forward primer was selected from the 3' coding sequence (bases 1688-1715, Tm 64.3 °C), which together with the original reverse oligomer, amplify a product of 205 bp. Amplifications using these primers are referred to as 3'205 in the Results. A primer pair was also chosen from the 5'UTS and 5'coding sequence, ME3-5T19: bases 1-17, Tm 64.2 °C and the reverse complement of bases 119- 97, Tm 62.2°C. The product from this amplification is 119 bp. Amplification of the whole gene (or pseudogene) was attempted using this forward primer (of ME3-5119) and the common reverse primer of both ME3-3'89 and 205. If the genomic sequence was intronless, the product would be expected to be 1892 bp. This primer set is referred to below as ME3-1892. Amplifications were performed as described in Methods and 5 \i\ of the product electrophoresed through 1.8% agarose in 10% TBE buffer. The product was viewed by ultraviolet illumination after staining with ethidium bromide. Results ME3-3'89: Results using the first primer pair and a limited panel of somatic cell hybrids indicated a locus on chromosome 18, Table 3.8.1. This table illustrates the need to have an adequate number discordances not only where the locus is amplified and a given chromosome absent (the upper frame of the table), but also where the locus is not amplified but the chromosome is retained (lower frame). These results do not exclude the possibility of additional loci amplified by these primers on one or more of chromosomes 5, 9, 13, 17 or 19. When more somatic cell hybrids were tested it became apparent that there was a second locus on chromosome 9. The PCR products using these primers were of identical size, whether amplified from chromosome 9 or 18. The possibility of further loci on any Ch. 3.8 Mitochondrial NAD+ -dependent malic enzyme 219 other chromosome was excluded: there is concordance between retention of either chromosome 9 or 18 (or both chromosomes) in the hybrids and amplification of the ME3-3'89 sequence (Table 3.8.2). There are at least six examples of discordance with each other chromosome. Amplification of hybrids containing characterised fragments of chromosome 9 permitted regional localisation of the chromosome 9 sequence to the interval 9pter- pl3. The ME3-3'89 primers amplify is present in hybrid 298-6 which contains 9pter-pl3 as the only chromosome 9 component, and in hybrid C10BU which has 9p. The sequence is absent from 640-63al2 (commonly known as CJ9q) which contains 9q. The somatic cell hybrids comprising David Markie's chromosome 18 mapping panel (Markie et al, 1992) were also amplified using these primers. All the hybrids were positive, suggesting that the chromosome 18 locus lies in 18qll.2-q21.3 (data not shown). However, it is not known which of these hybrids also contains chromosome 9. Selected hybrids were amplified using the four further sets of primers, TTR, ME3-3'205, ME3-5'119 and ME3-1892. Transthyretin, TTR, maps to 18qll.2~ql2.1 and primers amplifying part of the human gene were used to check hybrids for the presence of human chromosome 18 (Abbott et al, 1991). Product of the expected size (415 bp) was amplified in hybrids RVL13 and 298-18. Hybrids 640-63al2 (CJ9q), SIF4A31, C4A11, C10b2BU, 298-6, B4- 2, F4scl3cll2 were negative (data not shown). The results of the ME3 amplifications are shown in Table 3.8.3. The 3’205 primers amplify DNA from hybrids containing chromosome 18 and not those with chromosome 9. Conversely, the 1892 primers amplify some chromosome 9 hybrids and not those with chromosome 18. The 5'119 primers did not give consistent results. Discussion This study illustrates the importance of ensuring that primers do not amplify more than one locus before presuming that a unique assignment has been made. The data above indicate that there are at least two ME3- or ME3-like loci, one (or more) on chromosome 18 and one (or more) on Ch. 3.8 Mitochondrial NAD+ -dependent malic enzyme 220 chromosome 9, at 9pter-pl3. The preliminary results using the additional primer sets suggest that the locus on chromosome 9 may be a processed pseudogene. It is amplified by the ME3-1892 primers (in some hybrids containing chromosome 9 and not chromosome 18), but not by the 3'205 set, perhaps because of loss of the exon sequence to which the forward primer hybridises. The locus on chromosome 18 may be the true gene, which is too large to be amplified by the ME3-1892 primers (because of the presence of intron sequences). There is no evidence to suggest that there may be a locus either on chromosome 6 (to which ME1 and ME2 map) or on chromosome 11 (the human chromosome syntenic with the region of mouse chromosome 7 to which mod2 has been located). There are 17 examples of discordance with chromosome 6 and 15 with chromosome 11. However, the murine homologues of several genes which map to human lip 15 have been found to be separated on mouse chromosome 7 by segments homologous to human 6p, llq, 15q and 16 (Rinchik et ah, 1991). There is no evidence from this study of a locus on human chromosomes 15 or 16, either. There is confusion in the literature about the characterisation and nomenclature of the malate dehydrogenases and the malic enzymes. An example is the article by Loeber et ah, from which the ME3 primers were derived, which referred to this mitochondrial NAD+ -dependent malic enzyme with the EC number 1.1.1.40 (Loeber et al, 1991). This is the Enzyme Commission number reserved for the malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating) enzyme. The cytosolic enzyme with these properties is ME1 which has been mapped to 6ql2 (Povey et al, 1975; Meera Khan et al, 1984), and the mitochondrial enzyme ME2 to 6pter-p24 (Kompf et al, 1985). The cDNA cloned by Loeber et al., which is referred to here as ME3, encodes a malate:NAD+ oxidoreductase, activated by fumarate. The coenzyme specificity indicates that this enzyme m ay be EC 1.1.1.39, a malate:NAD+ oxidoreductase which does not decarboxylate added oxaloacetate. Oxaloacetate-decarboxylating activity was not reported by Loeber et ah. Subsequently, Loeber et al. have cloned a Ch. 3.8 Mitochondrial NAD+ -dependent malic enzyme 221 human "mitochondrial NADP+ -dependent malic enzyme (EC 1.1.1.39)" (Loeber et al., 1994). This is probably a malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating) enzyme, EC 1.1.1.40. Many of these loci have been mapped in the 1970's by demonstration of the presence or absence of enzyme activity in somatic cell hybrids, and their genomic localisation has not been re-examined subsequently. In the mouse, the hepatic cytosolic malate:NADP+ oxidoreductase (EC 1.1.1.40) is encoded by the Modi locus, which has been mapped to mouse chromosome 9. The activity of this enzyme is strain-dependent and is influenced by a regulator locus on proximal mouse 12, Modlr, one allele of which confers high Modi enzyme activity and the other, low activity (Coleman et al., 1991). If a similar modifier locus exists in humans, this calls into question the mapping of the Modi homologue ME1 to human chromosome 6 by enzymic techniques (Chen et ah, 1973; Povey et ah, 1975). Application of a consistent nomenclature will require a re-examination of the substrate and co-enzyme specificities of the malate dehydrogenase and malic enzymes and confirmation of any localisation obtained by identification of enzyme activity in somatic cell hybrids using DNA-based techniques such as those perfomed in this study or DNA hybridisation techniques, e.g. FISH. 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(d 0 f t £ 0) d 0 • • cn ■rl 3 13 1 1 i p - 00 | + + I + I £ ft! H cn £ P 0 ft •rl cn 1 1 I I + + + £ + + + 00 u ■£ f t .—. t£ + + U CO CD 1 + I + + + + I I r—1 (N £ o + + i 1 I £ — 0 TJ CO 0 l > 1 1 I + I + U1 ID ft I I £ 0) o ft id £ [> „_„ CD £ i + 1 1 l I 0 1 00 CD £ o o +1 1—1 cn — r0 H 0 cd x—1 £ >0 u LO id + i 1 + l p - £ i I + £ o -sh id U 0 > r~H cn c o ■rH Id cn + I I 1 1 1 O’ I + A ■rl r l Ch. 3.8 Mitochondrial NAD+ -dependent malic enzyme 226 LO f - c n 0 0 \— I 4-1 c n -—- -—- -—« -—- -—- O -—« rH CN CO LO CO CO X— 1 & rHLnLnini>00HO^rHr-|HrHrH^ t—I x—I *—I rH PC M fd o' fd ■U X (D d f d d) co co 0 ) d) fn 23 £ £ ■ H P4 «n o &r \ (D h g X 0) oq r H G - H ™ (d > a £ I I I I + + LO -— ' 0 0 o c n X— 1 X— 1 + + + a ) ti .. 1— 1 o X + + + + + I I o- + id fd a 0 0 c n c n 4 J I p * oq I + + + I X— 1 Q) 0 » rH a . CN I I + + + I I P - I ■rl 0) M r X a fd U a P3 o cd -U PQ CN I + I P> I g 03 Ch I + I I + •a !>i CO ■H Ch. 3.8 Mitochondrial NAD+ -dependent malic enzyme 227 C h a p t e r 3.9: Resolution of chromosome 11 - related mapping issues: assignment of RXRB to chromosome 6p21.3 Introduction Retinoic acid (RA) and retinoid X (RX) receptors are retinoic acid-inducible enhancer factors belonging to the superfamily of steroid/thyroid nuclear receptors. RXRB is a member of the Retinoid X Receptor RXR family, which includes three genes, the Retinoid X Receptors alpha, beta, and gamma, RXR A, RXRB and RXRG (Mangelsdorf et al., 1992). The RXR genes, together with the three members of the related RAR gene family (Retinoic Acid Receptor alpha, beta and gamma, RARA, RARB, RARG), bind to and mediate the effects of all-trans Retinoic Acid, RA and/or 9-cis RA. Both retinoids are derivatives of vitamin A and are involved in growth, development and differentiation. RARA was the first retinoic acid binding protein to be isolated (Giguere et al., 1987; Petkovich et al., 1987). The cDNA encoded a protein that bound retinoic acid with high affinity; the protein was found to be homologous to the receptors for steroid hormones, thyroid hormones, and vitamin D3, and functioned as a retinoic acid-inducible transacting enhancer factor. RARA was assigned to chromosome 17 (Bale et al., 1988) and regionally m apped to 17q21 (Brand et al., 1988; Mattei et al., 1988). RARA was later shown to be one of two genes involved in the (15;17)(q22;qll.2-ql2) breakpoint which is a characteristic cytogenetic feature of acute promyelocytic leukaemia (the other gene being MYL/PML). The 15q+ derivative PML-RARA gene was found to encode a chimeric fusion protein with oncogenic properties (Borrow et al., 1990). Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 228 Brand et al. also provided evidence for the existence of a second retinoic acid receptor RAR-beta RARB localised to 3p25-p21 (subsequently 3p24, Mattei et al, op. cit.). This had been identified previously as HAP, HBV- Activated Protein, a novel gene found at the site of integration of the hepatitis-B virus in a hepatocellular carcinoma (Dejean et al., 1986). A third type of RA receptor, the gamma receptor RARG, was first identified in the mouse where mRNA was detected predominantly in skin (a known target for retinoic acid). The human homologue was cloned using the mouse cDNA (Krust et al., 1989), and was shown to be the predominant RAR expressed in both mouse and human skin. RARG was assigned to human chromosome 12ql3 (Mattei et al., 1991). Mattei et al. (1991) m apped the RARA, RARB, and RARG gene loci in man, and also in mouse and rat. These confirmed and extended the syntenies between: human chromosome 17, mouse chromosome 11, and rat chromosome 10 (RARA); human chromosome 3, mouse chromosome 14, and rat chromosome 15 (RARB); and hum an chromosome 12, mouse chromosome 15, and rat chromosome 7, (RARG). The assignments also indicated that tight linkage between RAR genes and HOX gene clusters has been conserved during mammalian evolution (HOXB cluster, human chromosome 17, mouse chromosome 11, rat chromosome 10; HOXC cluster, human chromosome 12, mouse chromosome 15). Mangelsdorf et al. identified a nuclear receptor structurally and functionally related to the RAR family which was referred to as retinoid X receptor alpha, RXRA (Mangelsdorf et al, 1990; Mangelsdorf et al., 1991). This receptor differs from the three RARs within the ligand-binding domain and is incapable of high affinity binding of retinoic acid. It was subsequently found that the high affinity ligand for RXRA (and other RXR) is 9-cis retinoic acid, v.i. (Heyman et al., 1992). A second member of this family of retinoid X receptors was identified by sequential screening of expression libraries with a retinoic acid response element and RAR (Yu et al., 1991). The cDNA isolated encoded a coregulator highly related to RXR-alpha, which they named retinoid X Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 229 receptor beta, RXRB. This protein formed heterodimers with RAR, and increased DNA binding and transcriptional activity of RXRA on promoters containing retinoic acid-, but not thyroid hormone- or vitamin D-, response elements. However, RXRB also heterodimerised with thyroid hormone and vitamin D receptors, and increased both DNA binding to, and transcription from, their respective response elements (Hallenbeck et al., 1992; Marks et al., 1992). RXRA was also found to form heterodimers with these receptors. It was suggested that the retinoid X receptors target the high affinity binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate DNA response elements. The mouse rxrb gene had been cloned two years previously when it was recognised that H-2RIIBP (rxrb) was a member of the nuclear hormone receptor family which bound the regulatory element of major histocompatibility class 1 genes and the oestrogen response element (Hamada et al., 1989). A third member of the RXR family was isolated in mouse and a human homologue Retinoid X Receptor Gamma, RXRG, identified (Mangelsdorf et al., 1992). The RXR family is homologous to the XR2C and CF1 gene at the Drosophila ultraspiracle locus. This locus is involved in pattern formation, and the relatedness of RXR to XR2C and CF1 implies that similar transcriptional activators may be used in morphogenesis in both vertebrates and invertebrates (De Luca, 1991; Christianson et al., 1992). The genes for all the steroid/thyroid/retinoid receptors have a similar structure, with four domains or regions: A/B, C, D, and E (Robertson, 1987). The function of region A/B is not clear; C encodes the DNA-binding domain; D is thought to be a hinge region; and E encodes the ligand- binding domain. The DNA-binding domain is the most highly conserved, both within and between the groups of receptors. The ligand- binding domains show less homology (which may reflect the different specificities of each receptor, v.i.). The RAR and RXR nuclear receptor proteins are activated by binding to RA or 9 -cis RA, and control the expression of target genes by binding to Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 230 specific DNA sequences, hormone response elements. This sequence- specific binding ability is conferred by a highly conserved "DNA-binding" domain of 68 amino acids encoding a zinc finger. The retinoic acid, thyroid hormone, and vitamin D receptors, as well as the retinoid X receptors activate transcription from response elements containing two or more copies of the consensus motif AGGTCA. RA response elements have been identified in a number of different organisms and genes, e.g. rat growth hormone and phosphoenolpyruvate carboxykinase, mouse complement H and laminin Bl, human and mouse RARB/rarb, human osteocalcin and human alcohol dehydrogenease (De Luca, 1991). A more variable C-terminal domain, the E domain, of about 220 amino acids has been identified as the retinoid ligand-binding region. The RXR genes share only 20% nucleic acid homology with the RAR receptor family in this ligand binding domain. This is consistent with the finding that RARs and RXRs have different ligand specificities: RARs bind both all- trans RA and 9 -cis RA whereas RXRs bind 9-cis RA only (Heyman et al., 1992; Levin et al., 1992; Mangelsdorf et al., 1992; Allenby et al., 1993). The RXRs bind to response elements that are distinct from those regulated by RARs. However, RXR proteins may act cooperatively with RARs by dimerisation to enhance binding of RARs to their elements. Similar heterodimerisation has also been found to occur between RXRs and other members of the nuclear receptor gene family, such as the thyroid hormone receptor and the vitamin D3 receptor (Yu et al., 1991). Within a family, members show considerable similarity (by definition, of course): RXRB has more than 95% sequence homology with RXRA in the DNA-binding domain and more than 90% homology in the ligand- binding domain (Yu et al, 1991). However, comparison of the amino acid sequences of the six 6 human and mouse RARs suggest that the interspecies conservation of a given member of the RAR subfamily, alpha, beta, or gamma, is much higher than the conservation of all three receptors in a given species. This may indicate that each RA receptor type Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 231 may perform a specific function (or functions) which are conserved between species. The gene for the Retinoid X Receptor alpha, RXRA, was assigned to chromosome 9 and regionally mapped to 9q34 in the interval between D- segments D9S66 and D9S67 by PCR amplification of somatic cell and irradiation fragment hybrids containing characterised regions of 9q (Fitzgibbon et al., 1992; Zhou et al., 1992). The localisation was confirmed by FISH of genomic RXRA clones to human metaphase chromosome preparations (Jones et al., 1993). Since RXRA was found to lie in the TSC1 critical region, we wished to determine the chromosomal position of the RXRB gene in order to consider its involvement as a candidate for the second or third TSC loci. This work was performed before the localisation of TSC2 to chromosome 16pl3 (Kandt et al., 1992). Methods Primer design DNA from rodent parent cell lines, somatic cell hybrids and human controls was amplified by PCR. The oligonucleotide primer sequences were chosen from 5' untranslated region of the human RXRB cDNA (EMBL HSRXRB, M84820) forward, 5-CTC TCA GGG GCT TCC TCG TGC TC-3'; reverse, 5'-CCG CTG AGG GAG GAA GGG CG-3', maximising discrepancies between human and mouse rxrb sequence at the 3'end of each oligomer (Leid et al, 1992). These oligomers amplify a 131 bp product. Amplifications were performed as described in Methods. A single product of the expected size, 131 bp, was seen when human DNA was amplified using these RXRB primers. The amplification was specific for the human sequence; non-specific rodent products were not visible in any of the somatic cell hybrids tested (Figure 3.9.1). Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 232 A different forward primer (forward, 5'-CAG AGT CTT TCT CTC AGG GG- 3') together with the reverse primer as above, was used initially (resulting in a 141 bp product). The melting temperature, Tm, of this oligo is 58 °C, whereas the that of the reverse oligo is 72 °C. It was not possible to increase the annealing temperature, Ta, of the reaction above 58 °C. Two products were seen when human DNA was amplified using these RXRB primers at this Ta, one of the expected size, 141 bp, and a larger product of about 600 bp. Similar- or larger- sized products were also visible in some of the somatic cell hybrids but these non-specific products could be differentiated from the specific 141 bp band (Figure 3.9.2). This latter product was not observed when mouse, rat or hamster control DNA was amplified. This illustrates the one of the advantages of designing oligos which have a high Tm when amplifying human sequence in somatic cell or irradiation hybrids (see the introduction the Results chapter, "Use of resources generated: mapping of cofilin genes, CFL1 and CFL2"). Annealing of the oligos to rodent sequences having partial complementary sequence homology occurs with lower Ta and non-specific amplification may take place. This is minimised if one can design the oligos to be sufficiently long or GC-rich to ensure that they anneal at temperatures approaching the ideal extension temperature for the thermophilic DNA polymerase used in the PCR reaction. It also permits faster cycling since rapid extension (at a rate similar to that at 70-72 °C) takes place as soon as an oligo has annealed at 68 °C, and so the extension time may be reduced. In addition, less time is spent by the PCR reactor cooling the block from the denaturation temperature (commonly 93 °C) to 68 °C c.f. 58 °C, for example. Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 233 Results RXRB PCR of somatic cell hybrid panels, localisation of RXRB to 6p21.3-6q21: The results of the amplifications of the somatic cell hybrid panel are listed in Table 3.9.1. There is complete concordance between retention of chromosome 6 in the hybrids and amplification of the RXRB sequence, and there are at least six examples of discordance with each other chromosome. The FG10 hybrid is known to contain a fragment of chromosome 6, but the extent of this fragment has not been determined (Griffo et al., 1993). EDAG3R and MCP6 (both positive for RXRB) also contain a partial chromosome 6: EDAG3R 6pter-6q21, MCP6 6p21.3-6qter. This indicated that RXRB must lie in the interval 6p21.3-6q21 (Table 3.9.1). Dr John Boyle kindly provided aliquots of DNA from a panel of somatic cell hybrids, containing characterised fragments of chromosome 6 (Boyle et al, 1992). Hybrid Chromosome 6 RXRB C om ponent EDAG 3R 6pter- 6q21 + RAGMH 9.4.8 6pter- 6ql5(q21) + RAGSU 3.1.2.3 6pter- 6ql4 + 56.47 6pter- 6p21.1 + CALLA9 1.9.9 6pter- 6p23 - EDAG 2.9.8 6q21- 6qter - IJA9 2.21.14 6ql2- 6qter - MCP6 6p21.3- 6qter + CALLA9 1.13.10 6p23- 6qter + 2068RAG 22.2 6pter- 6q27 + ROHRAG 9.32 6q27- 6qter - Table 3.9.2: Segregation of RXRB in a human chromosome 6 mapping panel (Boyle et al, 1992). Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 234 PCR of these hybrids permitted regional localisation of RXRB to 6p21.3- p21.1 (Table 3.9.2, above). The critical hybrids are MCP6 and 56.47: RXRB was positive in MCP6 (6p21.3-6qter) and also positive in 56.47 (6pter- 6p21.1). Fluorescent in situ hybridisation with RXRB cosmids, localisation of RXRB to 6p21.3: Jude Fitzgibbon isolated RXRB cosmids by screening a human genomic cosmid library (Cachon-Gonzales, 1991) with a 141 bp PCR product generated from the 5' untranslated region of the RXRB gene (forward, 5'- CAG AGT CTT TCT CTC AGG GG-3'; reverse, 5’-CCG CTG AGG GAG GAA GGG CG-3') by amplification of human genomic DNA. The 141 bp product was excised from a gel, phenol-chloroform extracted, and oligo- labelled with 32P. Three cosmids RXRblO, RXRbll and RXRbl2 were identified. In situ hybridisation of biotinylated cosmid probe to human metaphase chromosomes confirmed the localisation to the short arm of chromosome 6 (Karen Woodward). Specific signals were seen on each chromatid of the chromosome 6 homologues in all 20 metaphase spreads analysed. A more precise localisation to 6p21.3 was obtained by simultaneous examination of fluorescent signals and R banding (Figure 3.9.3). Discussion During the preparation of our manuscript submitted to the Annals of Fluman Genetics (Fitzgibbon et al, 1993), Fleischhauer and colleagues published the localisation of RXRB to chromosome 6 within the 6pter-ql3 region (Fleischhauer et al., 1993). We confirmed the mapping of RXRB to chromosome 6 and refined the localisation to the short arm, 6p21.3, to the same band as the HLA complex. This is consistent with mapping studies in the mouse which have localised the rxrb gene to mouse chromosome 17, close to the mouse major histocompatibility H2 complex (Hoopes et al, 1992). This assignment was confirmed by two other groups and Nagata et Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 235 al. physically mapped RXRB to the HLA-DP region (Almasan et al., 1994; Nagata et al., 1995). The RXRB and RXRG genes are therefore unlikely to play a direct role in the pathogenesis of TSC since they map to regions which are not currently implicated as candidate regions of the genome for TSC. At the time of the mapping of RXRA in 1993, the gene was a possible candidate locus for TSC1, however. It is of interest that RXRB has been localised to the short arm of chromosome 6, where there is evidence for a locus for dominant, non- syndromic orofacial clef ting, OFC1. Retinoids are teratogenic in birds and mammals, and cause multiple malformations, including abnormalities in craniofacial and limb development. Cleft palate and absence of the upper beak has been reported in chick embryos exposed to RA and RA receptors have been shown to be involved in craniofacial development. In addition, the pattern of expression of one of the RAR receptor family, RARB (which has been mapped to 3p24) is altered (in chick facial primordia) by exogenous RA exposure (Rowe et ah, 1991). Eiberg et al. studied pedigrees with autosomal dominantly inherited, non- syndromic, orofacial cleft and showed linkage with the A subunit of clotting factor XIII, F13A1 (Eiberg et ah, 1987). F13A1 is itself linked to HLA (Zmax 11.44, at theta = 0.25 in males, theta = 0.35 in females, (Buetow et ah, 1994)). F13A1 has subsequently been m apped by in situ hybridisation telomeric to HLA at 6p24-p25 (Board et ah, 1988). The presence of a locus for cleft lip on the distal portion of 6p is supported by the reports of chromosomal rearrangements in the region in patients with OFC. A patient with a de novo terminal deletion of 6(p22.2->pter) had unilateral cleft lip and palate and a midline maxillary defect together with other congenital abnormalities (Sachs et ah, 1983). Kormann- Bortolotto described a 6p23 terminal deletion (del(6)(qter->p23)) in a patient with multiple anomalies including cleft lip and palate (Kormann- Bortolotto et ah, 1990). More recently, Donnai reported a three generation family in which there was association between a translocation Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 236 (6;9)(p23;q22.3) and cleft lip and palate (among other features) in three of the familily members in two generations (Donnai et al., 1992). The mapping of RXRB to 6p21.3 suggests that it is centromeric to the critical region identified by linkage studies and by the chromosome 6 deletion and rearrangement patients, and is therefore unlikely to be a candidate gene for OFC1. Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 237 CN 00 Cn cn \—1 LD o CN e' rH X—1 cn 00 en cn cn r - o i> *—i r- r- r-~ o r - r - M l> cn [> rH cn r - r - r - Cn l> r - 0000 CO 000000000000 (d 00 CD x—i 00 rH 000000 x—I 00 CO cn cn cn cn cn cn cn cn cn cn cn cn Cn O'! cn O'! T5 \—i x—i x—i i—i *—i x—i x—i x—i x—1x—i 4J x—I cn • x—I c x—Ix—1x—1 X—1 x—1 U (d CD x-H CD t'H M o • (d Cn fd 3 • u i— i i—I i—i 4-> i—i i—i i—i i— i i—1i—i —I rH G i—i i—1l—1 1—1i—1cn (D w (D (tf td (d CD rd td td X + + I + I + + + + I + + | + D_|L|_|+ + + + CM CN + + + + + I i I I i + + I I I + + I I I i + + + + i i + i + I + I + I + I + + + O CN + I + I + + i i i g + I I I + I ° N1 OO 00 M CTi x—I i i i + I I i i + + I I I I I 00 + + + + + i + i + + + I + + + + i + + I + I + i + + + I I l + l + + cn c- c- co 4- ^ i + + -y i i + i r~* + + I I I I I I h ^ h ^ cn + + i g I + i I + I + I CM + + 2 j ^ CD CD LD + + + + + + + + + + cn r- cd lo w° 3^ + + I I + + I O CO S 'H + + i + g i + i + I I + I I + + I + + + + + i + i + I I I I + I + u 'i I I ! I I I I I ! + + + I I + + + + + I + I + I I + + + + I I I + I co 0 0 CD CD cn MH + I I + I I I I CM I + I I I I I c n cn n ! oo rH co + + + I + I + I I + I + I + I I I o I + I g I + + I + I + + + I M-l I I r~-■=+ r~- o CD + + + + + + + + M-l M-l I I I I I I LD I + I + I I + + + + I + I + + + I + I + + + + + I I + I I I + I I I I I + + m + + + + + I + I + I =j + I I + + CN i i i i + + g! I I + I I I I I I + I *—i i + + + + i + I + I I + I I I I + I I cn CN CN \- 1 cn P 04 w o Q Cn D 00 X—1 H W ^}f CO rR + I I + TJ x—1 LD cn < cn 04 m cn CO X CN o 00 00 D r- U ■rl N W T—1 rH LD < m U o W W 1- 1 < Sh cn CN £ (D £ x—1 fD cn CD o P r^ CN Pd o o U o Si Eh 52 H H Cn 04 Cn <3 04 \—1 U Eh 52 H rH tH Cn X—1 C>i CO Q [g O H Eh 3 H Q U U X—1 CO Q 0 o s H s X Cn S PP CO U X—1 CO W £ Cn p Cn £ CN Cn Cn CD CO 0 Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 238 Fig. 3.9.1: PCR of RXRB in human , rodent and somatic cell hybrid DNA, 131 bp product Key: (from left)'marker (PhiXl73/HaeIII),*human,3r a G (mouse),^Wg3H (hamster) ? FAZA (rat) , ‘•MOG2C2 ,*DUR4 . 3 ,1FG10E8EP2 . 9 ,9 EDAG3R/%RAGSU 3.1.2.3,"56 .47 ,,<'CALLA9 1.9 .9 ,^MCP6/barker Fig. 3.9.2: PCR of RXRB in human, hamster and somatic cell hybrid DNA, 141 bp product Key: (from left) 1 human,lWG3H (hamster),3marker (PhiX173/HaeIII) , V FST7 , *SIF15P5, tFST9/10 * FG10, * MOG2E5 ,** MOG34A4, ,%MOG2C2 , MJ1CL4 . Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 239 - V, 1Cx Fig. 3.9.3: Human metaphase chromosomes showing fluorescent in-situ hybridisation of biotinylated RXRB cosmid probe localised to chromosome 6p21.3 Ch. 3.9 Assignment of RXRB to chromosome 6p21.3 240 C h a p t e r 4: Discussion The work described in this thesis was successful in generating useful resources on chromosome 11. Unfortunately, for reasons beyond my control, it made very little contribution to the cloning of the TSC1 and TSC2 genes. In this final chapter I shall discuss some of the advances made in radiation hybrid techniques and positional cloning since that time. The work on the mapping of the different cofilin genes, described in Chapter 3.6, suggested some general points about the 3' untranslated regions of genes which are relevant to current major EST mapping efforts and these form the final part of that section. I also consider the identification of the two TSC genes, some recent insights in the molecular pathogenesis of the disease and whether there might still be a third TSC gene. Radiation Hybrids "The fusion of somatic cells of different species produces hybrids which, in most cases, preferentially lose some or all of the chromosomes of one of the parental species on prolonged subculture. So far, however, there has been no general way of predetermining which of the parental sets will be preferentially reduced. I report here a technique to this end in the belief that it will constitute a useful tool in the use of somatic cells for formal genetic analysis." (Pontecorvo, 1971). The technique reported by Pontecorvo in that seminal Nature paper was the use of low dose, non-lethal gamma irradiation (600 - 1,600) to generate chromosome breaks in one parent cell line and the imposition of HAT selection on both irradiated and "recipient" parents to eliminate any non hybrid parental cells. The rescue of a lethally irradiated cell line by fusion to rodent recipient cells was demonstrated four years later by Goss and Harris (Goss et al., 1975). Pontecorvo, Goss and Harris laid the foundations of a technique which was exploited in single chromosomal gene mapping Ch. 4 Discussion 241 fourteen years later (Benham et al, 1989; Cox et al, 1989), and which subsequently proved to be a very "useful tool" in whole genome genetic analysis (Walter et al, 1994; Gyapay et ah, 1996). The relative advantages of radiation hybrids compared with linkage-based approaches and physical techniques (other than RH) have been well described by Peter Goodfellow (Walter et ah, 1994; Gyapay et ah, 1996). The former are constrained by the need for polymorphic markers and multiple meioses. The latter, YAC, BAC, PAC and cosmid contigs, can be used to integrate maps based on monomorphic STS's as well as polymorphic markers. However, the construction of complete contigs of the human genome using YACs and unordered STSs has proved difficult and not all regions of the genome are covered by contigs that are sufficiently robust to permit efficient mapping of ESTs (Chumakov et ah, 1995). Maps based on RH can overcome both these disadvantages. Single chromosome irradiation hybrids The early use of radiation hybrids (RH) in monochromosomal gene mapping has been considered in the Introduction and Results discussion of Chapter 3.1. By the end of 1993, a review identified 49 different single chromosome irradiation hybrid panels generated from 18 human chromosomes or chromosome regions (Walter et ah, 1995). More recent publications have used monochromosomal radiation hybrid panels not only to order loci but also to use the STS markers to identify other physical resources within the region, such as YACs. An example of the integration of RH and YAC maps using this approach is the human chromosome 9p study of Bouzyk et ah, 1997. The presence or absence of 114 STS on 9p were scored by PCR in 93 radiation hybrids derived from a chromosome 9-only somatic cell hybrid (Bouzyk et ah, 1996). These same STS (plus 18 further markers) were used to identify YACs from the CEPH mega-YAC library (Cohen et ah, 1993). Each of 89 of the 114 original markers identified at least one YAC. Eighty eight YACs (of average size 0.9 Mb) were ordered into seven contigs separated by 5 gaps of <1 to 3 Mb, representing coverage of about 90% of the short arm of the chromosome Ch. 4 Discussion 242 and providing data additional to those in the Whitehead and CEPH YAC maps (Chumakov et al., 1995; Hudson et al., 1995). Markers confidently placed on the YAC map were added to the low-resolution RH framework, and together used to assign additional markers to generate an integrated high-resolution RH/YAC map containing 65 loci. Fifty-one of these markers were placed uniquely and the average resolution was 0.79 Mb. The study demonstrated some of the advantages of focused, single chromosome resources compared with whole genome mapping attempts: the location of microsatellites common to both the Bouzyk and the Genethon genetic maps (Dib et al., 1996) was refined and several of these markers ordered, and some discrepancies in the Whitehead STS-RH/YAC whole genome map were resolved. Integrated maps such as this further the development of a high resolution map of the genome, facilitate positional cloning attempts and provide the initial resources to develop sequence-ready contigs of substantial regions of a chromosome (Kumlien et al., 1996). Whole genome irradiation hybrids. It has been estimated that a panel of between 100-200 single-chromosome radiation hybrids is required to construct a robust map of an average chromosome, or over 4000 clones to cover the human genome (Barrett, 1992; Gyapay et al, 1996). The Pontecorvo-Goss-Harris technique does not require that the donor parent should be a single chromosome somatic cell hybrid, however. In the early 1990's Hans Lerach prompted Peter Goodfellow to reconsider the application of radiation hybrids to genome m apping. The Cambridge-Genethon group generated an experimental panel of hybrids using a diploid human fibroblast line as the donor parent irradiated with 3,000 rads and fused with thymidine kinase (TK)-deficient hamster A23 cells. A low irradiation dose was used to ensure that a majority of the hybrids contained sizeable human chromosomal fragments and to minimise the possibility that there would be any regions of discontinuity in the map. Forty-four hybrids were derived, Ch. 4 Discussion 243 characterised and used to make a chromosome 14 map using PCR-able 40 markers. The resulting order of these 40 loci was consistent with the Genethon and NIH chromosome 14 maps (correcting an error in the latter); nine further markers were located more precisely. This same approach was the used to construct a larger panel of 220 hybrids. Twenty one hybrids either showing few inter-A/u-PCR products and or lacking human TK were discarded (the former screen to minimise non productive typing of hybrids containing little human chromosomal material). A subset of 168 hybrids was randomly chosen. These were typed with 711 microsatellite markers and a framework map generated from 404 of these, selected to cover the genome at 10 cM intervals and with pairwise LOD scores of >9 between adjacent markers, where possible. No gaps were detected in the framework map except at some centromeres and the pseudoautosomal region of the X chromosome. The panel was used to locate more precisely 374 Genexpress ESTs previously assigned to chromosomes 1, 2, 14 and 16 using somatic cell hybrid lines (SCH). All ESTs but two showed pairwise LOD scores of >10 with a framework marker and concordance with the prior assignment was 96%. A majority of the discordancies were known from the SCH study to map to more than one chromosome. The two ESTs with a pairwise LOD of <10 showed a very high retention frequency in the panel (>70%) and one of these mapped to chromosome 17. The TK gene maps to chromosome 17 and is retained in every hybrid. Loci in this region are retained with high frequency (range for chromosome 17 loci 40 - 94%, c.f. other chromosomes 11 - 71%), but this potential Achilles heel did not appear to degrade the chromosome 17 map (Foster et al., 1996). A subset of 93 hybrids from the panel with the highest retention of human fragments was regrown and aliquots made available as Genbridge 4. Mapping data derived from these hybrids is made available on the RHdb website (http://www.ebi.ac.uk/RHdb/). A larger framework map was developed from two radiation hybrid panels (including the Genbridge 4 hybrids) and a YAC library using 1000 polymorphic genetic markers, on to which 16,000 hum an EST clusters (one-fifth of the estimated total number of genes) had been mapped at Ch. 4 Discussion 244 publication (Schuler et al., 1996a; Schuleret al., 1996b). This has become the principal method of mapping ESTs and cDNAs and has facilitated positional candidate approaches to gene identification. Unfortunately, the quality of sequence data in the EST databases may need refinement: in one study on chromosome 9q34 20% of EST clusters in the Unigene resource contained chimaeric clones mapping to more than one chromosome (Boguski et al, 1995; Wolfe et al, 1997). Conservation of 3' untranslated sequence, implications for radiation hybrid mapping Another potential problem has been highlighted in the analysis of cofilin 3' untranslated sequence (UTS). A basic premise of mapping by amplification of 3'UTS of a human gene in a well-characterised somatic cell- or radiation- hybrid panel is that rodent and human 3'UTS is divergent. However, cross-species conservation of 3'UTS is not uncommon. A partial search of the Medline National Library of Medicine database identified several examples of genes in which regions of the 5 and 3'UTS show some homology between mammalian species, Table 4.2. This summary indicates that a number of other cytoskeletal proteins, like cofilin, have 3'UTS which is conserved between species: collagen, actin, laminin, myosin, dystrophin and elastin. Human and rat beta actin 3'UTS are 74% homologous, and the human and bovine gamma actin 3'UTS share 83% nucleotide identity. In one highly conserved gamma 3' sub- region of 164 bases there is 93% homology between human and bovine, and 97% between human and the mouse gamma actin pseudogene (Chou et al, 1987). Selection of primers from the 3'UTS of an EST which coincidentally amplified a rodent homologue would render that EST unmappable on a panel derived from a rodent of the same species, and would not result in a mis-assignment to another human chromosome. The multiple localisations of some ESTs seen using the Genbridge panel are more likely to be due to human pseudogenes and gene families with similar 3'UTS than cross-species 3'UTS conservation (Gyapay et al, 1996). That study selected for WGRH mapping ESTs which had previously been assigned to chromosomes 1, 2, 14 and 16 using somatic cell hybrids and this Ch. 4 Discussion 245 initial mapping would have excluded those primers which gave uninterpretable results, possibly because of cross-species 3'UTS conservation. Positional cloning Most of the work described in this thesis was carried out (and published) before 1993. By the end of 1993 there had been 26 successful positional cloning attempts of which 19 were aided by the identification of chromosomal rearrangements, see introduction (Collins, 1995). The twelfth edition of the McKusick catalogue published in 1997 lists over 600 genes associated with disease-producing mutations, the vast majority of which have been isolated by positional cloning and positional candidate approaches (McKusick, 1997). This has been possible because many more highly polymorphic markers have been introduced, genetic maps have been greatly improved and many more cDNAs have been placed on the map (Boguski et al., 1995; Dib et al., 1996; Schuleret al., 1996a; Schuleret al., 1996b). Consequently, candidate genes within a genetic interval can rapidly be identified by searching a public sequence database. The physical map has also developed: YAC contigs have been assembled for chromosomal regions and whole-genome maps attempted (Cohen et al., 1993; Chumakov et al., 1995; Green et al., 1995; Hudson et al, 1995). Many of the CEPH mega-YACs map to the expected region, but a substantial minority (9 of 20 tested by FISH in one study, (Bouzyk et al., 1997)) are chimaeric or have no signal on the expected chromosome. Defective YACs are unsuitable for use as mapping and sequencing reagents and much effort is expended in weeding them out. Third generation clone resources, PACs and PACs, with insert lengths of between 90,000 - 350,000 base pairs have been introduced. Their larger insert size compared with cosmids has assisted contig-building and sequencing projects particularly in genomic regions containing tandem repeats and they are less prone to artefacts than YACs (Nehlset al., 1995). BAC-PAC end sequencing has been suggested as a simple strategy to elongate sequenced regions and pilot Ch. 4 Discussion 246 end-sequencing projects have started to test the feasibility of using this approach to sequence the human genome (Venter et al., 1996). Individual well-characterised radiation hybrids may be regarded as similar, large-scale cloning vehicles incorporating between 0.3 and 30 Mb DNA, which may be used to screen cosmid, BAC or PAC libraries, as this thesis has demonstrated. This procedure is no more complex than the use of YACs as screening probes. These smaller clones from specific chromosomal regions are a very useful resource, which may be integrated into ordered, overlapping sets or contigs and incorporated into the chromosome database, as have the cosmid resources described in this thesis, (Pang et al., 1996; Lemmens et al., 1997). This is an essential step in a conventional genomic sequencing approach: DNA sequences obtained from clones within the contig may be located on chromosomes with minimal uncertainty. Sequence derived from contigs is increasingly being used as primary resources in cloning projects, as demonstrated by the isolation of TSC1, which was identified from the sequence of a 1.8 Mb contig (Hornigold et al., 1997; van Slegtenhorst et al., 1997). TSC TSC2, tuberin The TSC2 gene on chromosome 16 was identified in 1993 (Nellist et al., 1993) just a year after the initial mapping of the gene, partly because of a unique family with a translocation and because a significant proportion of patients have large deletions in the TSC2 region readily seen on pulsed field gels or Southern hybridisations (see Introduction). The gene extends over 45 kb and has 41 coding exons and a further 5' untranslated exon which was discovered recently (Kobayashi et al, 1997). The cDNA is 5,474 nucleotides in length and encodes a protein of 1807 aminoacids, tuberin, which has a molecular mass of about 200 kDa. Analysis of the predicted aminoacid structure indicates that there are several hydrophobic domains which may represent membrane spanning regions, a leucine zipper motif near the N-terminal, and a region extending over several C-terminal Ch. 4 Discussion 247 exons with homology to a GTPase (guanosine triphosphatase) activating protein (GAP) for rapl, a Ras-related GTPase. A coiled coil region at aminoacid positions 346-371 has recently been recognised which interacts with the coiled coil region of hamartin (van Slegtenhorst et al., 1998). Variable splicing of exons 25 and 31 occurs (Xiao et al., 1995; Xu et al., 1995). It is widely expressed: the transcript has been found in all tissues and cell lines tested (e.g. brain, kidney, skin, liver, adrenal, colon and white cells) and in developing rat brain from day 14 onwards (Geist et al., 1996; Kerfoot et al., 1996). High levels of expression have been shown in olfactory bulb, brain stem nuclei, cerebellar Purkinje cells, anterior horn motor neurons and small blood vessels of many organs (Wienecke et ah, 1997). Detailed immunochemistry shows co-localisation of tuberin and its proposed target protein rapl in the perinuclear Golgi apparatus (Wienecke et al., 1996). The demonstration of inactivating germline TSC2 mutations and of loss of heterozygosity for 16pl3 markers in 50% of TSC-associated hamartomas from patients suggested that tuberin might act as a tumour suppressor, and that these growths arose by sequential mutation in concordance with the Knudson hypothesis (in TSC2, at least) (Knudson, 1971; Green et al., 1994). Further evidence to support this role of tuberin has arisen from the Eker rat and tissue culture experiments. The Eker rat has an autosomal dominantly inherited susceptibility to renal cell carcinoma, due to a mutation in the rat homologue of TSC2 (Yeung et al., 1993; Yeung et al., 1994; Kobayashi et al, 1995). Rats homozygous for the Eker rTsc2 m utation die in utero. A TSC2 transgene composed of the C-terminal 760 aminoacids (exons 27-41) containing the rap-GAP domain is able to rescue this embryonic lethal phenotype and suppress ethylnitrosourea-induced renal carcinogenesis (Orimoto et al, 1996; Kobayashi et al, 1997). In addition, wild type tuberin can suppress anchorage-independent growth of TSC2 mutant cell lines and reduce tumour formation by these lines in SCID mice (Jin et al, 1996). Furthermore, reduction of tuberin expression with antisense oligonucleotides prevents normal GO arrest of normal cell lines when serum is withdrawn from culture medium and the cells re enter the S phase of the cell cycle (Soucek et al, 1997). This evidence has Ch. 4 Discussion 248 led to the suggestion that tuberin may act as a growth regulator by indirectly reducing cyclin D1 expression. Tuberin immunoprecipitation in vitro inactivates rapla but not other rap like molecules including rap2, H-ras and rho which indicates that the C- terminal region of homology with a GTPase activating protein for the GTP binding protein rapl is functional (Wienecke et ah, 1995). However, tuberin may not only bind rapl: there is evidence that the C-terminal part of the molecule may also interact with rabaptin 5, a cytosolic protein that is an effector which binds the GTPase-activating protein rab5 (Xiao et ah, 1997). Rab5 is one of a number of small GTPase molecules involved in the control of endocytosis and intracellular vesicular trafficking. Binding of rabaptin to tuberin may approximate rab5 to the tuberin GAP domain, reduce rab5 activity and reduce the rate of fluid phase endocytosis. Fibroblasts from Eker rat homozygote embryos show abnormally increased endocytosis which is normalised by the introduction of tubulin (Xiao et ah, op. cit.). Increased endocytosis in TSC may enhance growth factor internalisation and distribution down the axon (Grimes et al., 1996), which in turn may permit abnormal neuronal migration or inappropriate cell survival and so contribute to the heterotopias which are a feature of TSC. TSC1, hamartin Deletions or rearrangements similar to those which led to the identification of TSC2 have never been seen in patients with tuberous sclerosis linked to TSC1. This, together with conflicting data from recombinant individuals, delayed the cloning of TSC1. The gene was eventually located by systematic mutation screening of transcripts and putative genes identified within a 900 kb region of a cosmid, PAC and BAC contig (van Slegtenhorst et ah, 1997). The TSC1 gene extends over about 53 kb of genomic DNA. It has 23 exons and encodes an 8.6 kb messenger RNA which includes 4.5 kb of 3' untranslated sequence. The 21 coding exons predict a protein of 1164 aminoacids with a calculated molecular mass of 130 kDa. Computer analysis of the sequence suggests that the protein, hamartin, has a single transmembrane domain in exon 6 and an Ch. 4 Discussion 249 extensive coiled coil region near the C-terminus. There was no sequence homology to any vertebral protein, but there was a possible match with a predicted 103 kDa S. pombe protein; a homologue in the Drosophila sequence database has been recognised recently (Povery, pers. comm). Northern hybridisation analysis has shown a major 8.6 kb signal expressed in all fetal and adult tissues tested. Variant splicing has not been recognised but minor hybridisation signals of 2.5 and 4 kb can be seen on Northern blots. New insights into the function of both hamartin and tuberin have been provided by elegant experiments in which the proteins were shown to exist as a complex (van Slegtenhorst et al, 1998). Yeast two-hybrid studies using tuberin and hamartin contructs demonstrated that the newly- recognised coiled-coil domain in tuberin interacts with the equivalent domain in hamartin. The two proteins were shown by immunofluorescence microscopy to co-localise in unidentified discrete cytoplasmic structures (in addition to more diffusely within the cytoplasm) when co-expressed in mammalian cells. If expressed singly, hamartin is associated with the structures only, whereas tuberin is not, and shows the diffuse cytoplasmic pattern. Co-expression of hamartin and control proteins containing coiled coil domains did not show localisation within the cytoplasmic structures. Association between tuberin and hamartin was also demonstrated in vivo by co-immunoprecipitation from HeLa and fibroblast lysates: hamartin could be recovered from the immunoprecipitates of antisera specific for tuberin, and vice versa. The failure of formation of a functional protein complex due to inactivation of either hamartin or tuberin may be the common step in the molecular pathogenesis which contributes to the shared TSC phenotype. TSC1 and TSC2 mutational spectrum The cloning of both TSC1 and TSC2 permits systematic mutation analysis in affected individuals and their relatives. At a recent NTSA-sponsored conference over 300 distinct mutations in 389 individuals or families were collated from publications, the two mutation databases and personal Ch. 4 Discussion 250 communication (Kwiatkowski, 1998). These are summarised in the Table 4.1 below (taken from the report). Tvoe of m utation TSC1 No. % TSC2 No. % Large deletions/rearrangements 3 2% 43 17% Small deletions and insertions 77 56% 83 33% Point mutations: Nonsense 52 37% 50 20% Point mutations: Splice site 7 5% 16 6% Point mutations: Missense 0 58 23% Total 139 250 Table 4.1: Type of m utation seen in patients with TSC1 and TSC2. Key: No.: number of mutations seen. The different types of mutation found in TSC1 and TSC2 are intriguing. 17% of mutations identified in TSC2 are large deletions or rearrangements (> 1 kb) of which about half are very large (> 50 kb). 59% are small deletions or insertions, nonsense point mutations or splice site mutations which lead to truncation of the protein. The remainder, 23%, are missense mutations. In TSC1, however, large deletions account for only 3 of the 139 different mutations identified, and in each case the deletion was <4 kb. The majority of the mutations in TSC1 are due to small deletions or insertions, nonsense point mutations or splice site mutations. In this series, no missense mutations were recorded. The majority of studies have used a combination of PFGE and Southern analysis to detect the large deletions and rearrangements in the TSC2 gene coupled with one or both of SSCP or heteroduplex analysis to identify the Ch. 4 Discussion 251 small deletions, insertions and point mutations. When SSCP and heteroduplex analysis are performed together the predicted sensitivity is 75 - 90% of all small mutations (Kwiatkowski, 1998). Assuming that both TSC1 and TSC2 are tumour suppressor genes and that the mutation results in a loss of function, the protein truncation test may be the most rapid method of detecting the smaller mutations in TSC2 and the majority of m utations in TSC1 (Roest et al., 1993; van Bakel et al., 1997). PTT has the great advantage over SSCP of allowing analysis of fragments of RNA up to 2 kb as opposed to the 150 - 250 bp size range of SSCP. The TSC2 mRNA can be screened in only three PCR reactions compared with the large number of small PCRs required to cover TSC2 by SSCP or heteroduplex analysis. PTT may also be the method of choice for mutation analysis in TSC1 if the rarity of missense point mutations is confirmed, although conventional SSCP analysis of TSC1 is relatively easier than for TSC2. The sensitivity of the test is not defined (van Bakel et al. provide a crude estimate of 60%) and it may be difficult to perform on direct lymphocyte RNA preparations because of the low abundance of TSC2 mRNA in this tissue, however. In pooling mutation data such as the table above it is difficult to avoid ascertainment bias (in that patients who are more severely affected may be preferentially ascertained and will skew the observed mutation spectrum). One study has attempted mutation detection in 171 sequentially ascertained, unrelated cases, of whom 147 were sporadic and 24 familial (Jones et al, 1997). All 21 coding exons of TSC1 were screened by SSCP and heteroduplex analysis. No gross gene rearrangements were seen by pulsed field gel electrophoresis. Twenty two pathological mutations were identified, 19 of which were truncating (12 small rearrangements, 7 nonsense substitutions), and 2 involved splice-sites. One was initially reported as the sole missense mutation to be identified by any group in TSC1 (although three missense polymorphisms were identified in TSC1 coding regions in other individuals including non-TSC controls). It occurred in a sporadic case, it was not present in either parent and polymorphic marker studies confirmed the expected paternity. However, Ch. 4 Discussion 252 only about a quarter of the TSC2 coding sequence was screened by SSCP and heteroduplex analysis so the possibility remains that an unidentified mutation in the unscreened TSC2 exons was responsible in this patient. An abstract describing this work suggests that this mutation has been withdrawn (Sampson et al, 1998). In contrast to TSC1, 19 large deletions or rearrangements were seen by PFGE or Southern hybridisation. Twenty eight further mutations were detected by the screens of 11 of 41 TSC2 exons. In total, TSC2 mutations were found in 45 sporadic and 2 familial cases, whereas TSC1 mutations were seen in 13 sporadic and 9 familial cases: TSC1 mutations were significantly under-represented among sporadic cases, confirming the similar finding of the TSC1 Consortium (van Slegtenhorst et al, 1997). This could be the result of higher locus- specific mutation rates at the TSC2 locus; an alternative or additional explanation may be that mutations in TSC2 may be more severe than those in TSC1 (contributing to ascertainment bias and an under representation of TSC1 cases). Jones et al also demonstrated that mental handicap was significantly more common in sporadic cases with TSC2 mutations (33/45) compared with those with TSC1 mutations (4/13). The odds ratio increased when probands were excluded from the analysis. In TSC1, there was a non-significant trend for 3', but not 5', truncating mutations to be associated with mental handicap, which might indicate that mutations which result in a truncated protein may exert a dominant- negative effect. Genotype - phenotype correlation Significant differences between TSC1 and TSC2 phenotypes have not been observed in familial cases (Povey et al, 1994b) with the exception of the contiguous gene syndrome involving TSC2 and PKD1 (adult-onset polycystic kidney disease, PKD) at chromosome 16pl3.3 (Brook-Carter et al, 1994). The large genomic deletions or rearrangements which are a frequent mutation in TSC2 often involve PKD1 which lies immediately centromeric or 3' to TSC2. This results in a syndrome in which there are features of both TSC and PKD, clearly defined in a recent study . Of 27 unrelated patients with both TSC and renal cystic disease, 25 had large Ch. 4 Discussion 253 deletions which involved both genes in 22. Seventeen had accelerated PKD and developed renal failure by 20 years of age. Mosaicism in TSC Mosaicism for the TSC2-PKD1 deletion was observed in 4 of the 27 patients with the combined presentation in this study and in 3 parents. In these 7 individuals the frequency of the mutated allele varied from 15 - 54% in lymphocytes. Severity of TSC did not differ significantly between mosaic and non-mosaic patients but renal function may have been better preserved compared with those with non-mosaic deletions. Mosaicism has been observed in other unpublished series (Kwiatkowski, 1998). The occurrence of low-level mosaicism with typical severe TSC may contribute to the failure of mutation detection (including complete sequencing of all exons from both genes) if the mutation is not represented in the white blood cell lineage. Biopsy and fibroblast culture from skin lesions (such as angiofibromas or peri-ungual fibromas) may be required as an additional source of DNA for mutation analysis in these patients. Germline mosaicism There is increasing evidence from linkage and molecular studies to indicate that gonadal or germline mosaicism does occur. The recent NTSA conference was aware of 10 families in which there are two or more affected children in whom a TSC2 mutation has been defined where neither parent has any sign of TSC on extensive examination and the mutation has not been found in DNA from parental blood (Kwiatkowski, 1998). Linkage analysis indicates that gonadal mosaicism may occur in either sex, but definitive molecular confirmation will require single sperm PCR or ovarian biopsy. TSC3? There is now little evidence for a third tuberous sclerosis locus. The majority of families are linked to either TSC1 or TSC2 (Kwiatkowski et al., 1993; Povey et al, 1994a; Povey et al, 1994b). Of the three "problem families" in the Galton MRC series where haplotype analysis gave Ch. 4 Discussion 254 conflicting results with markers in both TSC1 and TSC2 regions (Povey et al., 1994b), two have now been shown to have mutations in TSC1 and in the third family the clinical phenotype is confused (Povey, personal communication). Several large mutation detection studies have been published or are in progress (Wilson et al., 1996; Jones et ah, 1997; van Bakel et al., 1997; Au et al., 1998; Young et al., 1998). None have been able to identify mutations in all familial or sporadic cases at either gene. The Cardiff study (Jones op. cit.), which is one of the largest, has now identified mutations in about 140 of their 171 unrelated patients. It is likely that the failure to detect mutations in the remaining 30 patients is due to the technical problems of screening the 41 coding exons of the TSC2 gene. In the 32 Galton MRC families mutations have been found in all the TSC1- linked families. Of the remainder, 10 families are clearly linked to TSC2, and in these a definite or putative pathological mutation or polymorphism has been found in all but one family. It is theoretically possible that mutations in a third TSC gene might invariably lead to such a severe phenotype that the reproductive success is nil (so familial cases would be very rare). It would not be possible to identify this locus by positional cloning if mutations are rare and the majority of patients are sporadic cases. However, candidate loci may emerge as proteins involved with tuberin and hamartin in the cellular pathways described above are identified (such as rapl, rabaptin 5 and rab5). If well documented "problem families" or sporadic individuals remain after comprehensive mutation analysis of TSC1 and TSC2 then genes for these proteins could be screened for mutations. Ch. 4 Discussion 255 0G ■ H CD P P a ■ H £ ■ H ^ - k CO - H G p CO CD P (D u CD P — CO 4*! 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Primary features: CNS cortical tuber (histological confirmation) subependymal nodule or giant cell astrocytoma (histological confirmation) multiple calcified subependymal nodules protruding into the ventricle (radiological confirmation) Retina multiple retinal "astrocytomas" or hamartomas* Skin facial angiofibromas* multiple ungual fibromas* Secondary features History affected first-degree relative CNS cortical (or extra-cortical) tubers (radiological confirmation) non-calcified subependymal nodules (radiological confirmation) giant cell astrocytoma (radiological confirmation) Retina single retinal hamartoma, or achromic patch or patches* Skin shagreen patch* forehead plaque* Kidney renal angiomyolipoma (radiological or histological confirmation) (multiple) renal cysts (histological confirmation) Heart cardiac rhabdomyoma (radiological or histological confirmation) Lung pulmonary lymphangiomyomatosis (histological confirmation) Appendix 1 Diagnostic criteria for TSC 297 Tertiary features History infantile spasms CNS cerebral white-matter "migration tracts" or "heterotopias" (radiological confirmation) Skin hypomelanotic macules* "confetti" skin depigmentation Kidney renal cysts (radiological confirmation) Lung pulmonary lymphangiomyomatosis (radiological confirmation) Teeth randomly distributed enamel pits in deciduous and/or permanent teeth Gingiva gingival fibromas* Rectum hamartomatous rectal polyps (histological confirmation) Bone bone cysts (radiological confirmation) Others hamartoma/s of other organs (histological confirmation) Table A.1.1: Diagnostic criteria for TSC. The Gomez criteria modified by the National Tuberous Sclerosis Association (NTSA) Professional Advisory Committee. Adapted from Gomez, 1988a and Roach et al., 1992. Key: • histological confirmation is not required if the lesion is clinically obvious. The principal differences between the Gomez Criteria and the NTSA revision (Gomez, 1988a; Roach et al., 1992) are as follows: (1) the recognition that with some features, histological evidence provides a more secure diagnosis than radiological evidence alone. Hence, histological evidence is required in order that cortical tubers, subependymal nodules and giant cell astrocytomas may be admissible as primary features. Similarly, where renal cysts or pulmonary lymphangiomyomatosis exist, radiological evidence alone is a tertiary feature, whereas histological confirmation promotes each to a secondary feature. (2 ) the relegation of the fibrous forehead plaques and multiple renal angiomyolipomas from primary to secondary features (even when Appendix 1 Diagnostic criteria for TSC 298 histology is available), confetti-like hypopigmentation from a secondary to a tertiary feature, and the removal of seizures (other than infantile spasms) as a tertiary feature. This takes account of the lack of specificity of each of these signs: e.g., angiomyolipomas are relatively common in the general population (albeit usually solitary). (3) the admission of significant family history of TSC as a secondary feature. (4) the attempt, for the first time, to define what combination of features constitutes a diagnosis, with differing levels of certainty. In the consensus report published in the previous year, Paul Rosman had stated, "In certain circumstances, the combination of two or more or these secondary (or non-definitive) criteria may be sufficient to establish a diagnosis of TSC with confidence." (Rosman, 1991). However, these circumstances were not delineated and the opportunity for ensuring international consensus was not pursued. The NTSA attempt may be criticised (does a single cardiac rhabdomyoma plus renal and bone cysts really constitute sufficient grounds for a definitive diagnosis of TSC in the absence of a family history?) but it has provided useful guidance for paediatric neurologists and geneticists who may not see patients with TSC frequently and it has formed a basis for discussion between clinicians and molecular biologists of patients entered into TSC mapping studies. The emphasis of the revision on histological confirmation is understandable, but could be criticised if it leads to over-invasive investigation (brain biopsy) simply to reach a definite diagnosis of TSC. Using these criteria (but avoiding biopsy), it is difficult to make a definite diagnosis in infancy, since several primary and secondary features do not appear until late childhood or adult life (facial angiofibromas and periungual fibromas, renal angiomyolipomas). This may encourage those who enroll patients in genetic studies to accept "probable TSC" as equivalent to affected in order to increase numbers, with the risk of misclassification. This pitfall is well-recognised, however, and its avoidance requires regular clinical review of all at-risk individuals in Appendix 1 Diagnostic criteria for TSC 299 family studies classified as probable, suspect or unaffected. Three neuropathological lesions are characteristic of TSC: cortical tubers, subependymal nodules and giant cell astrocytomas. The tuber from which TSC is named is a hamartoma of cortical tissue composed of small stellate neurons and astroglial cells. It is thought to be a "rest" or residua of prim itive tissue which has not differentiated properly (Gomez, 1992). Numbers of tubers in each patient are variable and range from zero to several dozen. Periventricular subependymal nodules or gliomas commonly abut the anterior half of the lateral ventricle and are very common (90% of patients). They consist of glial and vascular elements (but may contain large cells similar to those found in giant cell astrocytomas). Cerebellar subependymal nodules are found in 25% of patients. Both subependymal nodules and tubers may eventually calcify. The third type of lesion is the giant cell astrocytoma. This is a histologically benign tumour which develops in 6-14% of patients. It causes symptoms by enlargement leading to obstruction of the ventricular system (e.g. the foramen of Monro) and hydrocephalus. Increase in size may be gradual (due to hyperplasia during the first or second decade of age) or acute (because of haemorrhage within the tumour). Even in the absence of the lesions described above, the cerebral cortex may be abnormal. Failure of migration of neurones during formation of the cortex may lead to areas of hypoplasia, beneath which lie islands of heterotopic grey matter with little myelin formation. Cortex which looks normal on MRI or at autopsy may show neuronal disorganisation and gliosis on histological examination. These more subtle lesions may explain the behaviour problems or impaired intellect in those patients who do not have obvious abnormalities on cranial CT or MRI. Neurological outcome in TSC is probably determined by a combination of the age at onset of epilepsy and the response to treatment, and the number, size and location of the cerebral lesions. Anecdotal evidence suggests that the onset may be precipitated by a febrile convulsion, and parents must be given advice about cooling of the febrile infant. There is no clear Appendix 1 Diagnostic criteria for TSC 300 consensus about whether infants with TSC should be given pertussis (whooping cough) vaccine, although the British National Formulary recommends avoidance where there is evidence of an "evolving neurological problem" (George, 1996). In the general population, neurological complications are much more common as a sequella of pertussis infection than after vaccination. However, with increasing "herd" immunity where the majority of normal children receive vaccination, children with TSC are at relatively low risk of infection, and parents of children with multiple cerebral lesions could be advised to postpone vaccination until late childhood, or to omit it altogether. Older children and adults may develop complex partial or other focal seizures, having had more generalised attacks in childhood. The dermatological features of TSC can be divided into depigmentary lesions ("ash-leaf" patches, confetti-like spots and other hypomelanotic macules) and hamartomas (both primary skin signs and shagreen patches and forehead plaques). Bourneville first thought that the facial angiofibroma in his female teenage patient was acne rosacea, which it resembles. Also known as adenoma sebaceum, it arises in a similar "butterfly" distribution over the nose and adjacent cheeks, in the nasolabial folds and below the lower lip. It is present in 40-90% of patients with TSC, and usually appears in mid-childhood (and rarely after puberty in contrast to acne rosacea or systemic lupus erythematosis) (Fryer, 1991; Gomez, 1991). The fibrous plaque is histologically similar to the facial angiofibroma. This secondary feature of TS is not common (25%); it may be present from birth as a solitary, slightly, elevated, reddish-orange patch on the forehead, face or upper eyelid. Plaques on the scalp are hairless and surrounded by thin, white tufts of hair. Periungual fibromas are fleshy outgrowths of skin and subcutaneous tissue which arise adjacent to finger- and toe-nails during and after puberty in 30% of patients. Nail growth may be affected, and ridging of the nail may occur in the absence of an obvious fibroma. "Shagreen" patches are areas of discoloured skin which feel like untanned leather with a rough, granular surface or "permanent gooseflesh", and are present in 25%. Large, single lesions may be found in Appendix 1 Diagnostic criteria for TSC 301 the lumbosacral region, and smaller areas elsewhere. The most common skin sign in TSC is hypopigmentation: almost all patients have several macules when examined with long-wavelength ultraviolet light (a Wood's lamp) and these may be present from birth. Similar hypopigmentation is not uncommon in the general population and vitiligo or nevi depigmentosi may be confused with lesions in TSC, both clinically and histologically, hence hypopigmentation alone is only a "suggestive" diagnostic feature. The 1998 NTSA Annapolis conference has revised this criterion to require three or more macules at least 5 mm diameter (S. Povey, personal communication). "Showers" of small, hypopigmented macules called confetti spots are more characteristic of TSC (although this is not accepted in the NTSA revision of the Gomez criteria, see above). Primary or secondary hamartomatous lesions which may also be found on examination or investigation include retinal phakomas, cardiac rhabdomyomas and renal angiomyolipomas. Their significance depends on numbers of lesions found and their context: a single retinal hamartoma or "phakoma" is a secondary diagnostic feature, whereas multiple hamartomas are a primary feature. These lesions may be obvious on direct ophthalmoscopy as a mulberry-like mass protruding into the posterior chamber, or an indistinct semi-transparent plaque in the periphery. Reported prevalence varies between zero and 87%, which probably reflects the examination technique used, the expertise of the examiner and whether the study was performed in a randomised, single blind fashion. Hypopigmented retinal patches have also been found in some patients. Cardiac rhabdomyomas are common and their prevalence is inversely related to the age of the TSC patient: they are present in about 60% of children but less than 20% of adults (Fryer et al., 1990). They have been detected in utero at 22 weeks gestational age and they may regress rapidly after birth (Webb et al., 1993). Only a minority are symptomatic, resulting in hydrops fetalis, cardiac failure in infancy or cardiac murmurs or Appendix 1 Diagnostic criteria for TSC 302 dysrrythmia in older children or adults. Among children who present w ith cardiac rhabdomyomas, 80% prove to have TSC (Webb, op. cit.). In infants they may be evident on cardiac ultrasound before any of the primary intracranial radiological signs become apparent (because of normal delay in myelination), and if present can lend support to a diagnosis of TSC in an infant presenting with infantile spasms ("probable TSC", NTSA revision). Renal angiomyolipomas are histologically benign tumours present in between 50% and 65% of patients with TSC; they are more common in women. They contain various tissue elements including blood vessels, smooth muscle, fibrous tissue and fat. In contrast to cardiac rhabdomyomas, the prevalence of angiomyolipomas increases with age, and it is not uncommon to find bilateral tumours or multiple foci in one kidney in TSC adults. These tumours are often asymptomatic, or they may cause loin pain, haematuria, hypertension or renal functional impairment. There have been several reports of retroperitoneal haemorrhage arising from the vascular elements; this may lead to exsanguination and death if not recognised and treated urgently (Bernstein et al., 1991). Solitary renal angiomyolipomas occur in the general population, with a preponderance in women, but are indicative of TSC if multiple and present together with renal cysts (Bernstein, op. cit.). Patients with TSC and angiomyolipoma have a 7% greater risk of developing renal cell carcinoma than individuals with angiomyolipoma who do not have TSC (Bernstein, 1996). This is of interest given the increased susceptibility to renal carcinoma of the Eker rat, which has a mutation of the rodent homologue of TSC2 (Kobayashi et al., 1995). Renal cysts occur in about one-fifth of patients with TSC and they are usually small and asymptomatic. Solitary cysts occur are common in individuals without TSC and so they are not a very helpful diagnostic marker, (unless present together with angiomyolipomas). Multiple, large cysts may be a presenting feature of TSC in infancy or childhood and polycystic kidney disease has been misdiagnosed on the basis of the ultrasound appearances (Bernstein et al, 1991). In retrospect, these Appendix 1 Diagnostic criteria for TSC 303 individual cases might have suggested the chromosomal location of the TSC2 gene at 16pl3 adjacent to the PKD1 locus. TSC2 was eventually mapped by a random total genome search in TSC families not linked to TSC1 on chromosome 9q34 (see below). The other diagnostic features listed in Table A. 1.1 above are either rare (pulmonary lymphangiomatosis, angiomyolipomas of organs other than kidney) or relatively common in the general population (e.g. periosteal thickening, bone cysts, dental pits). They are reviewed in Gomez, 1988a. Appendix 1 Diagnostic criteria for TSC 304 Appendix 2: Exploitation of selected irradiation hybrids to isolate markers in the region of the AT locu s This work was performed by Drs Bird and McConville, Birmingham and is included in (Gillett et al., 1993). DNA (0.1 mg) from six of the radiation hybrids containing fragments of chromosome 11 including the llq22-23 region (Jo 6 ', 12, 13', 31, 33, 48' was (separately) amplified using the hum an specific Alu oligomer 451 (Dorin et al, 1992). After a primary round of 37 cycles the reaction product was diluted 1:1000 and 1 microl. of the diluted product was reamplified using the same primer and conditions. The product of the second round of amplification was end-filled and kinased (using Klenow enzyme and T4 polynucleotide kinase in 50 mM Tris-HCl pH 7.6, 10 mM MgCl 2 , 5 mM DTT, 10 mM ATP and 1 mM dNTPs). Nucleotides and primers were then removed by chromatography in Qiagen-20 columns. The blunt-ended Alu PCR fragments were ligated into phosphatased, Smal-cut pBluescribe and cloned in E. Coli DH5alpha. As a preliminary characterisation, 30 recombinant clones were selected at random from a library derived from hybrids 12, 13', 33 and 48'. The 5' end of the Alu primer 451 is 56 bp from the 3' end of the Alu sequence and is oriented to amplify outwards from the 3' tail of the repeat. All clones were therefore expected to contain 56 bp of Alu sequence at both 5' and 3' ends of the clone. Clones with inserts of less than 300 bp were discarded, since it was felt that single copy sequence of less than 189 bp would be insufficient to generate useful chromosome 11 markers. Plasmids were digested with BamHI and EcoRI or PstI and SstI to release the insert as a single fragment and the digests were electrophoresed through 3 % agarose. Insert sizes (determined by comparison with a standard size marker) ranged from less than 100 bp to 1700 bp, with an average size of approximately 400 bp. Fourteen clones (47 %) were of at Appendix 2 Isolation of markers in the region of the AT locus 305 lleast 300 bp in size, and 12 of these were analysed further. Restriction maps were prepared for each clone to enable identification of fragments llargely free from Alu sequence or other internal repeat sequences. Nine out of the twelve inserts contained single copy fragments which gave strong signals with low background when hybridised to genomic DNA. The other three gave little or no specific signal. To increase the efficiency of isolation of recombinants free from internal repetitive sequence, a further series of clones was isolated. In this experiment recombinant colonies were inoculated into 96 well plates and grown in 150 microl. LB containing 50 mg/ml ampicillin. Gridded arrays of colonies from the 96 well plates were made on Hybond N filters using a 96 prong replica plating tool. Colonies gridded onto the filters were lysed, denatured and hybridised with a 177 bp SauIIIa fragment from the Alu repeat (derived from a region 5' to that from which the 451 primers were selected), and with a 1.2 Kb LI repeat fragment to identify and exclude clones containing Alu and/or LI repetitive elements. This screen led to the isolation of a further seven clones (IB3, IB4, IC2, ID3, IE2, 2A2 and IH4). A wide range of sizes of insert was obtained in these experiments, but seven were between 650 and 730 bp (six from Jol2 and one from Jo48'). The degree of clone duplication was assessed by one or more of the following methods: comparison of restriction maps, cross-hybridisation between clones, the pattern of hybridisation to genomic DNA digested with a number of restriction enzymes and sequencing. The insert derived from Jo48! was unique. Six clones of similar size derived from Jol2 (J12.5, J12.6, J12E, J12EF-B2, J12EF-B11 and J12/IB3) were shown to contain overlapping, but not identical sequences. Clones J12.5 and J12EF-B11, for example, hybridised to identical fragments in human genomic DNA digested with EcoRI, MspI or PvuII. J12.6 hybridised to the same EcoRI and MspI fragments, but to a different PvuII fragment. J12.6 and J12EF-B11, but not J12.5, hybridised to the same Pst fragment. A probe prepared from J12.6 which did not contain Alu sequences hybridised to J12.5, J12EF-B2 and J12EF-B11 (but not J 12.8 and J13EF-C10). Appendix 2 Isolation of markers in the region of the AT locus 306 It is unlikely that these larger inserts arose as a result of coligation events since all hybridised to a single band in genomic DNA and all mapped to the same region of llq23. The shortest of this group of clones, J12.5, had Alu sequence at each end, as expected. It is possible that the larger clones were generated either by amplification from adjacent Alu-like sequences or from adjacent non -Alu target DNA with chance homology to the 451 primer sequence. J12/1C2 and J12/1D3 also showed very similar hybridisation patterns suggestive of significant overlap, despite the inserts differing in size by about 200 bp. In this case the discrepancy in size appears to have been due to the cloning procedure. DNA sequencing showed Alu sequence at each end of the larger insert (clone J12/1D3) but at only one end of the smaller (J12/1C2). J12/1C2 was identical to J12/1D3 at the Alu end but terminated within the unique sequence of J12/1D3. In summary, of the fourteen clones selected as having single copy inserts, nine were unique: six from Jol2, two from Jol3' and one from Jo48'. Electrophoresis through agarose of Alu PCR products generated from Jol2 using primer 451 and visualisation using ethidium bromide showed about ten visible bands. This suggests that at least half of the products from this hybrid were cloned as single copy sequence. Regional localisation of these nine Alu PCR clones was determined by hybridisation to a panel of six somatic cell hybrids. This panel (comprising the hybrid lines M11X (Chiang et al., 1984), IB5 (Wadey et ah, 1990), PG7, PG9, PG48 (McConville et al., 1991), and A3EW2 (Guerts van Kessel et al., 1985; Sacchi et al., 1986)) divided chromosome 11 into seven "bins" or regions: llpter-pl3, Ilpl3-llq22, Ilq22-q23.1, Ilq23.1-q23.2, Ilq23.2-q23.3, Ilq23.3-q24.2, and llq24.2-qter). The mapping of the eight Alu PCR- derived inserts on this hybrid panel is shown in Table 4 of Gillett et ah, 1993 (attatched), and their regional localisation is summarised in Figure 2 of the same paper. All of the six probes from irradiation hybrid Jol2 mapped back to the regions of llq originally detected in the hybrid by marker analysis and by FISH. The map positions of these were distributed Appendix 2 Isolation of markers in the region of the AT locus 307 between three intervals across the whole region from llq 22 to q23.3 (D11S35 to ETS). None of the inserts mapped to the region of lip detected in Jol2. Of the two clones from Jol3', J13'C10 proved to be of hamster origin, and J13'F mapped to llpl3-llcen, a region not detected in the marker analysis of this hybrid. J48'B6, mapped proximal to llq22, a region which was subsequently shown to be contained in this hybrid. The two most promising markers, J12.8 (D11S535) and J12.1C2 (D11S611) were further investigated by the Birmingham Department of Cancer Studies (McConville et al, 1993). A biallelic restriction fragm ent length polymorphism (D11S535) or a temperature gradient gel electrophoresis polymorphism (D11S611) was identified for each marker and pairwise linkage analysis confirmed that the markers were located close to existing markers in the AT region. The heterozygosity of these polymorphisms was between 0.40 and 0.44; in order to increase the information available from these loci a further RFLP (D11S611) and TGGE (D11S535) were identified by screening end-fragments of YACs containing the two marker sequences. Surprisingly, at each locus the two systems showed little linkage disequilibrium, despite being physically and genetically linked. When combined into a haplotype, the heterozygosity was significantly im proved (D11S535 0.69, D11S611 0.63). A series of three point analyses in AT families using markers in the AT region together with D11S611 and D11S535 allowed AT to be placed in an interval of 4.1 cM flanked by D11S611 (centromeric) and D11S384/D11S535 (telomeric). The odds against placement in the more proximal or distal intervals was 9 x 1012:1 or 6470:1 respectively. Haplotype analysis in individual families confirmed a location for AT distal to D11S611 and proximal to D11S384/D11S535. There were no families (of any complementation group) in which recombination placed the AT locus outside this interval. These findings reduced the size of the existing candidate region by more than half. The computed maximum location score for the AT locus favoured the more distal part of the interval, approximately 1 cM centromeric to D11S384/D11S535. This estimate was confirmed when ATM was identified: the 5' end of the coding sequence lies 1.04 Mb Appendix 2 Isolation of markers in the region of the AT locus 308 centromeric to D11S535 (Savitsky et al., 1995). Neither the linkage nor the haplotype analysis provided any evidence to refute the assumption that only one AT locus was segregating in the AT families studied. This assumption was also confirmed by the cloning of ATM, a single gene in which mutations were identified in patients previously assigned to each of the four complementation groups (A, C, D and E). Appendix 2 Isolation of markers in the region of the AT locus 309 Appendix 3: Standard solutions, buffers, media and primer sequences Standard solutions and buffers Church hybridisation buffer 0.5 M NaPi pH 7.2 (iu\), 7 % SDS, 1 mM EDTA, 1 % albumin. Denaturing solution 1.5 M NaCl, 0.5 M NaOH Formamide stop solution 98 % deionised formamide, 10 mM EDTA pH 8.0, 0.025 % w /v bromophenol blue, 0.025 % xylene cyanol. Hybridisation buffer 1 M NaCl, 10 % w /v dextran sulphate, 1 % w /v SDS. Loading buffer 0.25 % w /v bromophenol blue, 0.25 % w /v xylene cyanol; 25 % w /v Ficoll in distilled water. The ficoll was dissolved in water at 65 °C before addition of the dyes. Lysing solution 0.5 % SDS, 100 p g /m l proteinase K in STE. Neutralising solution 1.5 M NaCl, 0.5 M Tris-HCl pH 7.5. 10 x PCR buffer 100 mM Tris-HCl pH 9.0, 500 mM KC1, 15 mM MgCl 2 , 0.1% gelatin, 1% Triton X-100. The final PCR mix contained 10 % glycerol, in addition. Phosphate-buffered saline, PBS 150 mM NaCl, 10 mM Na2 HPC>4 / NaH 2 PC>4 pH 7.4. For 200 ml 1 x PBS one tablet (Sigma) was added to 200 ml dd water. Appendix 3 Standard solutions, buffers, media and primer sequences 310 RNase A This was purchased in a dessicated form and made up to a stock concentration of 10 mg/ml in sterile dd water. The solution was boiled for 10 minutes to destroy DNase activity and stored at -20 °C. 1 M sodium phosphate solution pH 7.2 Prepared by titration of 1 M solutions of Na2HPC>4.12H20 and NaH2P0 4 .2H2 0 . ssc 150 mM NaCl, 15 mM sodium citrate pH 7.0. STE 0.1 M NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0. TAE 40 mM Tris-acetate pH 8.0, 40 mM sodium acetate, 1 mM EDTA. TBE 90 mM Tris-HCl pH 8.0, 90 mM boric acid, 2 mM EDTA. TE 10 mM Tris-HCl pH 8 , 1 mM EDTA pH 7.6. TNE 10 mM Tris-HCl pH 8 , 100 mM NaCl, 1 mM EDTA pH 8.0. TKM 10:10:1 10 mM Tris pH 7.5, 10 mM KC1, 1 mM MgCl 2 - Bacterial cell culture Am picillin Prepared to a stock concentration of 50 m g/ml using sterile dd water, filter sterilised (pore size 0.22 pm) and stored at -20 °C. Working concentration 50-100 pg/ml. Kanamycin Prepared to a stock concentration of 25 m g/ml using sterile dd water, filter sterilised (pore size 0.22 pm) and stored at -20 °C. Working concentration 12.5-25 pg/m l. Appendix 3 Standard solutions, buffers, media and primer sequences 311 LB (Luria Bertani) medium 1 % w /v tryptone (Bacto, Difco), 0.5 % w /v yeast extract (Bacto, Difco), 1 % w /v NaCl. LB agar LB medium with 1.5 % w /v agar. LB glycerol medium LB medium with 5 % v/v glycerol. NZY agar NZY broth with 1.5 % w /v agar. NZY broth 1 % w /v NZ amine (casein hydrolysate); 0.5 % w /v yeast extract, 0.5 % w /v NaCl, 0.2 % w /v MgS 0 4 .7 H 2 0 . Adjust to pH 7.5 with NaOH. SM buffer per litre: 5.8 g NaCl, 2 g M gS04.7H20, 50 ml 1 M Tris-HCl pH 7.5, 5 ml 2 % w /v gelatin. 2 x YT medium 1.6 % w /v tryptone, 1 % w /v yeast extract, 0.5 % w /v NaCl. Mammalian cell culture Amphotericin B Dissolve 100 mg amphotericin (Sigma) in 40 ml water, filter sterilise and freeze in 1 ml aliquots. Dilute one 2.5 mg aliquot in 100 ml medium (working concentration 2.5 mg/1). Ciprofloxacin Dissolve 100 mg ciprofloxacin (Bayer) in 20 ml water, filter sterilise and freeze in 1 ml aliquots. Dilute one 5 mg aliquot in 500 ml medium (working concentration 10 m g / 1). Eagle's minimal essential medium plus fetal calf serum (FCS/E) For 500 ml: 390 ml dd water, 45 ml Eagles MEM (lOx), 50 ml fetal calf serum, 5 ml GPS, 5 ml 100 x sodium pyruvate, 5 ml non-essential aminoacids and 5 ml Hepes (1 M). Correct to pH 7.6 with 1-2 ml NaHCC >3 / NaOH. Appendix 3 Standard solutions, buffers, media and primer sequences 312 Glycerol medium for freezing For 100 ml: mix together 72 ml dd water and 10 ml glycerol and sterilise by autoclaving. Add 8 ml Eagles MEM, 1 ml Hepes (1 M) and 20 ml fetal calf serum to the sterile bottle and bring to pH 7.2 with 0.25-2.5 ml NaHCC >3 / NaOH. GPS For 100 ml: 200 mM L-glutamine (ICN Labs), 600 mg penicillin (UCLH Pharmacy), 1 g streptomycin (Sigma). Freeze in 5 ml aliquots and dilute to a working concentration of 1 % v/v in medium. Hanks’ balanced salt solution For 300 ml stock solution: 32 g NaCl, 1.6 g KC1, 0.36 g Na2HP04.12H20, 0.24 g KH 2PO 4 , 4 g glucose, 0.08 g phenol red. Made to volume with dd water, autoclaved and stored at 4 °C. Working concentration: dilute 1 in 12 with dd water, autoclaved and stored at 4°C. Brought to pH 7.0 - 7.2 with NaHCC>3 / NaOH. Hypoxanthine-Methotrexate-Thymidine, HMT ”HAT" 100 x stock solution diluted to a final concentration of 100 pM hypoxanthine, 10 pM methotrexate and 16 pM thymidine. NaHC0 3 / NaOH For 100 ml: 5.6 g NaHC03, 6 ml 10 M NaOH. Mix thoroughly and sterilise by membrane filtration. PEG solution Add 5.5 g polyethylene glycol PEG 4000 (Merck) to 5 ml PBS, microwave to dissolve, filter sterilise (pore size 0.22 pm). RPMI - 1640 medium For 100 ml: 76 ml dd water, 9 ml RPMI (x 10), 10 ml fetal calf serum , 1 ml GPS, 1 ml non-essential aminoacids, 3 ml 5.3 % w /v NaHC03, 1-2 ml 1 M NaOH. Trypsin - Versene solution For 100 ml: 0.02 g EDTA in 100 ml sterile Hanks' balanced salt solution (working concentration). Before use add 5-10 ml 1% trypsin solution (ICN Labs) and correct pH with NaHC03 / NaOH. Appendix 3 Standard solutions, buffers, media and primer sequences 313 Solutions for FISH Deionised Formamide For 100 ml: 5 g ion exchange Amberlite monobed MB1 mixed resin was added to 100 ml formamide, sealed in a Duran bottle, protected from light and agitated for 2 hours. The solution was filtered twice through W hatm an 1MM paper and stored at -20 °C. Formaldehyde buffer 50 mM MgCl 2 in 1 x PBS. Hybridisation mix 50 % v/v deionised formamide 2 x SSC, 10 w /v dextran sulphate. The mix was incubated at 65 °C for 2 hours to ensure that the dextran sulphate was completely dissolved and then stored at -20 °C. KC1 / EDTA hypotonic solution For 100 ml: 0.3 g KC1, 0.02 g EDTA, 0.48 g Hepes pH 7.4. Sterilised by autoclaving and stored at 4 °C. 10 x Phosphate buffer 0.5 M N aH 2P04 / N a2HPC>4 pH 7. Proteinase K Purchased in a dessicated form and diluted in sterile dd water to a stock concentration of 50 jig/m l. Proteinase K buffer 20 mM Tris-HCl, 2 mM CaCl2 pH 7.4. Appendix 3 Standard solutions, buffers, media and primer sequences 314 Primer sequences Oligonucleotide PCR primers used. (Ta = annealing temperature, °C = Centigrade, Size: as cited in reference or by calculation from sequence). Locus Size, bp Position & Ta °C Reference Sequence 15' to 3'. forward above reverse! APOC3 715-730 61 (Gillett et al., 1993) TGG CCC AGC AGG CCA GGT ACA CCC G c.f. (Zuliani et al., 1990) GGA GGC CGA GGC AGG AGG ATC CCC CAT 199 intron XII - exon 13, 52 (Gillett et al., 1993) AAA TAT CAC GTT GCT GCC CAT GAG G GAA TCT TCA TCC AGT GAT GAG CGG G CD3D 382 intron II - exon 3, 55 (Cotter et al, 1989) GAG CTT AAC TCA GCA AGA CAG GAG CAG AGT GGC AAT GAC ATC CD5 480 3' untranslated, 70 (Richard et al., 1991) AGA AGG ATC CGC AGG GGT GGA TGC T ACT GGG ATC CAT GAG CAA AAA GCC G CLG 447 5' untranslated, 55 (Gillett et al., 1993) GTA CAG GAGCCG AAC AGC CAT CAG G c.f. (Theune et al., 1991) GCC TCT TGC TGC TCC AAT ATC CCA G CFN1 179 3' untranslated, 60 (Gillett et al, 1996) ATC CCC ATT CCC CAC CTG G TCC TGC TTC CAT GAG TAG CCG T COX8 179 3' untranslated, 70 (Richard et al., 1991) TCC CTC ACA CTG TGA CCT GAC CAG C GGG GAC CCC ACC AAG CAG GGT CAG T D11S35 152-162 38 (Litt et al, 1990) ACA ATT GGA TTA CTA CTA GC TGT ATT TGT ATC GAT TAA CC D11S144 c. 550 59 (Gillett et al, 1993) TTC CCC AGT AGC TTC TGT CTC CTG C TCA CTC CCA GTT CAG TAT CCT GTC C D11S146 c. 1200 55 pers. comm. S. Leigh Sequence not provided. & Prof. RV Thakker D11S385 286 50 (Gillett et al, 1993) TTT TAT AGG GAC AGG ATC TTG C GGC TGT ATA ATC TTG TGT TCT C D11S420 188-208 49 (Luo et al. 1990a) AGT TAC ACC GGT TCT GCA GA GAT TAA TGA TAG TGC TAT CC Appendix 3 Standard solutions, buffers, media and primer sequences 315 D11S490 147-167 54 (Luo et al., 1990b) CAC AAA CAT TGG CGC AT TTC TGG GTC ACG GTG CTT CA D11S527 142-166 60 (Browne et al., 1991) ATG CGG CTC CAA GAC AAG TTC GCC CCT CTA CTT GTC TGG AG D11S528 73-91 (Hauge et al., 1991a) AAT GGT GTC CCC ACA CAT GT TCC TAC CTA CCG AGC TTA AA D11S533 300-900 66 (Eubanks et al., 1991) GCC TAG TCC CTG GGT GTG GTC GGG GGT CTG GGA ACA TGT CCC C D11S534 228-244 58 (Hauge et al., 1991b) ATA TGG AAA CTC TCC GTA CT GCA ACC ATG GAC AGT CTG GA D11S614 c. 158 8D11,57 (Sugiyam a et al, 1991) CCC CAA AAC AGA CCC ACC AGG ACT TCT TCT CTG ACC CCG GAT GTC TGC DRD2 80-86 58 (Hauge et al., 1991c) CAG GAG CAC GTT TCT CAT AC GGA GGG CGG TGC GGT CAT FGF3 415 exon 3-3' untrans., 55 (Cotter et al., 1989) AGT TTG TGG AGC GGA TCC ACG AAG ATG TCG CCA GGA GCT CTG FTH1 173 62 (Richard et al, 1991) GTC AAA AGA CGC GGA CAG AA AAT GGC CGG TGA CAG GTG AA FUT4 585 3' untranslated, 68 (Gillett, unpublished) GGA GCC TTG TTG GTG GAG AGT GGA c.f. (Goelz et al, 1990) CAG CAC TCC TGC AGG ACT GGC A GST3 496 intron VI-3'UTS, 62 (Richard et al, 1991) GAG GTT CAG TAA ACA CAG CC TTA GCA AAG CAG AGC AGA CC HBB 398 flanking exon 2,55 (Abbott et al, 1991) ACT GGG CAT GTG GAG ACA GAG AAG A TGT ACC CTG TTA CTT CTC CCC TTC C KRN1 229 3' untranslated, 62 (Richard et al, 1991) AGT GGG AAA CCT CAG GTA GCT CCC GA CAG TTT GGC TCA GAC ATA TGG GGG CA N CA M 179 5' untranslated, 50 (Abbott et al, 1991) TGG AAA TCT CTT CCA AAC ATC GGA G AAT TAG AAC TTT GGA GAG GGA TGG G PGA 293 exon 9-intron I, 60 pers. comm. D. Ogilvie CTC CAG AGC GAG GGG AGC TGC ATC & RV Thakker CGT TAT TGG TAG GAC AGG GAA CAG Appendix 3 Standard solutions, buffers, media and primer sequences 316 PYGM 704 intron R-exon 20, 65 (Richard et al, 1991) TGC TCA TGC ACC ATG ACC GGT GAG C CCA AGA GAG TGT GAC AGA CTC AAG G SEA 130 62 (Richard et al., 1991) CTC AAG GCC AGG CAT CAC T GGA CTC TTC CAT GCC AGT G THY1 314 exon 2,55 (Cotter et al., 1989) AGA AGG TGA CCA GCC TAA CGG TCT GAG CAC TGT GAC GTT CTG TYR1 478 exon 1 - intron 1, 52 (Gillett et al, 1993) TCA GAC TAT GTC ATC CCC ATA GGG c.f. (Giebel et al, 1990) TGC AAT GAG TGT TCA GGT GAG AAG TYR4 268 TYRL, flanking exon 4, 48 (Giebel et al, 1991) ACA ATA TGT TTC TTA GTC TG (Spritz et al, 1990) TGG TAA CAC TAG ATT CAG C ALU various AluTV, 57 (Cotter et al, 1990) CAG AAT TCG CGA CAG AGC GAG ACT CCG TCT C CFL2 271 3' untranslated, 58 (Gillett et al, 1996) ACA ATG AAT GAA GGA AAT ATC ATT TAT AAA TAA TAC TGA AAA AAG TTG ACC ATC ME3-3'89 89 coding seq. - 3'UTS, 62 (Gillett, unpublished) GG CCA GAA TCT GCA TCA AGC c.f. (Loeber et al, 1991) AA AAA GGG GTT CCC TGG AGC ME3-3'205 205 coding seq. - 3'UTS, 58 (Gillett, unpublished) CCT ATA TGC TAA TAA AAT GGC TTT CCG A c.f. (Loeber et al, 1991) AA AAA GGG GTT CCC TGG AGC ME3-5Y19 119 5'UTS and coding seq., 56 (Gillett, unpublished) GCT GAG CAT CGC CAG GG c.f. (Loeber et al, 1991) GGT GGA AAC TAC TCT TAA CCG GG ME3-1892 1892 5'UTS - 3'UTS, 58 (Gillett, unpublished) GCT GAG CAT CGC CAG GG c.f. (Loeber et al, 1991) AA AAA GGG GTT CCC TGG AGC TTR 415 flanking exon 2, 60 (Abbott et al, 1991) CAG TGT GTC TGG AGG CAG AAA CCA T TCT CTA CCA AGT GAG GGG CAA ACG G RXRB 141 5' untranslated, 58 (Fitzgibbon et al, 1993) CAG AGT CTT TCT CTC AGG GG c.f. (Leid et al, 1992) CCG CTG AGG GAG GAA GGG CG RXRB 131 5' untranslated, 68 (Fitzgibbon et al, 1993) CTC TCA GGG GCT TCC TCG TGC TC c.f. (Leid et al, 1992) CCG CTG AGG GAG GAA GGG CG Appendix 3 Standard solutions, buffers, media and primer sequences 317 Publications arising from this thesis Gillett, G. T., McConville, C. M., Byrd, P. J., Stankovic, T., Taylor, A. M., H unt, D. M., West, L. F., Fox, M. F., Povey, S. & Benham, F. J. (1993). Irradiation hybrids for human chromosome 11: characterization and use for generating region-specific markers in Ilql4-q23. Genomics 15, 332-341. [Appended]. McConville, C. M., Byrd, P. J., Ambrose, H. J., Stankovic, T., Ziv, Y., Barshira, A., Vanagaite, L., Rotman, G., Shiloh, Y., Gillett, G. T., Riley, J. H. & Taylor, A. M. R. (1993). Paired STSs amplified from radiation hybrids, and from associated YACs, identify highly polymorphic loci flanking the ataxia telangiectasia locus on chromosome llq22-23. Hum Mol Genet 2 , 969-74. Fitzgibbon, J., Gillett, G. T., Woodward, K. J., Boyle, J. M., Wolfe, J. & Povey, S. (1993). Mapping of RXRB to human chromosome 6p21.3. A nn Hum Genet 57, 203-9. Benham, F., Sugiyama, R., H unt, D., Gillett, G. & Smith, M. (1993). Identification and regional localization of a highly polymorphic dinucleotide repeat D11S614 to the interval in llq23.3 flanked by recurrent translocation breakpoints. Ann Hum Genet 57, 281-284. Vanagaite, L., Savitsky, K., Rotman, G., Ziv, Y., Gerken, S. C., White, R., W eissenbach, J., Gillett, G., Benham, F. J., Richard, C. W., James, M. R., Collins, F. S. & Shiloh, Y. (1994). Physical localization of microsatellite markers at the ataxia- telangiectasia locus at Ilq22-q23. Genomics 2 2 , 231- 233. Povey, S., Burley, M. W., Attwood, J., Benham, F., H unt, D., Jeremiah, S. J., Franklin, D., Gillett, G., Malas, S., Robson, E. B., Tippett, P., Edwards, J. H., Kwiatkowski, D. J., Super, M., Mueller, R., Fryer, A., Clarke, A., Webb, D. & Osborne, J. (1994). Two loci for tuberous sclerosis: one on 9q34 and one on 16pl3. Ann Hum Genet 58, 107-127. Pang, J. T., Lloyd, S. E., W ooding, C., Farren, B., Pottinger, B., H arding, B., Leigh, S. E. A., Pook, M. A., Benham, F. J., Gillett, G. T., Taggart, R. T. & Thakker, R. V. (1996). Genetic m apping studies of 40 loci and 23 cosm ids in chromosome Ilpl3-llql3, and exclusion of p-calpain as the multiple endocrine neoplasia type 1 gene. Hum Genet 97, 732-741. Gillett, G. T., Fox, M. F., Rowe, P. S. N., Casimir, C. M. & Povey, S. (1996). Mapping of human non-muscle type cofilin (CFL1) to chromosome llql3 and muscle-type cofilin (CFL2) to chromosome 14. Ann Hum Genet 60, 201-211. [Appended]. Publications arising from this thesis 318 Envoi ...On a huge hill, Cragged and steep, Truth stands, and he that will Reach her, about must, and about must go, And what the hill's sudde?tness resists, win so; Yet strive so, that before age, death's twilight, Thy soul rest, for none can work in that night. To will implies delay, therefore now do. Hard deeds, the bodies pains; hard knowledge too The mind's endeavours reach, and mysteries Are like the sun, dazzling, yet plain to all eyes. from John Donne, Satire III, lines 79 - 88 In memory of Jack, Charlotte and Dora. GENOMICS 1 5 , 332-341 (1993) Irradiation Hybrids for Human Chromosome 11: Characterization and Use for Generating Region-Specific Markers in 11q14-q23 G . T. G illett,* C . M . M c C o n v il l e , ! P. J. By r d , ! T. St a n k o v ic , ! A . M . T a y l o r , ! D . M . H u n t ,* L. F. W est,* M. F. Fox,* S. Povey,* and F. J. Benham*'1 *The Galton Laboratory, University College London and MRC Human Biochemical Genetics Unit, Wolf son House, 4 Stephenson Way, London NW1 2HE, United Kingdom; and t Department of Cancer Studies, Cancer Research Campaign Laboratories, University o f Birmingham, Birmingham B15 2TT, United Kingdom Received June 2, 1992; revised November 16, 1992 previously been reported (Smith et dl, 1990; Fahsold et High-dose irradiation hybrids containing fragments al, 1991). The possibility of a locus on chromosome 11 of chromosome 11 have been generated, with a view to was initially suggested by the finding of an unbalanced isolating new region-specific markers. Forty-seven 11;22 chromosome translocation in a neonate who was lines were scored for 34 markers: average retention the first case of TSC in the family. The mother of was 6%. Fourteen lines contain markers from llq l4 to the child had a de novo balanced translocation llq23. One of these, Jol2, has llq markers extending t(llq23.3;22qll.2), and the child had trisomy llq23.3- from tyrosinase (ql4-q21) to PBGD (q23.3) plus one qter (Clark et al, 1988). Subsequent linkage analyses marker (TYRL, p i 1.2) from lip . In situ hybridization with probes from llq in TSC families have neither con usingAlu PCR products from Jo 12 as probe confirmed firmed nor excluded an llq locus (Smith et al, 1990; that the human DNA is derived from two regions, one Haines et al, 1991). This is in part due to the relatively in proximal lip and a second, larger region in llq23. Plasmid libraries of Alu PCR products from this and few markers that are both well-mapped and highly poly three other hybrids have been made. Six of eight recom morphic in the Ilql4-q23 region. To improve the map of binants identified as having single-copy inserts were this region, we have constructed a panel of irradiation mapped back to the regions of Ilq22-q23 detected in fragment hybrids to provide resources from which to de the originating hybrid; only one mapped to a region not rive new, region-specific markers. originally detected, and one was of hamster origin. Generation of somatic cell hybrids by irradiation and These six clones provide new markers in Ilq22-q23 fusion was first proposed by Pontecorvo (1971). This that can be used directly for polymorphism studies. technique was subsequently used by Goss and Harris This series of hybrids is therefore a valuable resource (1975) for intrachromosomal gene mapping. It has been for the rapid generation of markers from specific, de exploited recently by several groups to provide mapping fined regions of chromosome 11. ©1993 Academic Press, Inc. resources within delimited regions of the genome (Ben ham et al, 1989; Cox et al, 1989; Nelson, 1991). In an example of this approach, a human-rodent hybrid con INTRODUCTION taining a single human chromosome is lethally irra diated. Irradiated cells are fused with an HPRT-defi- Following the first report of linkage of a gene for the cient rodent parent, and hybrid clones containing an ac dominantly inherited disease tuberous sclerosis (TSC) tive rodent HPRT gene are selected by growth in to human chromosome 9q34 (Fryer et al., 1987), there medium containing HAT. Retention of a specific region have been several studies indicating genetic heterogene of the human chromosome may be achieved by the im ity in this condition (Janssenet al, 1990; Sampson et al, position of selection of an expressed gene within that 1989). A very recent collaborative study (Kandt et al, region. However, even in the absence of selection for 1992) has reported the localization of a definite second retention of human chromosomal material, hybrids do locus for TSC on chromosome 16pl3, closely linked to D16S283. However, some TSC families do not show seg contain randomly integrated fragments of the chromo regation of the disease with either the chromosome 9q34 some from the irradiated parent, in which case the size or the chromosome 16pl3 markers (Short et al, 1992; and number of the fragments are related to the irradia tion dose (Benham et al, 1989; Benham and Rowe, 1992; Benham and Povey, unpublished observations). Some evidence for TSC genes on chromosomes 11 and 12 had Cox et al, 1990). Generation of human probes from human-rodent hy 1 To whom correspondence should be addressed. Telephone: 071- brids containing small amounts of human DNA has 387-7050, ext. 5026. Fax: 071-387-3496. been achieved in various ways. A method that exploits 332 0888-7543/93 $5.00 Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved. CHROMOSOME 11 IRRADIATION HYBRIDS AND MARKERS 333 species specificity (Brooks-Wilson et al, 1990; Ledbet digestion of the product (Giebelet al, 1991). To confirm these results, ter et al., 1990; Nelson et al, 1989) uses oligomer primers human-specific primers were designed to amplify theMbol polymor phism in exon 1 of TYR (Giebel and Spritz, 1990). PCR amplifications derived from the human Alu consensus sequence. The were performed in 25- to 100-jul volumes for 35 cycles of amplification Alu primers direct amplification of inter-A fit human se (93°C, 20 s denaturation, Ta as stated in Table 1; 20-30 s; 72°C, 30-60 quences, and this achieves very efficient recovery of hu s; extension depending on length of product). Each reaction contained man DNA, even when it is present in small proportions 0.25-1.0 fig genomic human DNA, 0.5-1.0 fiM each oligonucleotide or at low levels. We have used the somatic cell hybrid primer, and 0.8-1.0 unit of Taq polymerase. The enzyme was added to each reaction after an initial 5-min denaturation at 95°C. Final reac J1CL4 (gift of C. Jones), which contains chromosome 11tion mixes were as follows: as the sole human component, to generate a set of high- “Cetus” (“C” in Table 1): 210fiM each dNTP, 1.5 mM MgCl2, 50 dose irradiation hybrids containing fragments of chro mM KC1, 10 mM Tris-HCl, pH 8.3. INT2, CD3D, and THY1 were mosome 11. No selection was imposed for retention of also amplified using a mix containing 2.0 mM MgCl2. any particular human DNA. We have exploited these “Anglian” (“A” in Table 1): 10% DMSO, 1.5 mM each dNTP, 6.7 hybrids as a resource from which region-specific mM MgCl2, 16.6 mM (NH4)2S04, 67 mM Tris-HCl, pH 8.8, 6.7 fiM EDTA, 10 mM /3-mercaptoethanol, 170 mg/liter bovine serum al markers have been obtained. bumin. “Promega” (“P” in Table 1): 210fiM each dNTP, 1.5 mMMgCl2, 50 mMKCl, 10 mM Tris-HCl, pH 9.0,0.01% gelatin, 0.1% Triton X-100. MATERIALS AND METHODS Reactions using primers for D11S490 and D11S420 included 10% DMSO. Cell Lines PCR products: 5 /d of each reaction was electrophoresed in 2% aga rose gels in TBE buffer. J1CL4 is a human-hamster cell line containing a single human Southern hybridization. Southern blots and subsequent hybridiza chromosome 11 (Kao et al, 1976). Its hamster parent is CHO-Kl. tion were done using standard procedures as described (Benhamet al., Wg3H is a HPRT-deficient hamster cell line. 1989). Fluorescence in situ hybridization (FISH). DNAs to be used as Cell Culture probes (total human, total hybrid, and Alu PCR products) were la beled with biotin-14-dUTP by nick translation using a BRL kit (Cat. Cell lines were cultured in Eagle’s medium supplemented with 10% No. 82475A). Unincorporated nucleotides were removed using a Seph- fetal bovine serum, 2.0 mM L-glutamine, 105 U/liter benzyl penicillin, adex G-50 column, and the labeled probe was ethanol precipitated. 100 mg/liter streptomycin base-equivalent, and 10 mMHepes. Cipro Metaphase chromosome preparations from lymphocytes and so floxacin (10 mg/liter pure substance, Bayer UK, Ltd.) was added after matic cell hybrid lines were made by standard procedures. Slides were irradiation hybrid clones were picked. J1CL4 and the hybrids were freshly prepared and aged for 2 h at room temperature before use. The cultured in the above medium to which HMT was added (10~4 Mhypo- hybridization was adapted from Pinkel et al (1986). Slides were pre xanthine, 10-5 M methotrexate, 1.6 X 10-5 M thymidine). treated with RNase (100 fig/ml 2X SSC, pH 7.0) under a coverslip in a moist chamber for 1 h at 37°C, washed in 2X SSC, dehydrated through an alcohol series, and air-dried. Incubation in proteinase K buffer (20 Irradiation Fusion Procedure mM Tris-HCl, 2 mM CaCl2, pH 7.4) was followed by a 7-min treat ment with proteinase K (50 fig/ml) at 37°C and washes in PBS. The Approximately 5 X 106 J1CL4 cells were harvested and irradiated at slides were then postfixed in 50 mM MgCl2/PBS/4% paraformalde room temperature in a Pantak HF30 industrial X-ray unit at 320 kV, hyde for 10 min at room temperature, washed in PBS, dehydrated 10 mA, with a total X-ray dose of 40,000 rad at a dose rate of 1000 through an alcohol series, and air-dried. Prior to hybridization, 100 /xl rad/min in two fractions lasting 2 hin toto. These were fused with of 70% formamide/PSSC was placed onto the slide under a coverslip, recipient Wg3H cells using a standard PEG fusion procedure (as de and the DNA denatured in an oven at 70°C for 5 min. The coverslips scribed in Benham et al, 1989). HMT was added 24 h after fusion. were removed and the slides plunged into ice-cold 70% ethanol twice, Colonies appeared within 3 weeks. Two clones were picked from each then into 90 and 100% ethanol baths, and air-dried. 80-cm2 flask into separate 25-cm2 flasks. Each clone was grown up to When total human DNA was used as probe, for each slide, 100 ng of 75 cm2, LDHA analysis was performed on cells from the 25-cm2 flask, labeled total human DNA was added to a hybridization mix of 20 fil and half of the clones, selected at random, were cryopreserved without 50% formamide/2X SSC plus 10% dextran sulfate and denatured at further analysis. The remainder were grown up to 3X 165 cm2, DNA 75°C for 5 min. When total hybrid DNA or Alu PCR products were was extracted by a conventional phenol-chloroform method, and cells used as probe, for each slide, 200 ng of labeled DNA plus 10fig of were cryopreserved from the refed 75-cm2 flasks. human Cot-1 DNA as competitor were suspended in 20 fi\ 50% form- amide/2X SSC plus 20% dextran sulfate. The DNA mix was then Characterization of Hybrids denatured at 75°C for 5 min and preannealed at 37°C for 30 min. The hybridization mix was then placed onto the slides under a coverslip, Biochemical analysis. LDHA was assayed by horizontal starch gel and the coverslip was sealed with rubber solution. Hybridization pro electrophoresis (Harris and Hopkinson, 1976). ceeded in a moist chamber at 37°C for 6 days. After hybridization, the PCR. Sequences of oligonucleotide primers are listed in Table 1. coverslips were removed and the slides washed in 50% formamide/2X References are to publications in which the primer sequences are SSC at 42°C for a total of 15 min and then in 2X SSC at 42°C for listed or to DNA sequences in the EMBL database from which 10 min. primers were designed by comparison of human with rodent sequence For signal detection, two preincubation steps were performed, the (when available) in order to amplify the human and not the hamster first for 5 min in 4X SSC/0.05% Tween 20 detergent and the second sequence. D11S144 primers were selected from sequences obtained for 10 min in 4X SSC/5% nonfat dried milk (Marvel). Biotin detection from 5' and 3' ends of a 0.9-kb EcoRI fragment of probe MCT128.1 was facilitated by a layer of avidin-FITC conjugate and biotin-anti- (cloned into pUC9). Double-stranded Sanger sequencing was per avidin D conjugate (Vector Labs, USA) followed by one amplification formed using a proprietary kit (Sequenase 1, United States Biochemi of signal with avidin-FITC. In each case conjugate was applied at a cal). The TYR primers flanking exon 4 of the tyrosinase gene (Spritz concentration of 5 fig/m\ (in 5% milk/4X SSC) 100 /il/slide for 20 min et al, 1990) amplified both the TYR locus (Ilql4-q21) and the related under a coverslip and washed off with 0.05% Tween 20 in 4X SSC for a sequence (TYRL, llp ll.2 ). These loci were distinguished by MspI total of 15 min. Slides were finally washed in 0.9% saline and mounted 334 GILLETT ET AL. TABLE 1 PCR Oligonucleotide Primers Used in the Characterization of the Hybrid Panel Size Conditions Locus (bp) Sequence (5' to 3'; forward above reverse) (Ta, °C) Reference HBB 398 ACT GGG CAT GTG GAG ACA GAG AAG A A Abbott and Povey, 1991 Flanking exon 2 TGT ACC CTG TTA CTT CTC CCC TTC C (55) CAT 199 AAA TAT CAC GTT GCT GCC CAT GAG G A Unpublished; cf. Theune Intron XH-exon 13 GAA TCT TCA TCC AGT GAT GAG CGG G (52) et al., 1991. Seq. from Quan et al., 1986 PYGM 704' TGC TCA TGC ACC ATG ACC GGT GAG C P Richard et al, 1991 Flanking intron XIX CCA AGA GAG TGT GAC AGA CTC AAG G (65) FGF3 415 AGT TTG TGG AGC GGA TCC ACG C Cotter et al., 1989 Exon 3-3' untrans. AAG ATG TCG CCA GGA GCT CTG (55) D11S533 300-900 GCC TAG TCC CTG GGT GTG GTC C Eubanks et al, 1991 GGG GGT CTG GGA ACA TGT CCC C (66) TYR 268 ACA ATA TGT TTC TTA GTC TG P Giebel et al., 1991; Spritz Flanking exon 4 TGG TAA CAC TAG ATT CAG C (48) et al., 1990 TYR 478 TCA GAC TAT GTC ATC CCC ATA GGG P Gillett, unpublished; cf. Exon 1-intron 1 TGCAAT GAG TGT TCA GGT GAG AAG (52) Giebel and Spritz, 1990. Seq. from Giebel et al, 1991 D11S35 152-162 ACA ATT GGA TTA CTA CTA GC A Litt et al, 1990 TGT ATT TGT ATC GAT TAA CC (38) D11S385 286 TTT TAT AGG GAC AGG ATC TTG C A Byrd, unpublished GGC TGT ATA ATC TTG TGT TCT C (50) CLG 447 GTA CAG GAG CCG AAC AGC CAT CAG G A Unpublished; cf. Theune 5' Untranslated GCC TCT TGC TGC TCC AAT ATC CCA G (55) et al., 1991. Seq. from Angelet al., 1987 DRD2 80-86 CAG GAG CAC GTT TCT CAT AC C Haugeet al, 1991 GGA GGG CGG TGC GGT CAT (58) NCAM 179 TGG AAA TCT CTT CCA AAC ATC GGA G A Abbott and Povey, 1991 5' Untranslated AAT TAG AAC TTT GGA GAG GGA TGG G (50) D11S144 c. 550 TTC CCC AGT AGC TTC TGT CTC CTG C A/C/P Gillett, unpublished TCA CTC CCA GTT CAG TAT CCT GTC C (59) D11S490 147-167 CAC AAA CAT TGG CGC AT P Luo et al., 1990b TTC TGG GTC ACG GTG CTT CA (54) APOC3 715-730 TGG CCC AGC AGG CCA GGT ACA CCC G A Gillett, unpublished. Seq. GGA GGC CGA GGC AGG AGG ATC CCC (61) from Protter et al., 1984; Shelley et al, 1985; cf. Zuliani and Hobbs, 1990 CD3D 382 GAG CTT AAC TCA GCA AGA CAG C Cotter, et al, 1989 Intron II-exon 3 GAG CAG AGT GGC AAT GAC ATC (55) D11S614 160-180 CCC CAA AAC AGA CCC ACC AGG ACT P Sugiyamaet al, 1991 8D11 TCT TCT CTG ACC CCG GAT GTC TGC (57) THY1 314 AGA AGG TGA CCA GCC TAA CGG C Cotter et al., 1989 Exon 2 TCT GAG CAC TGT GAC GTT CTG (55) D11S420 188-208 AGT TAC ACC GGT TCT GCA GA P Luo et al., 1990a GAT TAA TGA TAG TGC TAT CC (49) Note. Conditions: A, Anglian buffer (see Materials and Methods); C, Cetus buffer; P, Promega buffer; Ta, annealing temperature. Size: as cited in reference or by calculation from sequence. in antifade medium containing propidium iodide and DAPI (1 mg/ml Alu primers through Qiagen columns and approximately fig1 of con p-phenylenediamine dihydrochloride in 90% glycerol/10% PBS, pH centrated products was labeled with biotin-UTP by nick translation. 8.0, plus 0.15 jig/ml DAPI and 1 fig/ml propidium iodide). Slides were Only products from one primer were used for any given hybridization viewed on a Nikon Optiphot fluorescent microscope and images cap experiment. tured on an MRC 600 Confocal laser scanning microscope. Human sequences were specifically amplified from J1CL4 and Jo 12 Cloning of Alu PCR Fragments from J1 Irradiation DNA using one of twoAlu sequence primers. A1IV is from the 3' end of Hybrids the Alu sequence (5'-CAGAATTCGCGACAGAGCGAGACTCCGT- CTC-3' (Cotter et al, 1990)) and 451 is 56 bp from the 3' end (5'- DNA (0.1 mg) from four irradiation hybrids containing chromo GTGAGCCGAGATCGCGCCACTGCACT-3' (Dorin et al., 1992)). some 11 fragments from Ilq22-q23 (i.e., 12,13', 33, and 48') was am The PCR reaction was carried out in a total volume of 50 jil with plified using the human-specificAlu primer 451 (sequence as above 100-300 ng of genomic DNA. The reaction mix contained 10 mM (Dorin et al., 1992)). Reaction conditions were as follows: 50 mM KCl, Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl2, 170 mg/ml BSA, and 10 mM Tris-HCl, pH 8.4, 1.5 mM MgCl2, 100 mg/ml gelatin, 0.01% 250 nM each dNTP. The Alu PCR products were separated from the Tween-20, 0.1% Triton X-100, 0.1% NP-40, 200 mM dNTPs, 250 ng Markers in Chromosome 11 Irradiation Hybrids O h SSJOOQhQfcQhPHflPO®OOQOZOP \ ft HrH rH CO I+ I+ + + I I I I + I I I I I I I I I I I I I I I I I I I I I I I I I I I I I £*- HrH rH rH Ol CM CD rH (N rH h ft CO Ol rH HrH rH I I I + I+ + + + I I + cr * u I I II I I I I I I I I I I I I I I I I I I I I I + I I I I I I +I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 o CO (72 ft 4 D t t ft ft ft + O C O C M C O C O C M C O C O C O C O C M C O C O C M C M C M C H r M C M C H r M C ^ + + + + • + + + I I I I I I I I I I +I I I I I I I I I I I I I I I I I I I I I I I I I I I +I I I i i i - i i ii . i i . i . Q > t rH C£> CM I I I I I I I I I I• .I 82 I I I I I I I I I I I I • ft ft ft ft M CM CM CO CM IO + + + + + + + + + + + + + + + + + + + + + + + ' cro' i i lO 0CO 00 O CO CD 82 tf ft ft ft h ft M CM CM CO CM cr c i i '-a 00 rH CM uo 72 s 0 ►o > t 0 1 I + I + cm rH CM + + + + I I I • I I • I I • I I • I I I • 82 + + + I I I II I II + II I II I I II I I I I I I + + + I I I I I ' ft ft ft M CM CM co ^ + + o* o• I I I I I I I I I I I I I I I I O Tf O O) © CO f T lO o r rv! rvi o r rvi o r o r rvi o r o r o r H O ^ rH 72 t ft ft CM CO cr o* o* i i ft] r> 72 ft t f t f t f 3 § 3 § 3 M O O O CO CO CO CO CM I+ I+ - f t - f t < 3 . 82 & U' && CO m (1) + I I I I I +I I II II + I II I i i i i i P • •i '• - 00 CM 82 I t f . I I I + i V I + I • 02 I I I I I I I I II +I I I I I I I I I I I I +I I I I I I I I I I I I ft I I I I I I I I I I CO U i P rd 1 "8 02 0 02 c ft (H “ 335 primer, and one unit of Taq DNA polymerase in a 50-jid reaction. PCR conditions were one cycle of 94°C, 3 min; 70°C, 1 min; 72°C, 1 min; 35 cycles of 94°C 45 s; 70°C, 1 min; 72°C, 1 min (increasing by 6 s per cycle); and one cycle of 94°C, 45 s; 70°C, 1 min; 72°C, 10 min. Follow ing this primary round of amplification, the reactions were diluted 1000-fold and 1 ^1 of each was reamplified as above usingAlu primer 451 again. The resulting PCR products were end-filled and kinased using Klenow enzyme and T4 polynucleotide kinase in 50 mM Tris- HCl, pH 7.6, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, and 1 mM dNTPs. Nucleotides and primers were then removed by chromatogra phy through Qiagen-20 columns. The blunt-endedAlu PCR frag ments were ligated to phosphatased Smal-cut pBluescribe and cloned in Escherichia coli DH5«. Recombinant colonies were picked to 96- well plates and grown in LB containing 50 mg/ml ampicillin. Gridded arrays of colonies from the 96-well plates were made on Hybond N filters using a 96-prong replica plating tool. Gridded colonies on the filters were lysed, denatured, and hybridized to a 177-bp SauSA frag ment from the Alu repeat (outside the region from which the primers were derived) and a 1.2-kb Ll repeat fragment to identify and exclude clones that contained Alu or Ll repetitive elements. RESULTS One hundred and seven irradiation hybrid clones (des ignated Jol, 2, 3, etc.) were picked, and the expression of human lactate dehydrogenase-A, which maps to chro mosome lip, was scored in all of these; 10% of clones were positive. Forty-seven clones, randomly chosen from the 107, were grown up, and DNA was extracted and analyzed for retention of additional chromosome 11 markers. The remaining 60 clones were cryopreserved from confluent 75-cm2 flasks. The presence of chromo some 11 DNA was assessed by Southern hybridization, PCR, and, in selected hybrids, FISH. A total of 34 markers were tested; of these, one-third map at intervals along the whole of the chromosome outside the main region of interest. The rest lie in Ilql4-q23. Fifteen loci were tested by Southern blotting (listed in Table 2) and 19 by PCR assays (Table 1). Tyrosinase and its related sequence (on lip) were distinguished by three assays (see Materials and Methods). Results on the 28 hybrids positive for one or more markers are listed in Table 2. In 4 of these 28, only centromeric sequences were detected. The 19 hybrids that were negative for all markers tested have been omitted from the table. The retention of any given marker in the hybrid panel was approximately 6% whether assayed by hybridiza tion or by PCR, with the exception of the centromeric sequence D llZ l (43%). Eleven of 107 hybrids expressed human LDHA, indicating a slightly higher retention at this locus (10%). The presence of a randomly retained fragment of hamster DNA originating from the irra diated donor line was detected by testing for rodent aden- FIG. 1. Fluorescence in situ hybridization. (A, B) Chromosome spreads of hybrid Jol2 showing the presence of human DNA frag ments (probe: total human DNA). Two representative cells are shown: the culture was heterogeneous with respect to number and type of human fragments. (C) Human chromosome spread probed withAlu PCR products from hybrid Jol2. A wide signal is present around llq23, and a smaller signal is present in proximal lip. CHROMOSOME 11 IRRADIATION HYBRIDS AND MARKERS 337 TABLE 3 Characterization of the Chromosome 11 Hybrid Panel Hybrid Marker Location0 A3EW2 M11X IB56 PG7 PG48 PG9 HRAS pl5.5 + + _ nd + + INS pl5.5 + + nd - + + FSHB pl3 + + - - + + CAT0 pl3 + + - nd + - D11Z1 cen + + + nd + - D11S5270 ql3.5 + + nd nd + - TYR ql4-q21 + + + nd + - D11S36 q22-qter + + + - + - D11S84 q22 + . - + + - + - D11S35 q21-q22 + + + - - nd STMY q22.3-q23 + + + - -- D11S424 q22 + + + + - nd DRD2 q22-q23 + + + + - - NCAM q23-q24 + + + + -- D11S144 q22.3-q23.3 + + + + -- D11S29 q23-qter + - + + - - CD3D q23 + - + + -- THY1 q22.3-q23 + - + + -- ETSl q23.3 + + — -— Note. +, Marker present; —, marker absent; nd, marker not tested. All markers tested by Southern hybridization except where indicated. ° Localization as cited in Junien and Van Heyningen (1991) or GDB. b The IB5 cell line used appears to have lost part of chromosome lip and therefore differs from the published description of this line (Wadey etal., 1990). 0 Determined by PCR. osine deaminase expression (ADA), which had been least two fragments of human DNA: one integrated into found to exist as different isozymes in J1CL4 and Wg3h. a hamster chromosome and one or two very small sepa Of 107 hybrids tested, 14 (13%) retained the J1CL4 rate fragments (Fig. 1).Alu PCR products from Jol2 ADA as well as the Wg3H ADA, confirming the hybrid generated using variousAlu primers and painted onto nature of the cells and suggesting that the frequency ofnormal human metaphase spreads hybridized to two re random retention of rodent and human fragments is of gions: a wide area at Ilq21-q23, and a smaller one in the same order. proximal lip (Fig. 1). These mapping results are en Fourteen hybrids contained markers from Ilql4-q23. tirely consistent with the marker analysis and indicate These are Jo6', 8', 12, 13', 14, 16, 17, 23, 31, 32, 33, 48', that most, if not all, of the human DNA retained in Jol2 49'", 50'. Of these 14, 7 retained markers in the llq l4 - derives from the two regions of chromosome 11 detected q23 region and were negative for most other regions by the markers. tested (8', 12,13', 14,16, 23,49'"). Hybrid Jol2 seemed to be the best hybrid for our purposes and so has been con Alu PCR Cloning and Mapping sidered in more detail. Jol2 was positive for all 18 llq markers tested extending from TYR (ql4) to PBGD Libraries were constructed of Alu PCR products gen (q23.3), a region about 40 cM in length. It hybridized erated from four of the hybrids that contain Ilql4-q23 weakly to the centromeric probe pLCllA (D11Z1). The markers: Jol2, 13', 33, 48'. The total PCR product pool only lip marker detected was the tyrosinase-related se amplified from each line using the human-specific quence. Evidence from the relative intensities of the hy primer 451 (Dorin et al, 1992) was blunt end cloned into bridization signals on Southern filters indicated that the the Bluescribe plasmid vector. As a preliminary charac llq markers do not lie on a single human fragment: the terization of the libraries, a total of 30 randomly selected more proximal llq probes gave mostly strong signals clones were analyzed. Fourteen recombinants (47%) relative to control J1CL4 tracks, whereas the more distal were found to contain inserts greater than 300 bp. (This markers gave weaker signals, suggesting that retention lower size limit was chosen since the clones were ex of these sequences in the hybrid is on at least two sepa pected to contain 56 bp of Alu sequence at either end.) rate fragments. Twelve clones were analyzed further by restriction map This impression of the human chromosomal content ping and identification of fragments which consisted of in Jol2 was confirmed by fluorescencein situ hybridiza non-Alu, nonrepetitive sequences. Nine of the 12 yielded tion. Using total human DNA as probe on Jol2 meta fragments that gave reasonably strong signals, with low phases, FISH showed that many of the cells contained at background, when hybridized to human genomic DNA. 338 GILLETT ET AL. TRANSLOCATION HYBRIDS MARKER PROBES A3EW M11XPG48 PG9 PG7 1B5 HRAS INS CAT 112 11 Z1 J48B6 S527 TYR S36 S84 S35, STMY" J12.8, J12/1B4, J12/1C2 S424 DRD2.NCAM J12.5 S144 S29 CD3D J12/2A2 J12/1H4 23.3 THY1 ETS 24 25 F IG . 2 . Diagram showing the chromosome11 translocations present in the hybrid mapping panel and the localization of the sixAlu PCR clones to the intervals defined. The remaining 3 gave little or no specific signal. To inlikely that they represent coligation events. The shortest crease the efficiency of isolation of probes not contain of these clones, J12.5, had the expectedAlu sequence at ing internal repetitive DNA sequence, Alu PCR clones both ends, so it is possible that the larger clones were from the Jol2 library were gridded out into microtiter generated by amplification from adjacentAlu-like se arrays and prescreened with Alu and Line probes. (The quences or non-Alu DNA showing chance homology to Alu probe was an internal fragment not containing the primer sequences. Two other clones from Jol2, which primer sequence.) A further 5 recombinants were se differed in size by ~200 bp, also showed overlapping lected as containing largely single-copy inserts. sequences. Since one of these had Alu sequence at one The degree of clone duplication within this set of 14 end only, this aberrant clone may be the result of a clon recombinants was assessed by one or more methods: ing artifact. Thus of the 14 clones selected as having comparison of restriction maps, cross-hybridization be single-copy inserts, 8 were unique: 6 from Jol2, 1 from tween clones, the pattern of hybridization to genomic Jol3', and 1 from Jo48\ WhenAlu PCR products gener DNA digested with a range of restriction enzymes, and ated from Jo 12 using primer 451 were visualized in ethid- in some cases, sequencing. Six clones of similar size ium bromide-stained gels, approximately 10 visible ( ~ 650-730 bp) from Jol2 were found to contain over bands were observed (data not shown), suggesting that lapping, though not identical sequences. Since these all at least half of the products from this hybrid were cloned hybridized to a single band in genomic DNA, it is un as near single-copy sequences. CHROMOSOME 11 IRRADIATION HYBRIDS AND MARKERS 339 TABLE 4 Regional Mapping ofAlu PCR Clones on the Chromosome 11 Hybrid Panel Mapping hybrid Clone D Segment A3EW2 M11X PG48 PG9 PG7 IB5 Location J12.5 D11S607 nd + _ + + q23.1-q23.2 J12.8 D11S535 nd + - - - + q22 J12/1B4 D11S609 + + --- + q22 J12/IC2 D11S611 + + - nd ±° + q22 J12/IH4 D11S612 + - - - + + q23.3 J12/2A2 + --- nd + q23.3-q24 J13C106 nd ----- Hamster J48B6 D11S613 nd + + - nd .+ p!3-q22 Note. +, Positive by Southern blotting; —, negative by Southern blotting; nd, marker not tested. ° Positive by PCR but negative by Southern blotting. 6 No human-specific signal, but a band observed in hamster control and hamster-human hybrids. Regional localization of these eightAlu PCR clones Jol2, possibly because it scored negatively for all but one was determined by hybridization to a panel of six so marker from outside the region of interest (several of matic cell hybrids containing translocations involving which were scored). However, the clone derived from a human chromosome 11. This panel, comprising the hy different hybrid, Jo48', apparently originated from a re brid lines M lIX (Chianget al, 1984), IB5 (Wadey et al, gion not originally detected in the hybrid DNA, empha 1990), PG7, PG9, PG48 (McConville et al, 1991), and sizing the need to map back all markers obtained from A3EW2 (Guertz van Kessel et al, 1985; Sacchi et al, irradiation hybrids. 1986), allowed chromosome 11 to be divided into seven By cloning pools ofAlu PCR products derived from regions: llpter-pl3, Ilpl3-q22, Ilq22-q23.1, llq23.1- irradiation hybrids and screening for single or near-sin q23.2, Ilq23.2-q23.3, Ilq23.3-q24.2, and llq24.2-qter. gle copy insert fragments, six new markers in Ilq22-q23 Table 3 and Fig. 2 show the characterization by marker have been obtained. Two of these, D11S535 and analysis of each of the translocation breakpoints. D11S611, detect polymorphisms that have helped to lo The mapping of the eightAlu PCR-derived probes on calize the ataxia telangiectasia gene to a region of about the hybrid panel is shown in Table 4, and their regional 5 cM in llq22 (McConville et al, 1991). To generate localization is summarized in Fig. 2. All six of the probes additional markers from specific regions of interest, al derived from hybrid Jol2 mapped back to the regions of ternative Alu primers that amplify different human se llq originally detected in the hybrid line by marker anal quences may be used. For example, the 3' primer A1IV ysis and FISH, which is consistent with the human geno (Cotter et al, 1990) generates at least 40 products visible mic content of this hybrid. The map positions of these on ethidium bromide gels from hybrid Jol2. Since some were distributed between three intervals from across the bias toward cloning smaller or particularAlu PCR prodr whole region from q22 to q23.3 (D11S35 to ETS). No ucts may occur when using the total product pool, it may clones mapped to the region of lip detected in Jol2. The be useful in some cases to clone the Alu PCR products probe from Jol3', J13'C10, turned out to be of hamster individually following excision from gels. origin. The probe from hybrid Jo48', J48T$6, mapped One-third of the 47 hybrid lines in this set scored posi proximal to q22, a region undetected in this hybrid. All tively for markers from one or at most two different re the mapped llq probes are being screened for polymor gions of chromosome 11 (not counting the centromere phisms suitable for linkage analysis. sequence, which tends to integrate independently of other markers in high-dose hybrids (Benham et al, DISCUSSION 1989)). Although characterizing this number of hybrids for markers from throughout the whole of a chromosome The irradiation hybrid Jol2 provides a valuable re is somewhat laborious, several of these lines will be valu source from which region-specific human sequences in able as resources for deriving new markers from these Ilq21-q23 have been easily and efficiently derived. All other regions of chromosome 11 (see Table 2). Further six of the human Alu PCR products obtained from this use of the chromosome painting techniques, which yield hybrid have mapped back to locations throughout the additional information about fragment number, size, region of chromosome llq originally detected in the hyand heterogeneity, will allow the choice of irradiation brid by marker analysis. A potential problem with using hybrids suitable as resources for specific regions with high-dose irradiation hybrids as a source of region-spe greater precision. With the advent of chromosome-spe cific DNA is the possible presence of undetected frag cific cosmid libraries arrayed in ordered grids (Nizeticet ments. This did not in fact prove to be a problem with al, 1991), the potential of irradiation hybrids such as the 3 4 U GILLETT ET AL. Jo series for generating large numbers of region-specific Cox, D. R., Pritchard, C. A., Uglum, E., Casher, D., Kobori, J., and markers will be greatly increased. By screening ordered Myers, R. M. (1989). Segregation of the Huntington disease region cosmid or YAC libraries with Alu PCR products from an of human chromosome 4 in a somatic cell hybrid. Genomics 4: 397- 407. irradiation hybrid containing one or a few fragments I (Monaco et al, 1991), a large proportion of the DNA Dorin, J. R., Emslie, E., Hanratty, D., Farrall, M., Gosden, J., and Porteous, D. J. (1992). Gene targeting for somatic cell manipula representing that fragment should be quickly identified, tion: rapid analysis of reduced chromosome hybrids by Alu-PCR already in cloned form. Use of irradiation hybrids in com fingerprinting and chromosome painting.Hum. Mol. Genet. I: 53- bination with these other resources will contribute 59. greatly toward the construction of long-range contigs Eubanks, J. H., Selleri, L., Hart, R., Rosette, C., and Evans, G. A. from specific chromosome subregions. (1991). Isolation, localisation, and physical mapping of a highly polymorphic locus on human chromosome llq l3. Genomics 11: 720-729. ACKNOWLEDGMENTS Fahsold, R., Rott, H. D., and Lorenz, P. (1991). A third gene locus for tuberous sclerosis is closely linked to the phenylalanine hydroxylase Carol Jones very kindly provided the hybrid J1CL4. We thank Ver gene locus.Hum. Genet. 88: 85-90. onica van Heyningen for helpful discussion and encouragement. Philip Byrd performed the Alu PCR cloning in the laboratory of David Fryer, A. E., Chalmers, A., Connor, J. M., Fraser, I., Povey, S., Yates, Porteous, and we are indebted to David Porteous for his help and for A. D., Yates, J. R. W., and Osborne, J. P. (1987). Evidence that the unpublished Alu primer sequences and conditions. We are grateful to gene for tuberous sclerosis is on chromosome 9.Lancet 1: 659-661. Peter Goodfellow for advice on the irradiation-fusion technique and Giebel, L. B., and Spritz, R. A. (1990). RFLP for Mbol in the human help with the irradiation, Finbarr Cotter for initial help with PCR, tyrosinase (TYR) gene detected by PCR. Nucleic Acids Res. 18: Cathy Abbott for the subclone of MCT 128.1, Darren Griffin for assis 3103. tance with microscopy, and Bayer UK, Ltd., for Ciprofloxacin pure Giebel, L. B., Strunk, K. M., and Spritz, R. A. (1991). Organisation substance. Godfrey T. 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(1996), 60, 201-211 ZOl Printed in Great Britain Mapping of human non-muscle type cofilin (CFL1) to chromosome llql3 and muscle-type cofilin (CFL2) to chromosome 14 G. T. GILLETT1-3, M. F. FOX1, P. S. N. ROWE2, C. M. CASIMIR4 a n d S. POVEY1 1MRC Human Biochemical Genetics Unit, The Galton Laboratory, University College London, Wolf son House, 4 Stephenson Way, London, NW1 2HE 2 University Colleqe London, Department of Medicine, The Middlesex Hospital, Mortimer Street, London WIN 8AA 3 currently, Department of Clinical Biochemistry, GOS Hospital NHS Trust, Great Ormond Street, London WC1N 3JH 4Molecular Immunology Unit, Division of Cell and Molecular Biology, Institute of Child Health, 30 Guilford Street, London WC1N 1EH (Received 10.11.95. Accepted 6.12.95) SUMMARY Cofilin is a widely-distributed, intracellular, actin binding protein which is involved in the translocation of actin-cofilin complex from cytoplasm to nucleus. We have cloned a non-muscle-type cofilin (CFL1) from a human promyelocytic cDNA library and mapped this to human chromosome 11 by PCR amplification of 3' untranslated sequence in a panel of rodent-human somatic cell hybrids, and to the interval Ilql2-ql3.2 in a chromosome 11 somatic cell hybrid mapping panel. Confirmation of regional localisation tollql3 has been obtained by fluorescent in situ hybridisation of genomic cosmid clones, by demonstration of the presence of both SEA (the human homologue of avian retrovirus proviral tyrosine kinase, llql3) and CFL1 in some of these clones and by close linkage of CFL1 to SEA in a panel of high-dose irradiation hybrids. We have identified human muscle-type cofilin sequences by comparison of human expressed sequence tags with M-type cofilins of other species and we have mapped the human M-type cofilin, CFL2, to chromosome 14. cytoplasm to the nucleus, (Ohta et al. 1989). INTRODUCTION Cofilin has recently been found to be involved in Cofilin is an intracellular actin-modulating pinocytosis in the thyroid cell, (Saito et al. 1994) protein which binds and depolymerises fila and in the platelet response to aggregating agents mentous, F-actin and inhibits the polymerisation such as collagen and thrombin, (Davidson & of monomeric, G-actin at a 1:1 molar ratio and in Haslam, 1994). It is also an essential component a pH-dependent manner (Nishida et al. 1984; of an accessory pathway of T-cell activation and Muneyuki et al. 1985; Yonezawa et al. 1985; the nuclei in the transformed T-cell lymphoma Nishida et al. 1987; Abe & Obinata, 1989). These cell line Jurkat' contain substantial amounts of functions are inhibited by phosphoinositides and dephosphorylated cofilin (Samstaget al. 1994). phosphoinositol through an interaction with one It is postulated that cofilin may be involved of the actin-binding motifs of cofilin (Yonezawa in preventing apoptosis in T-cells stimulated et al. 1990; Yonezawa et al. 1991). In fibroblasts, through this accessory pathway. heat shock or exposure to dimethyl sulfoxide Two cofilin isoforms have been identified in induces binding of cofilin to actin and trans mouse, M-type (‘muscle’) and NM-type (‘non location of the cofilin-actin complex from the muscle’) (Ono et al. 1994). In contrast, the 2 0 2 G . T. G il l e t t a n d o t h e r s Table 1. Alignment of predicted aminoacid sequences of M- and NM-type Cofilins 60 t35436 r ...... m ...... CHKCOF ...... P ...... K...... TR...... MUSCOFILIN MA S GVTVNDEVIKVENDMKVRKS S T QEEIKKRKKAVLECL SDDKRQ11VEEAKQILVGDI MUSCOF A. S. G...... P. . V. . . . : ...... E. . KN.. L. . G. E V CFL1 A. S. G...... P. . V ...... E. . KN.. L. . G. E V PIGCOFIL A. S. G...... P.. V i ...... E. . KN. . L. . G. E V RNCOFIL A. S .G ...... P. .V ...... E. .KN. . L. . G.E...... V 120 hsb35h081 t35436 . S. CHKCOF A. MUSCOFILIN GDTVEDPYTSEVKLLPLNDCRYALYDATYETKESKKEDLVEIEWAPESAPLKSKMIYASS MUSCOF . Q. . D. . . . T. . . M. . DK...... N ...... CFL1 . Q. . D. . . AT. . . M. . DK...... S ...... PIGCOFIL . Q. .D . . . AT. . .M. .D K ...... C...... RNCOFIL . Q. .D. . . . T. . .M. .DK ...... S ...... 166 hsdheb062 ...... N ...... t31361 ...... N ...... hsb35h081 ...... N ...... t35436 ...... CHKCOF ...... N. mmb613 ...... MUSCOFILIN KDAIKKKPTGIKHEWQVNGLDDI KDRSTLGEKLGGSVWSLEGKPL MUSCOF ...... L...... L. A. CYEEV. . . C. . A ..... A. I ...... CFL1 ...... L...... L. A. CYEEV. . . C. . A ..... A. I ...... PIGCOFIL ...... L...... L. A. CYEEV. . . C. . A ..... A. I...... RNCOFIL ...... L...... L. A. CYEEV. . . C. . A ..... A. I ...... Dots ‘. ’ indicate aminoacid identity with the MUSCOFILIN sequence (shown in full). Spaces are used to denote aminoacids where translation was not possible because of nucleotide sequence ambiguity. Aminoacids (one letter codes) are numbered on the right. GenBank sequence identifiers used (except for CFL1). Human M-type clones t35436, t31361 (Adams et al. 1995) clones hsb35h081, hsdheb062 (Genexpress, 1995) Mouse M-type clone mmb613 (Davies et al. 1994) CHKCOF Chicken M-type cofilin (Abe et al. 1990) MUSCOFILIN Mouse M-type cofilin (Ono et al. 1994) MUSCOF Mouse NM-type cofilin (Moriyama et al. 1990) CFL1 Human NM-type cofilin (Ogawaet al. 1990; this paper, EMBL X95404) PIGCOFIL Pig NM-type cofilin (Matsuzakiet al. 1988) RNCOFIL Rat NM-type cofilin (Shirasawa et al. 1991) Note: there appears to be a sequencing error in t35436 at nucleotide 290, and the frame of the last four aminoacids is altered. Similarly, the last five aminoacids of hsb35h081 are out of frame because of a possible error at base 177. chicken appears to have only a single cofilin, the bryonic skeletal), mouse and human (placental) primary structure of which is most similar to the have also been characterised (Abe et al. 1990; mouse M-type. The mouse M-type cofilin is Moriyama et al. 1990; Ogawaet al. 1990). They expressed in heart, skeletal muscle and testis, are highly homologous and have over 80 % amino whereas the NM-type is found in a wide variety acid identity. The recently cloned mouse M-type of tissues studied (including heart and testis). cofilin (Ono et al. 1994) has greater homology NM-type cofilin expression is minimal in mature with chicken cofilin (96% aminoacid identity) mammalian skeletal muscle (Ono et al. 1994). than with the previously published mouse cofilin The cDNA sequence of a cofilin, cloned from a amino acid sequence (81 % identity). This latter porcine brain cDNA library, was first reported in clone, (Moriyama et al. 1990), was obtained from 1988 (Matsufcaki et al. 1988). It encodes a 166 a mouse brain library, and has been designated a amino acid protein which has a molecular mass NM-type cofilin by Ono et al. A homologue of of about 18-5 kDa. Cofilins from chicken (em cofilin has also been identified in Saccharomyces Mapping of CFLI type cofilins show surprising inter-species se quence conservation but there is no similarity between the 3'UTS of the different isoforms. The coding sequence of the two types is homologous but each has preferred aminoacids at certain positions which are conserved between species, for example, at 12 sites in the C-terminal 40 aminoacids (Table 1). The characteristic amino acid sequence of the M-type cofilin in this part of the coding region and the nucleotide sequence of the 3'UTS permits identification of expressed sequence tags submitted to the sequence data Fig. 1. PCR amplification ofCFLl in human, rodent and bases which are likely to be derived from human somatic cell hybrid DXA. Key: (from left): Kb marker, M-type cofilin cDNA clones. The deduced amino Genomic Controls: Human ( + ), RAG mouse ( —), FAZA acid sequences of four of these, T35436. rat ( —). WG3H hamster ( —). Hybrids: J1CL4 ( + ). HORP9.5 ( + ), HORL411B6 ( + ), MOG2E5 (-), HSB35H081, T31361 and HSDHEB062 are TWIN19F9 (-). EDAG3R (-). FST9/5 (-), FST9/10 shown in Table 1 (GenBank identifiers, see Table ( —), CTP34B4 ( —), Kb marker. 1 for references). T35436 is entirely coding sequence; T31361. HSDHEB062 and cerevisiae with 35-41% identity to mammalian HSB35H081 span the 3' end of the coding cofilin (Iida et al. 1993; Moon et al. 1993). sequence. The putative 3'UTS of these differ at Two full length cDNA clones approximately only one nucleotide and are very similar to both 1059 bp in length were isolated from libraries the mouse M-type cofilin and the chicken cofilin. made from the human promyelocytic cell line We have designed oligonucleotide primers to HL60 following induction with DMSO (Rowe amplify the 3'UTS of human M-type cofilin. et al. unpublished; EMBL accession number 0FL2. in human-rodent somatic cell hybrids, X95404). The sequence of the longest open read exploiting the few discrepancies between the ing frame of these clones is identical to the conserved M-type cofilin 3'UTS of these species. published 498 bp coding sequence of the partial Using these primers we have localised human human placental clone, (Ogawa et al. 1990) and CFL2 to chromosome 14. displays close homology to pig cofilin and mouse NM-type cofilin (99-4 and 98-8% aminoacid METHODS identity). The 510 bp 3' untranslated sequence is CFL1 PCR of a somatic cell hybrid panel also identical or very similar to 19 cDNA expressed sequence tags (data not shown). We DNA from rodent parent cell lines, somatic have designed oligonucleotide primers from 3' cell hybrids, and human controls was amplified untranslated sequence (3'UTS) of the HL60 by PCR. The human chromosomal content of the clones to amplify the human gene in somatic cell somatic cell hybrids has been characterised hybrids and in a panel of irradiation hybrids extensively by two or more of the following (derived from a parent hybrid containing human techniques: karyotyping, isozyme analysis, chromosome 11 as its only human component). Southern hybridisation, PCR and FISH. The Genomic cosmid clones have been identified in a oligonucleotide primer sequences were chosen gridded chromosome 11 cosmid library (Nizetic from 3'UTS of the human CFL1 cDNA (EMBL et al. 1991) and these have been mapped to X 95404) forward. 5'-ATC CCC ATT CCC CAC chromosome llq 13 by fluorescence in situ hy CTG G-3'; reverse, 5'-TCC TGC TTC CAT GAG bridisation. FI8H. TAG CCG T-3'. The primers were selected by The 3' non-eoding sequences of the M and NM- maximising discrepancies between the human 204 G. T . G il l e t t a n d o t h e r s Table 2. Segregation of CFL1 in a somatic cell hybrid mapping panel Chromosome Hybrid 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1617 1819 20 21 22 X Y CFL1 Reference DUR4.3 ------1-----1------1- + + + + f 1- + h + + f 4- Wonget al. 1987 HORL411B6 + - + ------+ _ + _ + ---+ ------+ + + Wonget al. 1987 HORP9.5 1- + H h — — ------1- + + Wonget al. 1987 J1CL4 _____ ------+ — — — — ______|_ Kao et al. 1976 MOG2C2 + — + + + — + + + + + — — + + + + + + + + + + + Wonget al. 1987 853 f- — Burke et al. 1985 762-8a _____ ------4. ______)_ _ Fisher et al. 1987 CTP34B4 + ? + — + + + + — — — + + + — + + + — — — — + — Wonget al. 1987 EDAG3R -t- — + — + — + — — + — + + + + + + + + — + — — — Boyd et al. 1987 F4SC13CL12 f ------+ ------f - Wong et al. 1987 FG10E8EP2 — + — — + + — + — — — — — — + — — + — — + — + — Purdue et al. 1991 FG10E8EP2.6 ------1------1------+ ------+ ------H------1- — Purdue et al. 1991 FST9/5 — — + + — + + + + + — + — + + — — + — + — + + — Kielty et al. 1982 FST9/10 ----- + + - + - + f + - + + + + ------+ -+ - + + - Wonget a l 1987 GM10611 + - - NIGMS, 1994 GM10612 + - - NIGMS, 1994 MOG2E5 + - + + + + + + + + - + + + + + + + + + + - Wonget al. 1987 MOG34A4 + - + + + + ? + - + + +? ?- + + -+ + + - Solomonet al. 1979 POTB2/B2 ------1------1--- 1- + — Andrews et al. 1981 SIF15P5 _ Wong et al. 1987 TWIN19F9 + + + + - + - + + - - + - + - - + + - + + + - - Wong et al. 1987 Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16171819 20 21 22 X Y CFL/Chromosome C + / + 20312 01113 52232 12312 243 D +/- 35243 54442 03322 43243 311 C - / - 1012 9 11 10 8 10 9 11 9 16 8 11 7 8 12 11 7 1211 1011 7 D - / + 53756 85747 08597 35945 6582 Key: ?, not tested or equivocal result; f, hybrid contains a fragment of the chromosome; C, concordant; D discordant and the mouse and pig sequences at the3' end of each oligomer, using the LINEUP sequence GFL1 PCR of a chromosome 11 somatic cell editor program (GCG, 1994). These oligomers hybrid panel amplify a 179 bp product. DNA from somatic cell hybrids containing Amplifications were performed in 25 /d vol characterised fragments of chromosome 11 (Hunt umes in a Hybaid Thermal Reactor for thirty- et al. 1994) was amplified using the same CFL1 five cycles (denaturation 93 °C for 20 s, annealing primers, as described above. 60 °C for 20 s, extension 72 °C for 20 s). Each reaction contained 0-25 pg genomic DNA, 0‘5pM CFL2 PCR of a somatic cell hybrid panel each oligonucleotide primer, and 0*8 units of DNA from a panel of hybrids similar to those Advanced BiotechnologiesThermus species poly used in the chromosomal localisation of CFL1 merase. The enzyme was added to each reaction was amplified using primers selected from the after an initial 5 minute denaturation at 97 °C. 3'UTS of human EST sequences with homology Final reaction mixes contained, in addition, to mouse M-type cofilin and chicken cofilin to 210 each dNTP, 1-5 m l MgCl2, 50 mM KC1, maximise discrepancies between the consensus 10 mM Tris-HCl pH 9*0, 0*01 % gelatin, 0*1 % human CFL2 sequence and the mouse sequence. Triton X-100 and 10% glycerol. The primer sequences were, forward, 5'-AC A 5 p\ of each reaction product was electro- ATG AAT GAA GGA AAT ATC ATT TAT-3', phoresed in 2 % agarose gels in TBE buffer and reverse, 5'-AAA TAA TAC TGA AAA AAG TTG the product visualised by ethidium bromide ACC ATC-3'. The product of the amplification is staining under uv illumination. 271 bp. Reaction conditions were identical to Mapping of CFL1 & CFL2 205 ,—— Fluorescent In Situ Hybridisation Fluorescent in situ hybridisation to metaphase chromosomes was performed as described pre viously with the exception that 5-bromo- deoxyuridine (BrdU) was incorporated into the chromosomes (Gillett et al. 1993). The five cosmid clones isolated from the ICRF RLDB were studied. Whole cosmids were biotinylated by nick translation. Labelled cosmid probe was annealed with excess Cotl DXA (Life Tech nologies) to compete out human repetitive se quences, and hybridised to human metaphase Fig. 2. PCR amplification ofCFLl in human, rodent and somatic cell hybrid DNA (chromosome 11 mapping chromosome preparations overnight at 37 °C. panel). Key: (from left): Kb marker, Genomic Controls: The signal was detected using avidin conjugated Human ( + ). WG3H hamster ( —), Hybrids: A3EW3B to fluorescein isothiocyanate, and amplified as ( + ), A3RS12B ( + ), M11X ( + ). PG48 ( + ), MARI ( + ), Jl-11 (-), EJNAC (-), CJ52 ( - ) , J1CL4 ( + ), Kb described previously. The chromosomes were marker. counterstained with propidium iodide and di- aminophenolindole (DAPI) to obtain R-banding when visualised under LW illumination. The those used for CFL1 (except for an annealing images were collated by means of confocal laser temperature of 58 °C). microscopy (Biorad MRC 600). Identification of CFL1 genomic clones from the SEA PCR of cosmid glycerol stocks ICRF RLDB gridded chromosome 11 cosmid The CFL1 cosmids isolated were tested for the library presence of SEA sequences using published 32P-oligo-labelled 179 bp PCR product ampli primers (Richard et al. 1991), and the same fied from human DXA template was hybridised amplification conditions as for CFL1. These to a human chromosome 11-specific genomic primers amplify a 130-base pair product. 0-5 /d of cosmid library gridded onto nylon membrane. a glycerol stock of cosmid-containing E. coli was The filters were kindly provided by Dr G. added to the reaction mix in place of target Zehetner, Imperial Cancer Research Fund Ref DXA. erence Library DataBase (RLI)B. library num RESULTS ber 107. set 12) (Nizetic et al. 1991). The PCR A mplification of CFL1 and SEA product was electrophoresed through 1-2% aga rose in TBE buffer. The 179 bp band was excised A single product of the expected size, 179 bp, from the gel and centrifuged through siliconised was seen when human DXA was amplified using glass wool. The concentration of DXA in the the CFL1 primers. Larger-sized products were eluate was estimated by fiuorimetrv and 50 ng of also visible in some of the somatic cell hybrids DXA was random-prime labelled with 32P (Amer- but these non-specific products could easily be sham Megaprime kit). The whole labelling re differentiated from the specific 179 bp band. This action was hybridised to the filters at 65 °C latter product was not observed when mouse, rat overnight in 05 M sodium phosphate buffer, or hamster control TO A was amplified [\%. pH 7-2/7% 8DS/1 mM EDTA. The filters were 1). The results of the amplifications of the somatic washed at 65 °C in 40 mM sodium pho sp h ate/1 % cell hybrid panel are listed in Table 2. There is SDS for a total of 45 min. and were autoradio complete concordance between retention of chro graphed overnight at room temperature. mosome 11 in the hybrids and amplification of 206 G. T. G il l e t t a n d o t h e r s Table 3. Segregation of CFLI in a human chromosome 11 somatic cell hybrid mapping panel Chromosome 11 Hybrid Component CFLI Reference A3EW3B 11 pter-11 q24 + (Guerts van Kessel et at. 1985) A3RS12B 11 pter-11 q23 + (8acchi et al. 1986) M ll-X 11 pter-1 lq23 + (Chiang et al. 1984) PG48 11 pter-11 q22.2 + (Gillett et al. 1994) MARI 11 pter-1 lq21 + (Fletcher et al. 1993) .11-11 11 pter-11 q 12 - (Glaser et al. 1989) EJNAC 11 pter-1 lcen - (Porteous et al. 1989) CJ52 11 ql3.2-11qter - (Koeffler et al. 1981) Calculation of Lod score, recombination fraction and centiray 40,000 by the method described by Cox (Cox et al. 1990) indicated that CFLI and SEA are closely linked: LOD 6.92, 0 = zero, cRay40 000 = zero. SEA has been mapped pre viously to llql3 by in situ hybridisation (Williams et al. 1988). CFLI cosmids Hybridisation with labelled cofilin PCR prod uct identified five cosmids which were isolated from the ICRF human chromosome 11 gridded Fig. 3. PCR amplification of CFL1 3'UTS and SEA in cosmid library, number 107 (L4/FS11): genomic cosmid glycerol stocks from the ICRF human ICR Fc*107E 1288, ICRFcl07E1046, chromosome 11 library, number 107 (L4/FS11). Key: (from left): Kb marker, CFLI primers: Human genomic ICRFcl07E0622, ICRFcl07A1277 and control ( + ), ICRFcl07E1288 ( + ), ICRFcI07E1046 ( + ), ICRFcl07G07102. The 130 bp product was amp ICRFcl07E0622 ( + ), ICRFcl07A1277 ( + ), lified in four of these using the SEA primers; ICRFcl07G07102 ( + ) and ICRFcl07B0333 (negative control, —), SEA primers: Human genomic control ( + ), ICR Fc 107 El 046 was negative (Figure 3). ICR Fc 107 El 288 ( + ), ICRFcl07E1046 ( - ) , ICRFcl07E0622 ( + ), ICRFcl07A1277 ( + ), CFLI FISH ICRFcl07G07102 ( + ) and ICRFcl07B0333 (negative control. —), Kb marker. In situ hybridisation of biotinylated cosmid probe to human metaphase chromosomes the CFLI sequence, and at least six examples of localised the signal to chromosome I lql3: there discordance with each other chromosome. was little or no non-specific background (Figure PCR of somatic cell hybrids containing charac 4). The signal was sufficiently strong to be easily terised fragments of human chromosome 11 visible in the majority of metaphase spreads. For permitted regional localisation to the interval each of the five cosmids, at least ten spreads were 1 lql2—11 ql3.2 (Fig. 2, Table 3). examined and specific signals were seen on each Retention of CFLI in a panel of 47 high dose chromatid of the chromosome 11 homologues. irradiation hybrids was studied by PCR of hybrid Amplification of CFL2 DNA using the primers listed above (Gillett et al. 1993). CFLI product was obtained in five A single product of the expected size, 277 bp, hybrids: Jo2\ Jol5\ Jo31. Jo48', Jo50' (data not was seen when human DXA was amplified using shown). These same hybrids also gave positive the CFL2 primers, and was not observed when results with an amplification of part of the SEA mouse, rat or hamster control DNA was ampli oncogene (Richard et al. 1991). All the other 42 fied (Figure 5). The results of the amplifications hybrids were negative for both CFLI and SEA. of the somatic cell hybrid panel are listed in Mapping of CFLI 6c CFL2 207 Table 4. There is complete concordance between retention of chromosome 14 in the hybrids and 4 amplification of the CFL2 sequence, and at least six examples of discordance with each other ij iX L W* chromosome, in each case including at least one 11 l / i (; w * A »5 f i i example in which the chromosome was present f! # and amplification of CFL2 was not seen. ■ *i \ V' f k : DISCUSSION "•V^; We have mapped the gene for Cofilin, CFLI to chromosome 11 q 13 by PCR of two panels of somatic cell hybrids, and by fluorescent in situ hybridisation of genomic cosmid CFLI clones. The human homologue of avian retrovirus pro- viral tyrosine kinase, SEA, has previously been mapped to the same band. Ilql3, by in situ hybridisation (Williams et al. 1988). We have demonstrated that CFLI is closely linked to SEA bv amplification of DXA from a high-dose irradiation hybrid panel, and by demonstration of both CFLI and SEA sequences in each of four of the five cosmids isolated from the ICRF chromosome 11 gridded cosmid library. The Fig. 4. Human metaphase chromosomes showing fluores average size of the partial digest used to cent in situ hybridisation of biotinylated cofilin cosmid construct similar cosmid libraries was about probe localised to chromosome 11 q 13. Clone ICRFe 107E1288 shown: regions of hybridisation (11 q 13) 55 kb (Nizetic et al. 1991). CFLI and SEA arrowed. Upper: entire spread. Lower: partial spread, probably lie within an interval of approximately enlarged. this size. Loci in the 1 lql3 region have been ordered in a separate irradiation hybrid panel (Richard et al. 1991: Jam es et al. 1994) which places SEA m between PYGM (centromeric) and D11S913 (telomeric), separated by a centiray distance of about 54 cRay9000 (approximately 2-7 Mb). Their analysis shows SEA to be most closely linked to D11S1957E (centromeric) and D11S951E, ex pressed sequence tagged sites lying between PYGM and D11S913. D11S1957E and D11S951E are estimated to be 14-8 cRay apart, equivalent to approximately 750 kb. We an ticipate th at CFLI is close to these loci, within Fig. 5. PCR amplification of CFL2 in human, rodent and the Multiple Endocrine Neoplasia type 1 can somatic cell hybrid DNA. Key: (from left): Genomic didate region (MEN1, Larsson et al. 1988). Controls: Human ( + ), RAG mouse ( —). FAZA rat ( —), WG3H hamster ( - ) . Hybrids: GM10479 ( + ), C10B2BU Sequence comparison with other mammalian (+), HORP9.5 (+), COX2 ( - ) . 5647CL22 ( - ) . FW4V6- cofilins indicates that the cofilin which we have A ( - ) , HORL411 B6(X4) ( - ) , 762-8a ( - ) , GM07299 ( —), GM10612 ( —), GM10611 ( —), Kb marker. mapped to chromosome 11 is the widely-ex- 2 0 8 G. T. Gil l e t t a n d o th er s Table 4. Segregation of CFL2 in a somatic cell hybrid mapping panel Chromosome Hybrid 1 2 3 4 5 6 7 8 9 10 1112131415 1617181920 21 22 X Y CFLIL Reference 3W4C15 ——————+ —— ? + + - + + _ + ------+ — + + Wonget al. 1987 C10B2BU ——+ + —- + ———- 1----- 1— ------——— + Humphries et al. 1983 CTP34B4 + + + —+ + —+ ———+ + + - + + + ----- —— + + Wonget al. 1987 CTP412A2 —+ ——— + + ————- - + ? ? + ?----- —— + + Joneset al. 1976 DUR4.3 -—+ —+ ————+ + H—1—(■ f - + + - + + + f + Wonget al. 1987 EDAG3R + - + —+ —+ ——+ —+ + + ? ? + + + - +—— + Boyd et al. 1987 FST9/10 —- + + — + - + %+ —+ + + + -----+ - + — + + + Wonget al. 1987 GM10479 ———————————-----+ - f ------——— + NIGMS, 1994 HORP9.5 + + + - + - — + + + Wonget al. 1987 MOG2C2 + —+ + + —H—1—1—h + -----+ + + + + + + + + + + Wonget al. 1987 MOG2E5 + —+ + + + + + + + —+ + + + + + + ----- + + + + Wonget al. 1987 PLTI.S —+ + + — + + + —— + + - + - - + + - + + + 2 + Solomon et al. 1979 SIF15P5 —+ ——- + + ——+ —-----+ + ------(. —— + + Wonget al. 1987 TWIN19F9 + + + + — + —+ + —- + ? + - - + + - + + + — + Wonget al. 1987 laA9498(602 + ) ————— i —————+ ------+ — + — Wonget al. 1987 289 ————— — — f —— f f + - - — Zhonget al. 1992 762-8a + + — Fisher et al. 1987 2806H7 + ------— Mulley & Callen, 1986 5647CL22 ———+ + + + + —— + - + ----- + - ? - + + ——— Nagarajanet al. 1986 640-63al2 ————— ——— f ——------———— Jones & Kao, 1984 CON2 f — 2 + —————— 2 — 2 ------+ + f - — + + — Swallow et al., 1987 FG10E8EP2 —+ ——+ f —+ f ——------+ ------+ — + — Purdue et al. 1991 FG10E8EP2.6 ————+ — 2 + —— 2 _ 2 - + ------1_----- 2 2 + — Purdue et al. 1991 FG10E8EP2.9 —+ ——+ ——+ ———------+ ------y ----- + — + — Purdue et al. 1991 FW4V6-A —+ ——+ + —+ ———+ -----+ ------1------+ — + 2 — Griffo et al. 1993 GM07299 + + — NIGMS, 1994 GM10253 —— + ——— —————------? — ? —— —— NIGMS, 1994 GM10478 ——— + — + ——— + — ------1_ — — —— NIGMS, 1994 GM10611 + — NIGMS, 1994 GM10612 ------+ -— NIGMS, 1994 HORL411B6 N4 ——+ ——————— + - + ------1------— + + — Wonget al. 1987 J1CL4 + — Kao et al. 1976 MCP6BRA f f — Goodfellow et al. 1982 PgME25NU — + f — de Klein et al. 1982 SIF4A24E1 ——- + — -—————------+ ------+ - + - Wonget al. 1987 Chromosome 1 2 3 4 5 6 7 8 9 10 1112131415 1617181920 21 22 X Y CFL2 CFL2 / Chromosome C + / + 5 5 9 6 5 7 8 6 3 7 5 10 514 5 3 9 8 2 6 7 7 8 D + / - 9 9 5 8 9 7 6 8 10 6 9 4 8 0 6 8 5 5 12 8 7 7 4 C - / - 1918181716 1519151819 1518162117 1919131918 141710 D - / + 1 3 2 4 5 3 1 5 1 2 3 2 3 0 4 2 2 6 1 2 6 3 9 1 Key: as Table 2. 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Science genes in the human, with the exception of an 250, 245-250. abstract published but not presented at the D a v id s o n , M. M. L. & H a s la m , R. J. (1994). Dephospho Annual Meeting of the American Society of rylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+.Biochem. J. 301, 41-47. Human Genetics, Montreal, 1994. This abstract D a v ie s , R. W., R o b e r t s , A. B., M o r r is ,XA. J., G r i f indicated localisation to chromosome lq25 f i t h , G. W., J e r e c i c , J., G h a n d i, S., K a is e r , K. & S a v io z , A. (1994). Enhanced access to rare brain (Hung et al. 1994). cDNAs by prescreening libraries: 207 new mouse brain ESTs. Genomics 24, 456-463. d e K l e i n , A., van Kessel, A. G., G r o s v e ld , G., We thank Dr Nigel Spurr, Imperial Cancer Research B a r tr a m , C. R., Hagemeijer, A., B o o ts m a , D., Fund, South Minims, UK and Dr Veronica van Hey ningen, MRC Human Genetics Unit, Edinburgh, UK for S p u r r , N. 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