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Isolation and mapping of clones from human chromosome 5 Bernard, Lynn Elizabeth 1992

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ISOLATION AND MAPPING OF CLONES FROM HUMAN CHROMOSOME 5 by LYNN ELIZABETH BERNARD B.Sc., Simon Fraser University, 1987 A THESIS SUBMITI’ED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES GENETICS PROGRAMME  We accept this thesis as conforming to the required standard  Signature(s) removed to protect privacy  THE UNIVERSITY OF BRITISH COLUMBIA April, 1992  0 Lynn Elizabeth Bernard, 1992  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Signature(s) removed to protect privacy  DEPARTMENT OF MEDICAL GENETICS The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 April 22, 1992  ABSTRACT This thesis describes the isolation and mapping of DNA clones from human chromosome 5, with emphasis on the 5q11.2-q13.3 region. This region is of interest because of a Vancouver family in which trisomy for the 5q11.2-q13.3 region co segregates with schizophrenia and renal anomalies. The 5q11.2-q13.3 region is of additional interest since chronic spinal muscular atrophy has been linked to markers within this region. A novel technique, Alu PCR differential hybridization, was developed to isolate clones from the 5q11.2-q13.3 region. The somatic cell hybrid HHW1O64 contains a chromosome 5 with an interstitial deletion of 5q11.2-q13.3, derived from a carrier member of the family segregating for the segmental trisomy. Alu PCR differential hybridization was used to isolate twenty chromosome 5 clones absent from the HHW1O64 hybrid. Radiation hybrid mapping was used to determine a rough order of clones isolated byAlu PCR differential hybridization. Order information was used to select clones to screen for polymorphisms. Nine polymorphic systems were detected. Multipoint linkage mapping of two of the new polymorphisms (D5S257 and D5S268) placed them onto the p arm of chromosome 5. This result was unexpected, since D5S257 and D5S268 were isolated based on their absence from the HHW1O64 hybrid. The HHW1O64 somatic cell hybrid therefore contains a deletion within the p arm of chromosome 5 in addition to the expected interstitial deletion of 5q11.2q13.3. Analysis of D5S257 and D5S268 in the family segregating for the segmental trisomy indicated that the rearrangement in this family does not involve Sp. Multipoint linkage analysis of the polymorphism associated with D5S260  11  Abstract placed this marker between two chromosome 5 index markers (D5S76 and D5S21) which had been mapped outside the 5q11.2-q13.3 interstitial deletion in HHW1O64. This map position was unexpected, since D5S260 was isolated on the basis of absence from the HHW1O64 hybrid. The most likely explanation for the inconsistency between the linkage and somatic cell hybrid data is that the chromosome 5 present in the HHW1O64 hybrid is deleted for DNA both proximal and distal to D5S76. This complex 5q rearrangement is present in the family segregating for the segmental trisomy, based on the analysis of D5S260 and D5S76 in this family.  111  TABLE OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  viii  LIST OF FIGURES  x  DEDICATION  xii  ACKNOWLEDGEMENTS  xiii  1. INTRODUCTION 1.1 THE 5q11.2-q13.3 REGION OF THE HUMAN GENOME 1.2 CHROMOSOMAL REARRANGEMENTS AND SOMATIC CELL HYBRIDS 1.3 ISOLATION OF DNA MARKERS 1.4PCR 1.SALUELEMENTS 1.6ALUPCR 1.7 RADIATION HYBRID MAPPING 1.8 POLYMORPHISM SCREENING 1.9 LINKAGE MAPPING  4 6 6 7 9 13 14 17  2. MATERIALS AND METhODS 2.1 MATERIALS 2.1.1 Somatic cell hybrids 2.1.2 Phage libraries 2.2 METHODS 2.2.1 Restriction enzyme digestions 2.2.2 Gel electrophoresis 2.2.3 Southern blotting 2.2.4 Oligolabeling 2.2.5 Preannealing with human DNA 2.2.6 Prehybridization, hybridization and washing 2.2.7 (GT) tract detection and isolation 2.2.8 Phage maniulations 2.2.8.1 Phage plating 2.2.8.2 Plaque lifts and plaque purification  22 22 22 23 24 24 25 25 27 29 29 30 31 31 32  iv  1 1  Table of Contents 2.2.8.3 Small scale phage prep 2.2.8.4 Large scale phage prep 2.2.9 Plasmid manjpulations 2.2.9.1 Ligation 2.2.9.2 Production of competent cells and transformation 2.2.9.3 Ligation independent cloning (LIC) 2.2.9.4 Colony screens 2.2.9.5 Plasmid minprep 2.2.9.6 Plasmid large scale prep 2.2.10 Sequencing 2.2 10.1 Denaturation of template 2.2.1 0.2 Sequencing reactions 2.2.1 0.3 Sequencing primers 2.2.10.4 Denaturing polyaciylamide gel electrophoresis 2.2.1 0.5 Manipulation of sequence data 2.2.11 Polymerase chain reaction (PCR) 2.2.11.1 Standard PCR reaction conditions 2.2.11.2 Standard cycling conditions 2.2.11.3 PCR primers synthesis and purification 2.2.11.4 Primer end-labeling 2.2.11.5 Electrophoresis of radiolabeled PCR products 2.2.11.6 Specific PCR reactions and primer sequences 2.2.l2Alu PCR differential hybridization 2.2.13 Statistical analysis 2.2.13.1 Radiation hybrid typing and analysis 2.2.13.2 Polymorphism typing and analysis 2.2.14 Somatic cell hybrid maniulations 2.2.14.1 Cell culture 2.2.14.2 Somatic cell hybrid DNA preparation 2.2.14.3 Preparation and staining of metaphase chromosomes  33 34 36 36 37 38 40 41 42 44 44 45 46 46 48 49 49 49 50 51 52 52 57 57 57 57 58 58 59 60  -  62 3. RESULTS 3.1 CLONE ISOLA TIONAND LOCALIZATION 62 3.1.1 Alu PCR differential hybridization 62 3.1.2 Alu PCR products from clones isolated by differential hybridization.... 66 3.1.2.1 Identification of multiple isolates and Alu- T3, Alu-T7 PCR for clones 5PCR1 to 5PCRJ1 69 3.1.2.2 Identification of multiple isolates forphage A1u19 to A1u73 70 ..  V  Table of Contents 3.1.3 Confirmation of localization Alu PCR localization blots 3.1.4 Confinnation of localization Genomic localization blots 3.1.5 Confirmation of localization PCR 3.1.6 Majority of clones do not correspond to visible differences between hybrid Alu PCR products 3.2 RADL4 TION HYBRID MAPPING 3.2.1 Radiation hybrid typing 3.2.2 Twopoint radiation hybrid analysis 3.2.3 Fouipoint radiation hybrid analysis 3.3 POLYMORPHISMS 3.3.1 Polymorphism screening and typing 3.3.2D5S205 3.3.3 D5S253 3.3.4 D5S257 3.3.5D5S260 3.3.6D5S262 3.3.7D5S265 3.3. Zi System AluS2A 3.3. Z2 System A1uS2B 3.3.8D5S266 3.3.9D5S268 3.4 LINKA GE ANALYSIS 3.4.1 Twopoint linkage analysis 3.4.2 Multipoint linkage analysis 3.4.2.1 ChromosomeS markers 3.4.2.2A1u52A andAluS2B 3.5 H13W1064 CHARACTERIZATION 3.5.1 HHWi 064 Sp deletion 3.5.2 HHW1O64 Sq deletion 3.5.3 Cytogenetic analysis ofHRW1064 3.6 CHARACTERIZATION OF CHROMOSOMAL REARRANGEMENT SEGREGATING IN TRISOMY FAMILY .3.t5.lsp 3.6.15q -  -  -  -  4. DISCUSSION 4.1 CLONE ISOLATION 4.2 RADIATION HYBRID MAPPING  vi  73 78 78 80 82 82 87 89 93 93 101 103 107 110 113 117 121 129 131 136 139 139 147 147 148 154 154 157 159 161 161 162 164 164 169  Table of Contents 173 4.3 POLYMORPHISM SCREENING AND LINKAGE ANALYSIS 181 4.4 COMPARISON OF MAPPING METHODS 4.5 CHARACTERIZATION OFHHW1O64AND SEGMENTAL TRISOMY..184 193 4.6 CONCLUSIONS 194 4.7SUMMARY 196 4.8 PROPOSALS FOR FURTHER RESEARCH REFERENCES  199  APPENDIX 1. ALGORITHMS 1. Radiation Hybrid Mapping 2. Mapping Functions Haldane And Kosambi 3. Polymorphism Information Content (PlC) 4. Effective Number Of Informative Recombinants And Meioses  209 209 211 213 214  APPENDIX 2. RADIATION HYBRID DATA  215  -  vii  LIST OF TABLES Table 1. Clone isolation and localization  64  Table 2. Alu PCR products obtained from clones exhibiting differential signals  67  Table 3.Alu-T3 and Alu-T7 amplifications for clones 5PCR1 through 5PCR11  70  Table 4. Multiple Isolates  72  Table 5. Localization of clones by hybridization to somatic cell hybrids GM1O114 and HHW1O64 75 Table 6. Radiation hybrid retention frequencies  86  Table 7. Twopoint radiation hybrid scores for markers linked at lod >3  88  Table 8. Polymorphisms  94  Table 9. Sequence of Alu polyA tails  98  Table 10. D5S205 Allele frequencies  101  Table 11. D5S253 Allele frequencies  104  Table 12. D5S257 Allele frequencies  108  Table 13. D5S260 Allele frequencies  111  Table 14. D5S262 Allele frequencies  113  Table 15. Observed matings Alu52A  122  -  Table 16. Allele frequencies for polymorphic locus within A1u52A system assuming a single polymorphism 125 Table 17. Expected versus observed genotypes for polymorphic loci within A1u52A system assuming a single polymorphism 125 Table 18. Expected versus observed offspring phenotypes assuming a single polymorphism model for Alu52A  127  Table 19. Alu52B Allele frequencies  129  Table 20. D55266 Allele frequencies  132  Table 21. D5S268 Allele frequencies  137  viii  List of Tables  Table 22. Twopoint meiotic linkage analysis  142  Table 23. Effective number of informative meioses (N) for new polymorphic markers  146  Table 24. Multipoint linkage analysis  149  Table 25. Marker localization to 5p or 5q11.2-q13.3  156  Table 26. Multipoint linkage analysis involving index markers D5S21, D5S76, D5S6, and D5S39 159 Table 27. Physical location of index markers  ix  188  LIST OF FIGURES Figure 1. Family segregating for segmental trisomy  3  Figure 2. Polymerase chain reaction (PCR)  8  Figure 3. Schematic representation ofAlu PCR differential hybridization procedure 11 Figure 4. Representation of human chromosome present in somatic cell hybrids HHW1O64 and GM1O114  12  Figure 5. Detection of clones derived from 5q11.2-q13.3 by differential hybridization Figure 6. Clone localization by hybridization to Southern blots of hybrid Alu PCR products 77 Figure 7. Clone localization by hybridization to Southern blots of hybrid genomic DNA 79 Figure 8. Alu PCR products  81  Figure 9. Alu PCR products for radiation hybrids 1 through 20  84  Figure 10. Probes from D5S260 and D5S262 hybridized to radiation hybrids 1 to 20 Figure 11. Fourpoint Radiation hybrid analysis Group #1  91  Figure 12. Fourpoint Radiation hybrid analysis Group #2  92  Figure 13. Sequence of D5S257 (GT) 11 tract  96  -  -  Figure 14. Sequence of D5S254 Alu polyA tract and flanking sequence and alignment with Alu consensus sequence  100  Figure 15. D5S205 TaqI polymorphism  102  Figure 16. D5S253 (GT) tract and flanking sequence  105  Figure 17. D5S253 (GT) polymorphism  106  Figure 18. D5S257 (GT)n tract and flanking sequence  108  Figure 19. D5S257 (GT)n polymorphism  109  x  List of Figures Figure 20. D5S260 (GT)n tract and flanking sequence  111  Figure 21. D5S260 (GT)n polymorphism  112  Figure 22. D5S262 (GT)n tract and flanking sequence aligned with Alu consensus sequence 114 Figure 23. D5S262 (GT)n polymorphism  116  Figure 24. D5S265 Alu polyA tract and flanking sequence compared with Alu consensus sequence  119  Figure 25. D5S265 Alu polyA tract polymorphisms  120  Figure 26. AIu52A and A1u52B polymorphisms  130  Figure 27. D5S266 (GT)n tract and flanking sequence compared with Alu consensus sequence 133 Figure 28. D5S266 (GT)n polymorphism  135  Figure 29. D5S268 (GT)n tract and flanking sequence  137  Figure 30. D5S268 (GT)n polymorphism  138  Figure 31. Chromosome 5 multipoint linkage analysis Summary  153  Figure 32. Metaphase chromosomes from somatic cell hybrid HHW1O64  160  -  Figure 33. Family segregating for segmental trisomy typed for D5S268, D5S257, D5S76 and D5S260 163 Figure 34. Comparison of radiation hybrid map and meiotic linkage map  183  Figure 35. Derivative chromosome 5 present in HHW1O64 and carrier female from whom HHW1O64 was derived 192  xi  DEDICATION  To the memory of Paul Douglas Leigh Bernard.  xli  ACKNOWLEDGEMENTS  I would like to thank my supervisor, Dr. Stephen Wood, for making this thesis possible through his encouragement, advice and support. I would also like to thank the members of my supervisory committee, Dr. Ann Rose, Dr. Fred Dill, Dr. Robert McMaster and Dr. Paul Goodfellow, for helpful discussions and advice during the course of my research. I would like to thank the members of my lab, Mike Schertzer, Craig Kreklywich, Heather Mitchell and Karen Henderson for advice and support, with special gratitude to Craig Kreklywich for assistance with sequencing and typing of microsatellite repeats. I am also grateful for the technical advice provided by the members of the Rose and Goodfellow labs, especially from Dr. Terry Starr and Angela Brooks-Wilson. I would also like to thank Dr. Fred Dill for assistance with metaphase chromosome preparation and karyotyping. I wish to express my gratitude to my parents and my brother, Gord, for their love and their belief in my abilities. I would like to thank my husband, Bruce, for his love, patience and interest in my research, and the members of his family for their unfailing support. I would also like to thank my friends for their patience and encouragement, with special thanks to Naazlin Devji. This thesis was supported in part by a studentship from the Medical Research Council of Canada.  xlii  1. INTRODUCTION A long-term goal in the field of human genetics is the development of methods for the localization and isolation of genes causing human disease. This goal has been complicated by the large size of the human genome, 3X10 9 base pairs (bp), the complexity of the human body, and by our long generation times with relatively few offspring. To counter these difficulties, geneticists studying humans have made various adaptations to conventional genetic methods. Since the human genome is so large, it has been subdivided into more manageable pieces through various cloning technologies. Variability at the DNA level has been utilized for the study of gene transmission in both non-affected and disease-carrying families. Extensive statistical methods for analyzing this variability have been developed. The research goal of this thesis was to isolate DNA markers from a specific region of human chromosome 5, qll.2-q13.3, and to provide information on the physical and genetic location of these new markers. These new markers would then provide additional tools for the investigation of disease loci which lie within this region.  1.1 THE 5q11.2-q13.3 REGION OF THE HUMAN GENOME A Vancouver family of Asian descent with a chromosomal rearrangement involving 5q11.2-q13.3 presented the first clue that genes within this region may play a role in the etiology of certain cases of schizophrenia, and may also be involved in kidney development (Bassett et al, 1988, McGillivray et a!, 1990). Schizophrenia and renal anomalies were observed in the two members of this family who are trisomic for the 5q11.2-q13.3 region. An unaffected carrier family member has a  1  1. Introduction balanced direct insertion of chromosome 5 material into chromosome 1 (46, XX, mv ins (1;5)(q32.3;q13.3-qll.2)). A pedigree of this family is shown in Figure 1. The suggested association between chromosome 5q and schizophrenia was originally supported by a linkage study involving 7 families of British and Icelandic descent (Sherrington et al, 1988). This study demonstrated linkage between 5q markers and schizophrenia, with maximal lod scores for the disease locus between the markers D5S39 and D5S76. A variety of modes of inheritance and criteria for schizophrenia were investigated, along with various levels of penetrance. The maximal lod scores were obtained if individuals with schizophrenia, schizophrenia spectrum disorders and various fringe disorders were classified as affected, and the disease was considered dominantly inherited with 86% penetrance. This study published by Sherrington et a! (1988) has thus far been the sole report of linkage between chromosome 5q markers and schizophrenia. Four studies demonstrating strong evidence against linkage between this region of chromosome 5 and schizophrenia have been published (Kennedy et a!, 1988; St. Clair et a!, 1989; Detera-Wadleigh et a!, 1989; McGuffin et a!, 1990). Taken together, the published linkage data seem to indicate that a major gene predisposing to schizophrenia does not lie within the 5q11.2-q13.3 region. However, the possibility still exists that a small proportion of cases of familial schizophrenia involve gene(s) within 5q11.2q13.3. The members of the Vancouver family who carried the segmental trisomy for 5q11.2-q13.3 had renal abnormalities. The proband has an abnormal left kidney, while his maternal uncle lacks a left kidney. These abnormalities are consistent with a diagnosis of hereditary renal adysplasia, an autosomal dominant condition  2  1. Introduction Figure 1. Family segregating for segmental trisomy  48, XV, der(1) mv ins (1;5)(q323;q13Sqll2) 48, XX, mv ins(1:5)(q32.3;qlS.3q11.2)  D  48, XV  3  1. Introduction  (MIM #191830; McKusick, 1990). Genetic linkage studies involving markers within the 5q11.2-q13.3 region and hereditary renal adysplasia have not yet been performed. The 5q11.2-q13.3 region became of additional interest due to reports of linkage between markers within this region and childhood-onset proximal spinal muscular atrophy (SMA; Brzustowicz et a!, 1990; Melki et at, 1990; Gilliam et a!, 1990). Proximal SMA is characterized by the progressive degeneration of the lower motor neurons in the spinal cord and brain stem motor nuclei, leading to paralysis of the limbs and trunk (Wessel, 1989). SMA is the second most common fatal recessive disorder after cystic fibrosis (Pearn, 1980). SMA has been subdivided into three types on the basis of the age of onset and severity of the disease: acute Werdnig-Hoffmann disease (type I), intermediate Werdnig-Hoffmann disease (type II) and Wohlfart-Kugelberg-Welander disease (type III). All three types are iuherited in an autosomal recessive fashion, and together account for over 90% of childhood SMA. Genetic evidence currently suggests that all three forms map to the same locus on chromosome 5q (Brzustowicz et a!., 1990; Gilliam et a!., 1990). The most likely position for the 5q SMA is between markers D5S39 and D5S6 (Sheth et a!., 1991).  1.2 CHROMOSOMAL REARRANGEMENTS AND SOMATIC CELL TIYBRJDS  Cytologically visible chromosomal rearrangements often provide the first clues for positioning human diseases. Confirmation of gene localization can then be performed by the isolation of additional rearrangements involving the same chromosomal region or by genetic linkage studies. Detection of chromosomal  4  1. Introduction rearrangements has served as a starting point for the localization of many disease genes, including Duchenne muscular dystrophy (Jacobs et a!., 1981), Wilms’ tumor/aniridia (Riccardi et a!., 1978), Prader-Willi syndrome (Ledbetter et al., 1982), and retinoblastoma (Francke, 1976). Somatic cell hybrids are cell lines formed by the fusion of human cells with immortalized rodent cells. These cell lines randomly lose human chromosomes until only one or a few human chromosomes are present in a relatively stable state. The retention of a human chromosome can often be maintained through the use of selection. Somatic cell hybrids therefore allow the separation of the human genome into the naturally occurring division of the chromosome. The segregation of chromosomes from an individual with a chromosomal rearrangement often affords a method for further subdividing large amounts of DNA. Somatic cell hybrids containing a single rearranged human chromosome can often be useful not only in delineating the extent of the rearrangement, but also for positioning DNA markers. This positioning can be confirmed using somatic cell hybrid mapping panels, which consist of a number of somatic cell hybrids containing a variety of chromosomal segments. The balanced carrier in the family in which trisomy for 5q11.2-q13.3 segregates with schizophrenia and renal anomalies carries a chromosome 5 deleted for 5q11.2-q13.3 (Figure 1). This deleted chromosome was segregated into the somatic cell hybrid HHW1O64 by Gilliam et a! (1989). Markers absent in the HHW1O64 hybrid were therefore assigned to the 5q11.2-q13.3 region. Gilliam et a! (1989) assigned the markers D5S21, D5S76, D5S71, 0B7 and G21 to outside the 5q11.2-q13.3 region, while the markers D5S6, D5S39, D5S78, HEXB, DHFR,  5  1. Introduction D5S63 and D5S51 were assigned to within the deleted region.  1.3 ISOLATION OF DNA MARKERS The initial portion of my project involved the isolation of DNA fragments from within the 5q11.2-q13.3 region defined by the segmental trisomy. As indicated previously, this region is of interest because it potentially contains genes predisposing to schizophrenia and hereditary renal adysplasia. The area is of additional interest due to the localization of SMA to the D5S6 D5S39 interval. -  The generation of additional markers from within the 5q11.2-q13.3 region also serves to delineate the trisomic area. A variety of techniques had been devised for the isolation of human DNA fragments from chromosomal subregions prior to the development of polymerase chain reaction (PCR) techniques. Subtractive DNA cloning (Kunkel et a!., 1985; Nussbaum et a!., 1987) has been used for construction of recombinant libraries containing regions of interest. This technique requires large amounts of starting DNA. Libraries have been constructed by the physical microdissection of metaphase chromosomes (Kaiser et a!., 1987), which involves complex DNA manipulations. Libraries have also been made using DNA from somatic cell hybrids containing part or all of a single human chromosome (for example; Scambler et a!., 1987). These libraries necessarily contain a large amount of rodent background, and must therefore be screened to isolate the human DNA sequences.  1.4PCR The polymerase chain reaction (PCR) involves the exponential amplification  6  1. Introduction  of the region of DNA found between two primers located in reverse orientations (Saiki et al., 1985; Mullis et aL, 1986; Mullis and Faloona, 1987). A diagrammatic representation of this procedure is shown in Figure 2. The procedure involves repeated cycles consisting of: (1) denaturation of target DNA, (2) annealing of DNA primers and (3) production of new DNA by extension from each primer. A thermostable DNA polymerase is used to enable the cycling to be carried out without the continual addition of polymerase (Saiki et a!., 1988). The first cycle consists of a linear increase in the amount of target DNA, but each subsequent cycle results in an exponential increase in the target sequence.  1.5 AL U ELEMENTS  A/u elements are human repetitive elements belonging to the SINE (short interspersed repeat) family of mammalian repetitive elements. A/u elements are approximately 300 bp in length and consist of two directly repeating monomer units. A/u elements have a copy number between 7X10 5 and 9X10 5 in the human genome  (Hwu et al., 1986). A/u elements have homology with the 7SL RNA, and it has been suggested that A/u elements may represent defective 7SL RNA molecules that have been reverse-transcribed and inserted into the genome (Ulla and Tschudi, 1984; Chen et a!., 1985). A large number of individual A/u elements have been sequenced,  and were found to be highly conserved, allowing the generation of a consensus A/u sequence (Deininger et al., 1981; Kariya et a!., 1987). A/u-like elements have been described in the genomes of other primates and in rodents. However, a high degree of sequence divergence has occurred between the primate and rodent repeat units (Jelinek and Schmid, 1982).  7  1. Introduction Figure 2. Polymerase chain reaction (PCR)  -  Cycle 1  J Cycle 2 -1-1-  Cycle 3  11-  -  Exponential amplification of sequence between primers using the polymerase chain reaction. For simplicity, amplification is shown as starting from a single strand of DNA. Primers are shown as arrows.  8  1. Introduction 1.6ALUPCR The development of polymerase chain reactions using primers from human specific sequences such asAlu elements (Alu PCR) greatly facilitates the isolation of human fragments from mixed DNA sources such as cell hybrids. Alu PCR involves the use of primers corresponding to the consensus sequence of the Alu family of repetitive elements in order to amplify the human DNA found between two adjacent elements (Nelson et al., 1989, Brooks-Wilson et a!., 1990). Sufficient divergence has occurred between human Alu elements and their rodent Alu-like homologues to allow human-specific amplification between adjacent Alit elements in somatic cell hybrid DNA. Alit PCR can be used on a variety of DNA sources, with (Brooks-Wilson et al., 1990) or without (Nelson et al., 1989) the production of recombinant libraries. Several different procedures have been reported involving the use ofAlit mediated PCR to isolate DNA fragments from specific chromosomal subregions. Regional localization of DNA fragments from the X chromosome was performed by Alu PCR amplification of YAC and phage isolates followed by hybridization to DNA from somatic cell hybrids (Nelson et a!., 1989). Fragments from chromosome 10 were amplified byAlu PCR synthesis from a somatic cell hybrid and cloned (Brooks-Wilson et a!., 1990). Fragments from Xq28 have been isolated by detection of differences visible on ethidium bromide stained gels between the PCR products obtained from two related somatic cell hybrids using primers specific to Alit and to the Li family of repetitive elements (Ledbetter et al., 1990). A fragment from l7pii.2 was isolated by hybridizing sub-fractions of the Alu PCR product from a chromosome 17 hybrid to detect differences between the Alu PCR products from  9  1. Introduction several chromosome 17 hybrids (Patel et a!., 1990). An additional procedure for the isolation of region-specific fragments, Alu PCR differential hybridization, was developed as a part of this thesis (Bernard et a!., 1991a). Alu PCR differential hybridization is based upon the differential hybridization of the Alu PCR products from two related somatic cell hybrids to a chromosome specific phage library. A schematic representation of this technique is shown in Figure 3. Alu PCR differential hybridization was used in conjunction with  two chromosome 5 hybrids, HHW1O64, the somatic cell hybrid which contains chromosome 5 deleted for qll.2-q13.3, and GM1O114, which contains an intact chromosome 5 as the sole human component. A diagrammatic representation of the karyotype of the human chromosome present in each of these hybrids is shown in Figure 4. GM1O114 and HHW1O64 were used as substrates for anAlu PCR reaction. Differential hybridization of these Alu PCR products to a chromosome 5 phage library allowed the isolation of clones from the 5q11.2-13.3 deletion region. Clones were first identified on the basis of their hybridization to the Alu PCR product from the chromosome 5 hybrid. Clones from within the deletion were then detected by differential hybridization, since they hybridized to the Alu PCR product from the chromosome 5 hybrid but failed to hybridize to the Alu PCR product from the deletion chromosome 5 hybrid. This technique should be generally applicable to any somatic cell hybrid- deletion hybrid pair.  10  1. Introduction Figure 3. Schematic representation of Alu PCR differential hybridization procedure  phage Hbrary hybridize Alu POR product from Intact chrom hybrid  pick positives  h y b rid I ze  h y b r i d ze Alu POR product  Mu POR product  from  from  intact chrom hybrid  deletion hybrid  purify  11  Introduction Figure 4. Representation of human chromosome present in somatic cell hybrids HHW1O64 and GM1O114  15.3 15.2 15.1 15.3 14  15.2 15.1  13.3 13.2 13.1  14  12 11 11.1  13.3_ 13.2 I 13.1  11.2  12 11  12  11.1  13.1  11.2 13.3  13.2 13.3  I  I  14 14 15 21 21 22 22  23.1  23.1 23.2  23.2 23.3  23.3  31.1  31.1  31.2  31.2  31.3  31.3  32  32  33.1 33.2 33.3  33.1 .  .,  34 34  35.1 35.2 35.3  35.1 35.2 35.3  GM1O114  HHW 1064  12  1. Introduction 1.7 RADL4 TION HYBRID MAPPING Ordering of human loci using radiation-induced breakage of human chromosomes was introduced by Goss and Harris in 1975. This technique involves the production of human-rodent cell hybrids by the irradiation of human lymphoblasts, followed by fusion with rodent cells and growth on selective media. The order of human loci near the selectable marker can then be deduced from locus retention data. This technique was used to determine the order of four loci on the human X chromosome (Goss and Harris, 1975, 1977a). A modification of this method which removed the selective pressure for radiation hybrid growth was used to produce a map of 8 loci on human chromosome 1 (Goss and Harris, 1977b). Radiation hybrid mapping was developed as an extension of the method of Goss and Harris (Cox et a!., 1990). Radiation hybrid mapping involves two major modifications of the method of Goss and Harris. A somatic cell hybrid containing a human chromosome of interest is irradiated as a first step in radiation hybrid mapping, rather than the human lymphoblasts used by Goss and Harris. Secondly, analysis of large quantities of marker retention data was made possible by the development of a series of algorithms (Cox et a!., 1990). The production of a radiation hybrid mapping panel involves the irradiation of a somatic cell hybrid line containing the human chromosome of interest followed by fusion of the irradiated cell line with a rodent cell line. Fragments of the human chromosome present in the parental line are non-selectively retained in each of the radiation hybrids. Physical maps of markers in the region of interest are made using algorithms to analyze marker presence or absence in the radiation hybrid panel (Cox et a!., 1990). A pair of markers which are close together will be co-retained in a large proportion  13  1. Introduction of radiation hybrids. Markers further apart will be co-retained in a smaller fraction of radiation hybrids. Radiation hybrid mapping provides an alternate procedure for the production of long range maps of the human genome, and complements other mapping procedures such as meiotic linkage mapping, in situ hybridization and pulse field gel electrophoresis (PFGE) (Stephens et a!., 1990). Radiation hybrid mapping also affords several advantages when compared to alternate methods. In contrast to meiotic linkage mapping, radiation mapping can be performed using nonpolymorphic, moderately repetitive probes. Radiation mapping is not constrained by restriction site placement, nor by meiotic cross-over frequencies. Radiation hybrid mapping was therefore felt to be the best method for rapidly determining the order of the chromosome 5 clones obtained byAlu PCR differential hybridization.  1.8 POLYMORPHISM SCREENING The majority of loci on the human linkage map are defined on the basis of polymorphisms detected within DNA, rather than the visible phenotypes most often followed in other organisms (Donis-Keller et a!., 1987). The more polymorphic a system, the more information which can be gained from a given number of meioses. For a fully informative system, the allele which each parent has passed to their offspring can be determined. The informativeness of a polymorphic system is reflected by the polymorphism information content, or PlC of the system (Botstein et a!., 1980). The PlC value of a system is equivalent to the fraction of informative meioses observed when a large number of unrelated individuals are scored. The probability that a given meiosis will be informative is therefore equal to the PlC for  14  1. Introduction the system typed. The algorithm for calculation of PlC is discussed in Appendix 1. The first polymorphisms detected within DNA were differences in restriction enzyme digestion patterns commonly known as restriction fragment length polymorphisms or RFLPs (Botstein et a!., 1980). This variation in restriction fragment length can occur through the production or removal of a recognition site for a restriction enzyme, or through the insertion or deletion of DNA within a restriction fragment. Such DNA polymorphisms are common in human DNA, and can be detected by hybridization with a DNA segment from within the variable restriction fragment. While RFLPs are common in the human genome, they are unlikely to be highly polymorphic, since the production of more than two alleles requires the presence of multiple events involving the same restriction fragment. Another type of DNA polymorphism has recently been detected which involves variations in the number of simple sequence repeat units, such as (dC dA)n(dGdT)n ((GT)n; Weber and May, 1989). Simple sequence repeats are interspersed throughout the genome, with a copy number of between 50,000 and 100,000. Simple sequence repeats have been postulated to be enhancers of gene expression (Hamada et a!., 1984a), hot-spots for recombination (Slightom et a!., 1980) or involved in formation of a left-handed conformation of DNA (Z-DNA; Hamada et a!., 1984b). Alternatively, simple sequences have been postulated to have no general function with respect to gene expression (Tautz and Renz, 1984; Levinson and Gutman, 1987). Slippage of DNA strands during replication, repair or recombination is the most likely mechanism for expansion of simple sequence elements throughout the genome (Tautz and Renz, 1984; Levinson and Gutman, 1987). Variation of repeat number within an element is also postulated to occur  15  1. Introduction through strand slippage (Weber, 1990). Amplification of a (GT) tract can be accomplished with PCR using primers immediately flanking the tract (Weber and May, 1989). (GT) tracts with a repeat number (n) of between 11 and 15 are usually polymorphic, with the PlC increasing as the number of uninterrupted repeats increases (Weber, 1990). Tracts with 16 or more repeats are invariably moderately to highly informative, with PlC values in the 0.4 to 0.8 range (Weber, 1990). These sequences therefore serve as a valuable source of highly informative DNA markers. These polymorphisms have the added advantage that they are PCR based, and can therefore be analyzed rapidly with a minimum amount of DNA. Multiple systems can be analyzed simultaneously if the PCR conditions are the same for all systems and the amplification products are nonoverlapping in size. The majority of polymorphisms which were identified and typed during the course of this thesis are of the (GT)n type. Alu elements are present in numerous copies in the human genome and a large degree of sequence variation is present between elements (Kariya et al., 1987; Jurka and Smith, 1988). The dinucleotide CpG is a frequent site of polymorphic change in mammalian genomes (Barker et al., 1984). Alu elements are CpG rich, and have therefore been suggested as readily available sources of polymorphisms (Orita et a!., 1989). A variety of PCR-based techniques have been developed to detect potential polymorphisms inAlu elements. The detection of polymorphisms within Alu elements has been accomplished by amplification ofAlu elements using unique flanking primers, followed by detection of differences by non-denaturing polyacrylamide gel electrophoresis (Orita et a!., 1989, 1990) or conventional denaturing gels (Epstein et a!., 1990; Xu et a!., 1991). Polymorphisms involving the  16  1. Introduction 3’ polyA tail of Alu elements have been detected by amplification of the polyA tail region using a unique end-labeled primer and a primer specific to the 3’ end of an adjacent Alu element (Economou eta!., 1990; Zuliani and Hobbs, 1990). The hypothesis that amplification of potentially polymorphic Alu polyA tails would be possible using a unique primer flanking the polyA tail together with a primer specific to the Alu consensus sequence was tested as a part of this thesis.  1.9 LINKAGE MAPPING  Genetic crosses and backcrosses cannot, for obvious reasons, be performed by geneticists studying humans. Information on marker or disease locus recombination rates and position must rely on available pedigrees. A variety of methods have therefore been developed to obtain the maximal amount of information from observed marker segregations. Human linkage data are generally evaluated using a sequential probability ratio, or likelihood test (Morton, 1955). The likelihood of obtaining an observed segregation of two markers within a family is calculated for various values of recombination between the two markers. An odds ratio can then be calculated for the various recombination fractions between the two markers. The numerator in each odds ratio is the likelihood at each recombination fraction and the denominator is the likelihood if the markers are unlinked. Since the number of children in human families is generally quite small, it is usually difficult to obtain a meaningful odds ratio from a single family. However, since segregation within unrelated families can be considered independent, the odds ratio at a given recombination fraction for several families is simply the product of the odds ratio at  17  1. Introduction that recombination fraction for each of the families. This method was first used prior to the availability of calculators, and therefore calculations were simplified by taking the logarithm of the odds ratio, or lod score (Morton, 1955). The lod score for several unrelated families typed for the same marker pair is therefore the sum of the lod scores obtained for each family. A lod score of 3, or odds of 1000:1 for linkage, is generally considered sufficient evidence to state that two markers are linked. A lod score of 2, or 100:1 odds for linkage, is considered to be suggestive of linkage, and a lod score of -2, or 1:100 odds against linkage, is considered evidence that two markers are unlinked. To calculate recombination distances and lod scores for any pair of markers, the markers must be typed in the same families. The Centre d’Etude du Polymorphisme Humain (CEPH) was therefore organized to provide a set of reference families (Dausset et al., 1990). CEPH families were selected on the basis of large sibship size and living parents and grandparents such that a maximal number of informative meioses could be obtained for each marker typed. CEPH provides DNA from reference families to collaborating members and collects genotype data for markers typed. Lod scores for various values of recombination fractions between two loci can readily be determined. However, lod score calculations become somewhat tedious when large numbers of loci are compared in pairwise fashion and calculations involving multiple loci are rather complex. A variety of computer programs have therefore been developed to perform linkage calculations. Linkage calculations in this thesis were performed using the computer program package LINKAGE version 4.7 (Lathrop et a!., 1984, 1985; Lathrop and Lalouel, 1988).  18  1. Introduction To accurately report recombination distances between loci, and to construct linkage maps spanning several loci, a mapping function must be used to convert the observed recombination frequency (e) to map distance, which is expressed in units of Morgans. One Morgan is the distance over which one cross-over event occurs per 100 meioses (KIug and Cummings, 1983). The conversion from recombination fraction to map distance is necessary for two major reasons. The first results from the fact that observable recombination events result from an odd number of exchanges between loci, while an even number of events will be scored as absence of recombination. Secondly, cross-over events are not independent of one another, a phenomenon which is referred to as interference (Ott, 1985). The conversion to map distance allows the production of linkage maps spanning multiple loci, since map distances are additive across contiguous segments. Various mapping functions have been proposed to convert recombination fractions to map distances. The first mapping function was proposed by Haldane in 1919, and was based on the assumption of no interference. This model therefore assumes a random, independent distribution of chiasmata along a chromosome. A variety of more complex mapping functions have since been derived to account for interference. The Kosambi mapping function assumes that interference is directly proportional to the recombination fraction between two loci (Kosambi, 1944). More complex estimates of interference have been incorporated into a variety of mapping functions (Ott, 1985), however these functions are generally rather cumbersome and are not often used. Either the Haldane or the Kosambi mapping function is therefore commonly used for the analysis of human data. Derivations of the Haldane and Kosambi mapping functions are discussed in Appendix 1.  19  1. Introduction The use of reference families such as the CEPH panel, together with computer program packages such as LINKAGE allow the placement of new genetic markers on linkage maps. If the markers are typed in disease families, information can be obtained as to the position of the disease gene relative to the markers. Linkage maps can then be used to predict the genetic distance between the disease gene and flanking markers. If physical localization information is available for the flanking markers, a general estimate can be made as to the physical distance a disease gene can be positioned within. Suitable steps can then be taken towards the isolation of the disease gene. The probability of positioning a disease gene on the linkage map increases as the density of markers on the linkage map increases. The use of highly informative markers such as (GT) microsatellites also increases the probability of detecting linkage. Linkage markers were developed during the course of this thesis to increase the density of the linkage map of human chromosome 5 and to provide highly informative PCR based polymorphic systems. The research described in this thesis was designed to investigate a region of human chromosome 5, qll.2-q13.3, defined by a segmental trisomy. This region was of interest due to co-segregation of the 5q11.2-q13.3 trisomy with schizophrenia and renal anomalies in a Vancouver family of Asian descent (Bassett et a!., 1988, McGillivray et a!., 1990). The region became of further interest due to reports of linkage between markers within this region and chronic spinal muscular atrophy (SMA; Brzustowicz et a!., 1990; Melki et a!., 1990; Gilliam et a!., 1990). The initial  objective of this project was to isolate DNA fragments from within the 5q11.2-q13.3 region. The next objective was to obtain order information on these DNA fragments. Polymorphism screening would then be carried out on clones of interest.  20  1. Introduction Linkage data obtained from the polymorphism screening would serve to position the markers on the chromosome 5 linkage map. The linkage position of the new markers would then be used to delineate the breakpoints of the segmental trisomy and provide tools for the investigation of disease loci which lie near the new markers.  21  2. MATERIALS AND METhODS  2.1 MATERIALS  2.1.1 Somatic cell hybrids The somatic cell hybrid GM1O114 contains an intact human chromosome 5 as the sole human component in a Chinese hamster background, and was obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). The hybrid HHW1O64 contains a human chromosome 5 with an interstitial deletion of 5q11.2-q13.3 as the only human material in a Chinese hamster background (Gilliam et al., 1989). The derivative chromosome 5 in HHW1O64 was derived from a balanced carrier member of a family in which trisomy for the 5q11.2-q13.3 region cosegregates with schizophrenia and renal anomalies (Bassett et a!., 1988; McGillivray et a!., 1990). The hybrid HHW213 contains a derivative chromosome 5 deleted for approximately 95% of the q arm (Overhauser et a!., 1986a). The only detectable human DNA in HHW213 is an intact 5p, the centromere, and part of band 5q11 (Overhauser et a!., 1986a). The human chromosome 5 is retained in the somatic cell hybrids by complementation of the temperature sensitive rodent leucyl tRNA synthetase gene (leuS) (Dana and Wasmuth, 1982). DNA samples from a chromosome 5 radiation hybrid mapping panel consisting of 150 radiation hybrids were obtained from Dr. Ellen Solomon (ICRF, London, England). This panel was produced by irradiation of the somatic cell hybrid PN/TS-1, which contains an intact chromosome 5 as the only human component in a Chinese hamster background, with approximately 50,000 rads of X  22  2. Materials and Methods rays. The radiation hybrids were produced by the fusion of the irradiated PN/TS-1 cells with A23 cells, a Tk Chinese hamster ovary cell line.  2.1.2 Phage libraries LAO5NSO1 is a flow sorted, chromosome 5, complete EcoRI-digest phage library in Charon 21A (Deaven et a!., 1986). LAO5NLO3 is a flow sorted, chromosome 5, partial Sau3A-digest phage library in Charon 40 (L.L. Deaven, unpublished results). Both phage libraries were constructed at the Life Sciences Division, Los Alamos National Laboratory (Los Alamos, NM) under the auspices of the National Laboratory Gene Library Project, which is sponsored by the U.S. Department of Energy.  23  2. Materials and Methods 2.2 METHODS  2.2.1 Restriction enzyme digestions Restriction enzyme digestions were carried out using 50 ng to 4 ug DNA, 1X restriction enzyme buffer, 0.1 mg/mi Bovine serum albumin (BSA), and ito 2 units/ag restriction enzyme. Digests were performed by incubation of samples for 1 hour to overnight at the temperature recommended for the restriction enzyme. Reactions were stopped by the addition of 1/4 volume stop buffer or by incubation at 65°C for 10 minutes.  Restriction enzyme buffers (lOX) NaC1  Tris-HC1  MgC1  DTr  100mM  100mM  100mM  500 mM  100 mM  100 mM  100 mM  1M  500 mM  100 mM  100 mM  200 mM  50 mM  1M  100 mM  low medium  -  high react4(BRL) reactl0(BRL)  -  1.5 M  -  Buffers were filter sterilized by passing through 0.2 m filters.  Stop buffer 0.25% bromophenol blue 0.25% xylene cyanol 40% w/v sucrose in dH O 2 60 mM EDTA  24  KC1  -  500 mM  2. Materials and Methods 2.2.2 Gel electrophoresis DNA samples were size fractionated by loading onto agarose gels and subjecting the gels to a constant voltage. To produce a gel, agarose was dissolved in lx TBE buffer by boiling, then allowed to cool to approximately 40 C before being poured into a mold and allowed to solidify. Ethidium bromide was added to the gel at a concentration of 1 pg/mi to allow the visualization of DNA using UV illumination. Alu PCR products or restriction digested genomic DNA samples were run on 0.8% agarose gels overnight at 30 V. Plasmid digests were run on 0.7% to  1.0% agarose gels for 1 to 16 hours at 30 to 100 V. PCR products from (GT) microsatellites were run on 3% Nusieve, 1% agarose gels for 1 to 3 hours at 100 V. Lambda DNA digested with Hindill and SstII was used as a size standard for agarose gels. ØX-174-RF DNA digested with HaeIII (Pharmacia) was used as a size standard for Nusieve gels. DNA samples were photographed after electrophoresis using a 302 nm transilluminator.  lox TBE buffer 54 g Tris base 27.5 g boric acid 20 ml 0.5 M EDTA (pH 8.0) O to 11 2 dH  2.2.3 Southern blotting Restriction digested DNA or PCR products were transferred from agarose gels to nylon membranes using the .method of uthern (Southern, 1975). A  25  2. Materials and Methods  Polaroid or negative was taken of the gel with a ruler present to determine the distance of migration. Excess agarose was then trimmed away, and the gel was soaked in 0.25 M HC1 for 15 minutes, 0.5 M NaOH/1.5 M NaC1 for 30 minutes and 1 M Tris/l.5 M NaC1 for 30 minutes. The gel was washed with distilled water O) between each solution. 2 (dH The denatured DNA was then transferred from the agarose gel to a nylon membrane. Either Genescreen (New England Nuclear) or Hybond (Amersham) was used for plasmid blots and Nytran (Schleicher & Schuell) or Hybond was used for blotting genomic DNA or PCR products. Unidirectional blots were made as follows. Two pieces of identically sized Whatman 3 mm filter paper were placed into a dish containing lox SSC. A glass plate was then put across the dish, and the filter paper was folded over the glass plate such that both ends of the paper remained in the solution to create a wick. The treated agarose gel was then placed on top of the wet filter paper. A prewetted piece of membrane cut to the shape of the gel was placed on top of the gel. Two pieces of filter paper cut to the size of the gel were then placed on top of the membrane. Any excess area of wick uncovered by the gel was covered with Saran wrap. A stack of paper towels approximately 20 cm high was then placed on top of the gel. This transfer was done for 2 hours to overnight. Bidirectional blots were made by sandwiching the treated gel between two membranes in an analogous fashion to the unidirectional blotting. Four pieces of filter paper and two pieces of membrane were cut to fit the gel, and pre-wetted. A piece of Saran wrap was placed on the bench and 6 to 7 paper towels were placed on top of the Saran wrap. Two pieces of filter paper, then a piece of membrane, then  26  2. Materials and Methods the treated gel, then another piece of membrane, then two additional pieces of filter paper were placed onto the paper towels. All of the paper towel unoccupied by gel was covered with Saran wrap and a stack of paper towels was placed on top. A glass plate and a weight were placed on top of the paper towels. The transfer was allowed to proceed for 2 hours to overnight. After transfer was completed, membranes were rinsed with dH O to remove 2 any agarose, placed between two pieces of filter paper, and baked for 1 1/2 to 2 hours at 80°C.  2.2.4 Oligolabeling PCR products or phage DNA were labeled with P-dATP 32 using the a random primer method (Feinberg and Vogelstein, 1984a,b). DNA samples were diluted to approximately 1 ng/il and boiled for 10 minutes, then placed on ice. A standard labeling reaction comprised 30 j.Ll (30 ng) boiled DNA, 10 l OLB-A, 5 jil 1 mg/mi BSA, 1 unit Kienow and 50 /.LCi P-dATP. 32 Reactions were also a performed using (1/2)X and 2X the standard reaction. Samples were held on ice until the addition of the P-dATP, 32 and then incubated at room temperature a overnight. The labeling reaction was stopped by the addition of 1 volume NTSB, and the unincorporated nucleotides removed by passing the reaction mixture through a Sephadex G-25 spin column. Spin columns were made by placing a 1 ml pipette tip into a collar formed by cutting the bottom and lid off a 1.5 ml eppendorf tube, then placing the collar and tip into a 12 X 75 mm culture tube. The bottom of the tip was plugged with siliconized glass wool, and G-25 Sephadex (equilibrated in 1/5 TE)  27  2. Materials and Methods was added until the tip was full for a full reaction or until half full for a half reaction. The column was spun in a clinical centrifuge for 3 minutes at 1000 rpm, and then transferred to a fresh culture tube. The sample was added and the column O and spun again. 2 spun again. The column was then washed with 2 volumes of dH Reactions which had been doubled were divided in half and passed through 2 spin columns. Final volumes of reactions were 200 l for full reactions, 100 l for half reactions and 400 ul for doubled reactions.  OLB-A Solutions A:B:C mixed in ratio of 100:250:150.  Solution A  Solution B  1 ml 1.25 M Tris (pH 8); 0.125 M MgC1  2 M Hepes (pH 6.6)  18 j.L1 2-mercaptoethanol  5 l 100 mM dTTP (Pharmacia)  Solution C  5 jil 100 mM dGTP  (“  “)  Hexadeoxyribonucleotides  5 ul 100 mM dCTP  (“  “)  (Pharmacia) suspended in TE at 90 OD/ml.  NTSB  1XTE  20 mM EDTA  10 mM Tris (pH 8.0)  2 mg/mi salmon sperm DNA  1 mM EDTA  0.2% SDS  28  2. Materials and Methods 2.2.5 Preannealing with human DNA Repetitive probes were prearmealed with a vast excess of sheared nonradioactive human DNA (Litt and White, 1985). Typical prearinealing conditions consisted of 30 ng probe DNA and 200 j.g sheared nonradioactive human DNA in a total volume of 270 l and a final buffer concentration of 2X SSC. Samples were boiled for 5 minutes, and then preannealed at 65°C for 15  minutes to 1 hour.  20X SSC 175.3 g NaC1 88.2 g sodium citrate O to 11 2 dH  2.2.6 Prehybridization, hybridization and washing Southern blots, plaque lifts or colony lifts were placed in heat-sealable bags and prehybridized with hybridization solution at 65°C for 1 to 4 hours to block non specific probe binding. Prior to hybridization, probes were denatured by boiling and then quick chilled on ice. Repetitive probes were preannealed with an excess of nonradioactive human DNA (section 2.2.5). The probe was then added to the hybridization solution, and hybridization carried out overnight at 65°C. Hybridization and wash solutions were pre-warmed to 65°C before use. Filters were washed twice for 15 minutes in lx SSC, 0.1% SDS and then twice for 30 minutes in 0.2X SSC, 0.1% SDS. If substantial background signal remained after these washes, filters were washed for a further 30 minutes to 1 hour in 0.2X SSC, 0.1% SDS. After  29  2. Materials and Methods washing, filters were dried briefly, then re-sealed in bags and exposed to Kodak X RP or X-AR film with DuPont Lightning Plus intensifying screens at -70°C or at room temperature for varying time periods. Hybridization signal was stripped from blots by rinsing in 0.4 N NaOH at 43°C for 30 minutes, followed by neutralization for 30 minutes at 43°C in 0.2 M Tris pH 7.5, 0.2X SSC, 0.2% SDS. For the majority of blots, this treatment removed all signals. Blots were then dried and re-used for hybridization. Blots were stripped and re-hybridized a maximum of 10 times.  Hybridization solution 6X SSC 0.3% (w/v) SDS 5X Denharts 100 g/l salmon sperm DNA  2.2.7 (GT) tract detection and isolation Poly(dA-dC)(dG-dT) (Pharmacia) was labeled using nick translation (Rigby et a!., 1977) with P 32 dATP and a BRL nick translation kit. Labeling conditions a were as specified by BRL. (GT)n tracts were detected by hybridization to this probe. Colony filters or blots were hybridized with nick translated probe overnight at 55°C, using hybridization solution lacking any competitor DNA. Blots were washed for 1 hour at 65°C in 1X SSC, 0.1% SDS. Filters or blots were exposed to Kodak RP film for 2 hours to overnight using DuPont Lightning Plus intensifying screens at room temperature.  30  2. Materials and Methods Hybridization solution used for (GT) 11 tract detection 6X SSC 0.3% SDS 5X Denharts  2.2.8 Phage manipulations  2.2.8.1 Phage plating E.coli strain LE392 (Sambrook et aL, 1989) was used as host cells for phage growth. A 5 ml L-broth culture containing 0.2% maltose was inoculated using a single colony from a freshly streaked plate of LE392 cells. The culture was grown overnight at 37°C, then the cells sedimented by spinning at 2500 rpm for 5 minutes in a clinical centrifuge. The bacterial pellet was resuspended in 1/2 volume 10 mM MgCl. For phage plating, 100 l of host cells were combined with up to 10 phage and incubated at room temperature for 15 minutes to allow the phage to infect the bacteria. The infected cells were mixed with 3 ml of soft NZY top agarose (47°C) and spread onto a freshly poured NZY plate. The plate was then incubated at 37°C overnight to allow plaques to develop. Confluently lysed plates (all bacteria on plate lysed) were produced using approximately  31  io phage per plate.  2. Materials and Methods  L-broth  NZY broth  5g yeast extract  lOg NZamine  lOg tryptone  5g yeast extract  5g NaC1  5g NaC1  lg D-glucose  2g MgCl  O 2 lldH  O 2 lldH  3001.Ll iON NaOH (pH to 7.2-7.4)  il iON NaOH (pH to 7.2-7.4) 1 300  Autoclave 20 minutes at l5lbs pressure  Autoclave 20 minutes at l5lbs pressure  Top agarose Add 7 g/l agarose to broth prior to autoclaving  Plates Add 12 g/l agar to broth prior to autoclaving  2.2.8.2 Plaque lifts and plaque purification  Plaque lifts (Benton and Davis, 1977) were made using the following procedure. Phage plates were cooled at 4°C for at least 1 hour prior to making lifts. A nitrocellulose circle was placed on top of the plate, and allowed to become completely wet. The filter and plate was marked three times by stabbing with the needle of a syringe containing India ink. The filter was removed from the plate and placed DNA side up on top of Whatman filter paper soaked with the following solutions:  32  2. Materials and Methods 1) 1.5M NaC1, 0.5M NaOH; incubate for 4 minutes 2) 1.5M NaC1, 1M Tris pH8; incubate for 4 minutes 3) 2X SSC; rinse (approximately 1 minute) Filters were air dried and baked under vacuum for 1 1/2 to 2 hours at 80°C. Up to 7 plaque lifts were made from a plate. Duplicate plaque lifts were made by placing 2 filters simultaneously on top of a plate. Phage were screened by hybridization of probes to plaque lifts. Plaques which were positive were picked with a toothpick, diluted with ) diluent, replated and re-screened. Three successive screens were carried out to obtain purified phage.  ), diluent 10 mM Tris, pH 7.5 10 mM MgCl  2.2.8.3 Small scale phage prep A confluently lysed NZY agarose plate was overlaid with 5 ml ice cold SM solution and left at 4°C overnight. The overlay was collected, a few drops of CHC1 3 added, and the tube spun at 2500 rpm for 5 minutes in a clinical centrifuge. The supernatant was transferred to a fresh tube and a few more drops of CHC1 3 added. The supernatant was then stored at 4°C until the phage prep was performed. Small amounts of phage DNA were obtained using the following procedure (Davis et al., 1980). 10 jil 20% SDS was added to 1 ml of phage overlay solution, mixed, and incubated at room temperature for 10 minutes. 100 l of 2 M Tris; 0.2 M EDTA  33  2. Materials and Methods was then added, the tube mixed and incubated at 70°C for 10 minutes. 100 ul of 5M CH COOK, pH4.8 was added and the tube cooled in an ice-water bath for 30 3 minutes. The tube was spun for 10 minutes in a microcentrifuge and the supernatant decanted. Two phenol extractions and one Sevags extraction were performed. Two volumes of EtOH were added to the aqueous fraction and the tube spun for 5 minutes in a microcentrifuge. The supernatant was discarded, and the pellet rinsed in 70% EtOH and spun again. The pellet was resuspended in 50 j.Ll TE containing 0.1 g/jil RNase and incubated at 37°C for 15 minutes to degrade the RNA.  SM solution  Sevags  100 mM NaC1  24 volumes CHC1 3  8 mM MgSO4  1 volume isoamyl alcohol  50 mM Tris (pH 7.5) 0.01% (v/v) gelatin  2.2.8.4 Large scale phage prep Twenty confluent plates were made for each phage stock from which large quantities of DNA were required. The plates were cooled at 4°C for at least one hour and then overlaid with Sml/plate ice cold Adiluent. Plates were left at 4°C overnight. The overlay was collected into large centrifuge tubes, a few drops of 3 added and the tubes centrifuged at 10,000 rpm for 10 minutes in a Sorvall CHC1 centrifuge. One to two mis of the clear supernatant was saved for titering and the remainder transferred to clean centrifuge tubes. Large amounts of phage DNA  34  2. Materials and Methods were prepared using the following procedure (Davis et al., 1980). Solid NaC1 was added to the supernatent in the centrifuge tubes to a final concentration of 1 M (29.2 g/500 ml of culture) and dissolved by swirling. Solid PEG 8000 was then added to a final concentration of 10% w/v and dissolved by slow stirring. The solution was then cooled in ice water for 1 hour to precipitate the phage particles. The phage particles were collected by centrifugation at 11,000 rpm for 10 minutes at 4°C in a Sorval centrifuge. The supernatant was discarded and excess supernatant removed by standing the centrifuge bottles upside down for 5 to 10 minutes. Any residual supernatant was then removed by gently wiping the sides of the bottles with Kimwipes. The pellet was then gently resuspended in 9 ml )diluent using a wide bore pipette. Residual bacterial debris was removed by centrifuging at 2500 rpm for 10 minutes. The supernatant at this stage typically exhibited a bluish tinge due to the suspended phage particles. 0.75 g/ml solid CsCl was added to the suspended phage particles and mixed gently to dissolve. The phage suspension was transferred to ultracentrifuge tubes and heat sealed. The tubes were spun in the ultracentrifuge at 60,000 rpm for 16 to 20 hours at 20°C. After centrifugation, the phage particles could be visualized as a blue-white band. This band was collected by removing the top of the ultracentrifuge tube to allow air into the tube, and then puncturing the side of the ultracentrifuge tube with a syringe and drawing up the phage particles. The phage in CsCl were stored at 4°C. DNA was extracted from phage using a formamide extraction method (Davis et a!, 1980). One volume of phage in CsCl was mixed with 1/10 volume 2 M Tris; 0.2 M EDTA in a microcentrifuge tube. One volume of formamide was added and the mixture incubated for 30 minutes to several hours at room temperature. One  35  2. Materials and Methods volume of H 0 then 6 volumes of 95% EtOH were added. The DNA was 2 precipitated by centrifugation for 1 minute in a microcentrifuge. The supernatant was discarded and the DNA pellet resuspended in 1X TE.  2.2.9 Plasmid manipulations  2.2.9.1 Ligation DNA fragments were subcloned into Bluescriptil by ligation. Vector and insert DNA were restriction digested to produce complementary sticky or blunt ends. Digests were stopped by heating at 65°C for 10 minutes. Ligations were performed by mixing vector and insert DNA in a 1:1 to 1:4 ratio in 1X ligation buffer and 1 unit T4 DNA ligase in a reaction volume of 10 to 20 l. 50 to 100 ng of plasmid DNA were typically used for a ligation reaction. Samples were ligated overnight at 14°C.  lox ligation buffer 500 mM Tris 100 mM MgC1 10 mM ATP 100 mM DTT 1 mg/mi BSA  36  2. Materials and Methods 2.2.9.2 Production of competent cells and transformation DH5a cells (Sambrook et al., 1989) were made competent using the following procedure (Hanahan, 1983). A culture tube containing 5 mIs of L-broth was inoculated with a DH5a colony from a freshly streaked plate and grown overnight. The overnight culture was added to 300 ml of SO media in a 21 flask. The 300 ml culture was grown at 37°C at 275 rev/mm until 0D 600 was approximately 0.5. The culture was cooled in a salt/ice-water bath for 5 minutes, then poured into pre-cooled centrifuge tubes and spun at 2500 rev/mm for 10 minutes at 4°C in a Sorval centrifuge. The supernatant was removed, and the pellet washed with 150 ml pre-cooled 10 mM MgC1;10 mM MgSO4. The cells were then spun at 2500 rev/mm for 10 minutes at 4°C in a Sorval centrifuge. The supernatant was removed and the pellet resuspended in 100 ml pre-cooled FSB solution. The cells were placed on ice for 15 minutes, then spun for 10 minutes at 2500 rev/mm at 4°C in a Sorval centrifuge. The supernatant was removed, and the pellet resuspended in 24 ml of FSB solution. 0.84 ml of dimethylsulphoxide (DMSO) was added, the cells were mixed gently and incubated on ice for 10 minutes. An additional 0.84 ml DMSO was added, the cells mixed gently and incubated on ice for 10 minutes. Aliquots of competent cells were frozen in liquid nitrogen and stored at -70°C. Cells obtained from this procedure generally had an efficiency of io6 to colonies/Lg supercoiled Bluescriptil. Ligated plasmids were transformed into competent DH5a cells using the following procedure. Competent cells were thawed on ice, then 50 ul of cells aliquoted into a chilled 17 X 100 mm polypropylene tube. 1 to 100 ng DNA was added to the cells and incubated for 30 minutes on ice. L-broth was added to a total  37  2. Materials and Methods volume of 100 l and the cells heat-shocked at 42°C for 45 seconds. The cells were placed on ice for 2 minutes, then 0.1 to 1 ml L-broth was added and the cells incubated at 37°C for one hour. Appropriate amounts of cells were plated onto XJA plates.  SO media  FSB Solution  20 g bactotryptone  10 mM CH COOK 3  5 g yeast extract  100 mM RbC1  10 mM NaCl  45 mM MnCl 2  2.5 mM KC1  10 mM CaCl2  10 mM MgCl  3 mM hexamine cobalt chloride  10 mM MgSO4  10% v/v glycerol  O to 11 total volume 2 dH  Adjust pH to 6.4 using  Autoclave for 20 minutes at 15 lbs pressure  concentrated HC1. Filter sterilize into an autoclaved bottle.  XJA plates Plates were made with L-broth media plus 12 g/l agar added before autoclaving. The following media supplements were added after autoclaving and cooling media to approximately 50°C: 50 g/ml ampicillin, 40 ag/mi X-Gal (5-bromo-4-chloro-3indolyl-B-D-galactoside) and 120 tg/ml IPTG (isopropyl-B-thiogalactopyranoside).  2.2.9.3 Ligation independent cloning (LIC) The PCR product from 5PCR1 (D5S205) was subcloned using ligation independent cloning (LIC; Aslanidis and de Jong, 1990). This procedure generates  38  2. Materials and Methods recombinants without using restriction enzymes or T4 DNA ligase. A PCR reaction is performed on both the vector and insert, followed by digestion with T4 DNA polymerase to produce long overhanging complementary tails. Vector and insert are then combined and transformed into bacteria. The PCR products generated by Alu-LIC PCR on 5PCR1 and plasmid-LIC PCR on pUC19 (see section 2.2.11.6) were purified by running samples out on an 0.7% agarose gel and cutting out the PCR products using long wavelength UV illumination. The DNA was then separated from the agarose by spinning the agarose plug through glass wool for 1 minute in a microcentrifuge. Single stranded tails were generated by treating the vector and insert DNA with 2 units T4 DNA polymerase in 1X T4 DNA polymerase buffer with 0.5 mM dGTP present for the Alu-LIC PCR product and 0.5 mM dCTP present for the vector. Samples were incubated at 37°C for 20 minutes and then heated to inactivate the enzyme for 10 minutes at 65°C. The T4 polymerase treated product was purified by a phenol/Sevags extraction. DNA samples were then OAc and 2 volumes 95% 4 precipitated by the addition of 1/2 volume 7.5 M NH EtOH followed by centrifugation for 10 minutes at 4°C in a microcentrifuge. The pellet was washed with 70% EtOH, reprecipitated by centrifugation in a microcentrifuge for 5 minutes at 4°C, dried for 5 minutes in a vacuum desiccator and resuspended in TE. Vector and insert were mixed at a 1:3 ratio in 1X ligation buffer and incubated for 1 hour at room temperature. Transformations were carried out as usual.  39  2. Materials and Methods 5X T4 DNA polymerase buffer 165 mM Tris pH 8 COOK 3 330 mM CH 50 mM MgCl 2.5 mM DTT 0.5 mg/mI BSA  2.2.9.4 Colony screens Colony screens for plasmids (Grunstein and Hogness, 1975) were made as follows. A nitrocellulose filter was labeled, placed on top of a XIA plate and allowed to sit until the filter was thoroughly wet. A XIA plate for stock storage was labeled in the same fashion as the nitrocellulose filter. Colonies were streaked using a toothpick onto the nitrocellulose filter and the stock plate. Both plates were incubated overnight at 37°C to allow growth of colonies. The stock plate was stored at 4°C. The nitrocellulose filter was removed from the MA plate and placed colony side up on top of Whatman filter paper soaked with the following solutions: 1)1.5 M NaC1, 0.5 M NaOH; incubate for 4 minutes 2)1.5 M NaCl, 1 M Tris pH 8; incubate for 4 minutes 3) 2X SSC; rinse (approximately 1 minute) Filters were then air dried and baked under vacuum for 1 1/2 to 2 hours at 80°C. Colonies were screened by hybridization of probes to filters.  40  2. Materials and Methods 2.2.9.5 Plasmid minzrep Small amounts of plasmid DNA were generated using an alkaline lysis miniprep procedure (Birnboin and Doly, 1979; Ish-Horowicz and Burke, 1981). 5  mis of L-broth was innoculated with the colony of interest and then grown overnight at 37°C in the presence of 50 g/ml ampicillin. 0.5 ml of culture was added to 0.5 ml of glycerol and saved as a stock. The remainder of the culture was centrifuged at 2500 rpm for 10 minutes in a clinical centrifuge. The supernatant was removed, the pellet resuspended in 100 J.Ll solution I, transferred to an eppendorf tube and incubated at room temperature for 5 minutes. 200 jil of freshly prepared Solution II was added, the tube inverted several times to mix the contents and then incubated on ice for 5 minutes. 150 l Solution III was added, the tube inverted several times to mix and then incubated on ice for 5 minutes. One volume of buffer-saturated phenol was added and mixed and the tube was spun for 15 minutes in a microcentrifuge. The aqueous layer was removed and transferred to a fresh tube. One volume of Sevags was added, the tube mixed and then spun for 5 minutes in a microcentrifuge. The aqueous layer was removed and transferred to a fresh tube. 1/10 volume of 7.5 M NH OAc and 2 volumes of 95% EtOH were added and the 4 DNA precipitated by centrifugation for 10 minutes at 4°C in a microcentrifuge. The pellet was washed in 70% EtOH, then spun for 5 minutes at 4°C in a microcentrifuge. The pellet was air dried for 10 to 15 minutes, then dessicated for 5 minutes. The pellet was resuspended in 40 to 100 ul TE containing 0.1 g/l RNase. Samples were then incubated at 37°C for 15 to 30 minutes to remove RNA. Samples were stored at -20°C.  41  2. Materials and Methods Alkaline lysis solution I  Alkaline lysis solution H  50 mM glucose  0.2 N NaOH  10 mM EDTA  1%SDS  25 mM Tris pH 8.0 4 mg/ml lysozyme (Add powdered lysozyme to solution just prior to use)  Alkaline lysis solution III COOK 3 60 ml 5M CH 11.5 ml glacial acetic acid 28.5 ml dH O 2  2.2.9.6 Plasmid large scale prep To produce large quantities of plasmid DNA, a large scale alkaline lysis plasmid prep was performed (Birnboin and Doly, 1979; Ish-Horowicz and Burke, 1981). Alkaline lysis Solutions I, II and III are as indicated for the small scale plasmid prep. 500 ml L-broth containing 50 jg/ml ampicillin was inoculated with a colony containing a plasmid of interest. The culture was grown at 37°C for approximately 5 hours until the 0D 600 was between 0.4 and 0.5, then 1 ml of 80 mg/ml chloramphenicol was added and the culture incubated at 37°C for 12 to 16 hours. The culture was centrifuged at 4000 rpm for 10 minutes at 4°C in a Sorval centrifuge, the supematant discarded and the pellet resuspended in 35 ml STE. The solution was transferred to smaller centrifuge tubes and re-centrifuged at 4000 rpm for 10 minutes at 4°C in a Sorval centrifuge. The pellet was resuspended in 10 ml  42  2. Materials and Methods ice cold Solution I, then incubated at room temperature for 5 minutes. 20 ml of  freshly prepared Solution II was added, the contents mixed by inverting rapidly several times and the tube stored on ice for 10 minutes. 15 nil of ice cold Solution III was then added, the tube inverted several times to mix and stored on ice for 10 minutes. The bacterial debris was removed by centrifugation at 15,000 rpm for 25 minutes at 4°C in a Sorval centrifuge. The supernatant was transferred into 2 centifuge tubes, and 0.6 volumes of room temperature isopropanol was added to each tube. The tubes were mixed gently and placed at room temperature for 15 minutes. The DNA was precipitated by centrifugation for 10 minutes at 2500 rpm at room temperature in a clinical centrifuge. The supernatant was discarded and the pellet air dried. The pellet was dissolved in 10 mi TE (pH 8.0) and 1 g/mi CsC1 and 0.8 ml 10 mg/mi ethidium bromide added. The sample was placed in the dark for 15 minutes, then transferred to a 16X76 mm polyallomer ultracentrifuge tube and heat sealed. The sample was then centrifuged at 60,000 rpm for 18 to 20 hours at 20°C in an ultracentrifuge. The plasmid DNA band was visualized with a long wavelength UV transilluminator and extracted using a syringe. Ethidium bromide was removed from the plasmid DNA by several extractions with butanol saturated with dH O. The DNA was precipitated by adding 2 volumes of dH 2 O and 6 2 volumes of EtOH, placing the sample at -20°C for 1 hour to several days and centrifuging for 10 minutes at 2500 rpm at room temperature in a clinical centrifuge. The pellet was washed with 70% EtOH, then centrifuged for 10 minutes at 2500 rpm at room temperature. The supernatant was removed and the pellet air dried. The plasmid DNA was resuspended in 0.5 to 1 ml of TE.  43  2. Materials and Methods STE 100 mM NaC1 10 mM Tris, pH 8.0 1 mM EDTA, pH 8.0  2.2.10 Sequencing Sequencing was performed on alkaline lysis miniprep DNA or M13mp18 single stranded DNA by the dideoxy chain termination method (Sanger et al., 1977) using S 35 dATP and a Sequenase sequencing kit (U.S. Biochemical Corp.). The a protocol used was a modification of standard techniques to allow sequencing through regions of high secondary structure.  2.2.1 0.1 Denaturation of template Plasmid miniprep DNA was denatured and precipitated prior to sequencing in the following fashion. 18 j.Ll (5 to 10 J.Lg) of plasmid miniprep DNA was denatured by the addition of 2 jil of 2 N NaOH; 2 mM EDTA and incubation at room temperature for 5 minutes. The sample was then neutralized by the addition of 2 l of 2 M NH OAc. 55 jil ice cold 95% EtOH was added and the sample placed at 4 -70°C for 5 minutes. The denatured DNA was then precipitated by centrifugation in a microcentrifuge for 15 minutes. The supernatant was removed and the pellet washed with 200 1 ice cold 70% EtOH. The sample was spun for 5 minutes in a microcentrifuge and the pellet dried for 5 minutes in a desiccator. The pellet was left dry for up to 2 hours at -20°C before the sequencing reaction. The pellet was resuspended in 6 to 6.5 jil dH O immediately preceding the sequencing reaction. 2  44  2. Materials and Methods 2.2.1 0.2 Sequencing reactions Sequencing reactions were carried out on 5 to 10 ,.g denatured plasmid miniprep DNA or 2 g single stranded M13mp19 DNA. Plasmid or M13 DNA was mixed with 5 to 10 pmoles sequencing primer in a total volume of 7 jLl. 1 ,ul of dimethyl sulfoxide (DMSO) was added and the sample was boiled for 3 minutes. The sample was placed in liquid nitrogen for 5 minutes, then thawed quickly and spun for 1 second in a microcentrifuge. 2 l of sequencing buffer was then added and the sample incubated at room temperature for 5 minutes. 6.3 l of labeling mix was added, and the sample incubated at room temperature for 3 to 5 minutes.  3.5 l of the sample was then aliquoted into each of 4 tubes containing termination mixes pre-warmed to 37°C. The termination tubes were then incubated at 37°C for 3 to 5 minutes. The reactions were stopped by the addition of 4 l sequencing stop mix. Samples were stored at -20°C.  Labeling mix 1.025 jillOOmMDTT 2.05 l 0.75 M dGTP; 0.75 jM dCTP; 0.75 M dTTP 1 j1a SdATP 35  0.525 jil DMSO 1.8 l 10 mM Tris pH 7.5; 5 mM DTT’; 0.5 mg/mi BSA 0.25 j.Ll Sequenase add just before ready to begin -  45  2. Materials and Methods Termination mixtures  Sequencing stop mix  80 jM dGTP  95% formamide  80 j.MdCTP  20 mM EDTA  80 M dATP  0.05% Bromophenol Blue  80 jtM dTl’P  0.05% Xylene Cyanol FF  50 mM NaC1 8 M of one of ddGTP, ddCTP, ddTFP or ddATP  2.2.10.3 Sequencing primers Universal primers used for sequencing plasmids were T3 and T7 primers. M13mp19 was sequenced using the universal -40 primer. Universal sequencing primers were obtained from Stratagene. Primer sequences are indicated below. T3 primer: 5’ ATfAACCCTCACTAAAG 3’ V primer: 5’ AATACGACTCACTATAG 3’ -40 primer: 5’ G’ITI’I’CCCAGTCACGAC 3’ Sequencing was also done using the TC65A primer, which is homologous to the consensus sequence of the Alu family of repetitive elements, and with unique primers flanking (GT)n tracts orAlu 3’ tails. The sequences of these primers are listed in section 2.2.11.6 (Specific PCR reactions and primer sequences).  2.2.10.4 Denaturing polyaciylamide gel electrophoresis Sequencing reactions were subjected to denaturing gel electrophoresis using a 6% Hydrolink (AT Biochemicals) sequencing gel. Sequencing gel systems were  46  2. Materials and Methods obtained from BioRad. One sequencing system consists of two 21 cm X 50 cm plates, one of which contains a buffer container, two 0.4 mm spacers, a 0.4 mm comb, two clamps, a casting tray, a base, a stabilizer bar and electrical leads.  6% Hvdrolink sequencing gel 25.2 g urea 3.6 ml lOX TBE 6 ml 50% Hydrolirik Long Ranger solution O 2 30 ml dH The gel ingredients were added to a flask in the order given and the urea was dissolved by placing the flask in warm water. Sequencing plates were cleaned, spacers were sandwiched between the plates at either side and the plates clamped together on the sides. The bottom of the gel was sealed by placing 13 ml gel solution, 52 1 N,N,N’,N’-tetramethylethylenediamine (TEMED) and 208 l 25% (w/v) ammonium persulfate (APS) in a casting tray, then pressing the gel firmly into the solution for approximately 1 minute such that the solution moved between the plates by capilliary action. The gel was then left for 2 minutes to ensure that the bottom seal was completely polymerized. To pour the gel, 26 j.Ll of TEMED and 100 l of 25% APS was added to the remaining gel solution, and the solution was pipetted between the sequencing plates using a 25 ml pipette. The top of the gel was then covered with the flat side of a sequencing comb, and the gel allowed to polymerize for 1 hour. The base was filled with 350 to 500 ml of 0.6X TBE and the gel placed in the base and propped in a vertical position using the stabilizer bar. The buffer chamber was then filled with 0.6X TBE and the comb reversed such that  47  2. Materials and Methods the teeth were placed slightly into the gel, producing wells for sample loading. The gel was pre-run at 55 W until the gel temperature reached 50°C (approximately 1 hour). Sequencing reactions were denatured by heating at 95°C for 3 to 5 minutes, then loaded onto the gel and run at 55 W for various time periods. Since sequencing proceeds in aS’ to 3’ direction, the sequence closer to the 3’ end can be obtained by increasing the run time. A second loading of certain samples was therefore used to obtain sequence spanning a longer region. Up to 250 bp of sequence could be obtained from a single load and up to 400 bp from a double load.  2 2.1 0.5 Maniulation of sequence data Sequence information was stored and manipulated using the computer program ESEE sequence editor (E. Cabot, Access Biosystems Inc., Burnaby, B.C.).  48  2. Materials and Methods 2.2.11 Polymerase chain reaction (PCR) PCR reactions (Saiki et at., 1985; Mullis et at., 1986; Mullis and Faloona, 1987) using  Taq DNA polymerase (Saiki et at., 1988) were performed using plasmid DNA, human lymphoblast DNA or somatic cell hybrid DNA.  2.2.11.1 Standard PCR reaction conditions 10 ng plasmid DNA, 40 ng human lymphoblast DNA or 40 ng somatic cell hybrid DNA 50 mM Tris pH 8.0 0.05% Tween 20 0.05% NP4O 2 mM MgCl 0.2 mM each of dATP, dCTP, dGTP and dTT’P primer(s) see below -  0.625 units of Taq DNA polymerase O to a total reaction volume of 25 j.1 2 dH 25 l mineral oil overlay  2.2.11.2 Standard cycling conditions Thirty cycles of 1 minute denaturation at 94°C, 30 second annealing at 58°C, and 1 minute extension at 72°C were performed. After cycling was finished, the tubes were held at 72°C for 10 minutes and then stored at 4°C until removed from the thermal cycler. A Perkin-Elmer Cetus thermal cycler was used.  49  2. Materials and Methods 2.2.11.3 PCR primers synthesis and punfication -  Unique primers for PCR were determined using the following criteria: 1) Primer was approximately 20 bp in length and contained roughly equivalent fractions of A + T and G + C, such that Tm was approximately 58°C. Oligo T was estimated using the formula Tm(°C)  =  4(G + C) + 2(A+ T) 4. -  2) Primer exhibited a minimal amount of self-complementarity and did not have a tendency to form primer dimers. Primers were screened for self-complementarity and dimer formation using the NAR program (Rychlik and Rhoads, 1989). Once a pair of primers (primerl, primer2) had been determined for a given system, their sequence was compared to ensure that a high frequency of primerl-primer2 dimer would not occur. 3) Primer did not have significant homology with sequences available through the European Molecular Biology Laboratory (EMBL) sequence database version 27. The computer program FASTA version 1.2 (Pearson and Lipman, 1988) was used to search the primate portion of the EMBL database for sequence homologies with each primer. Primers were purchased either from Bio-Synthesis Inc. (Lewisville, Texas) or the UBC Oligonucleotide Synthesis Laboratory. Primers obtained from Bio Synthesis Inc. had been gel filtration purified, desalted and lyophilized and were therefore not purified further. Bio-Synthesis primers were resuspended in 1X TE, an 0D 260 measurement made of a fraction of the sample, and the concentration adjusted to 10OM assuming 1 0D 260  33 j.Lg/ml. Primers were stored at -20°C.  Primers obtained from the UBC Oligonucleotide Synthesis Laboratory were  50  2. Materials and Methods  made using an Applied Biosystems 380B oligonucleotide synthesizer. Primers from the UBC Oligonucleotide Synthesis Laboratory were purified by passing through a C-18 Sep-Pak cartridge in the following manner. The dried primer was resuspended in 1.5 ml 0.5 M NH4OAc and then loaded onto a C-18 Sep-Pak cartridge which had been pre-washed with 10 ml HPLC grade acetonitrile and 10 ml 2 dH O . The cartridge was washed with 10 ml 2 dH O . The primer was then eluted with 1 ml 20% acetonitrile. A fraction of the sample was used to determine the quantity of primer eluted by reading the 0D 260 and the remainder of the sample dried using a Speed Vac. The purified primer was then resuspended in 1X TE at a concentration of 100 M assuming 1 0D 260  =  33 g/ml and stored at -20°C.  2.2.11.4 Primer end-labeling  PCR reactions used for the detection of polymorphisms were performed in the presence of radioactive primers. Primers were radioactively end-labeled using T4 polynucleotide kinase (PNK; Sambrook et a!., 1989). A typical end-labeling reaction consisted of 5 l 10 M primer, 1X PNK buffer, 25 j.Ci ( P) 3 2 dATP, and 0.67 units of PNK in a total volume of 10 j.Ll. Reactions were also performed for larger quantities of primer by increasing the amounts of all components by the same factor. Primers were end-labeled for 45 minutes at 37°C and the reaction stopped by incubation at 65°C for 15 minutes. End-labeled primers were then added directly to PCR reactions.  51  2. Materials and Methods lox PNK buffer  500 mM Tris, pH 7.5 100 mM MgCl 50 mM DT 1 mM spermidine  2.2.11.5 Electrophoresis of radiolabeled PCR products After radiolabeled PCR reactions, 8 ul of sequencing stop mix were added. 8 l from each reaction was run on a 6% Hydrolink sequencing gel. Sizes of alleles were determined relative to the sequenced allele using an M13 sequencing reaction as a secondary size standard.  2.2.11.6 Specific PCR reactions and primer sequences 1) Alu PCR. Alu PCR involves the use of primers corresponding to the consensus sequence of the Alu family of repetitive elements to amplify the human DNA found between two adjacent elements (Nelson et a!., 1989; Brooks-Wilson et a!,. 1990). The primer used was the A1S primer of Brooks-Wilson et aL (1990) which has the sequence: 5’TCATGTCGACGCGAGACTCCATCTCAAA3’. The 3’ 18 nucleotides of the A1S primer are homologous to the extreme 3’ end of the Alu consensus sequence. Alu PCR reaction conditions were the standard conditions using 0.4 j.M A1S primer. Alu PCR cycling conditions consisted of 25 cycles of 1 minute denaturation at 94°C, 1 minute annealing at 58°C, and 3 minutes extension at 72°C, with an additional 10 second extension increase at 72°C each cycle. A final  52  2. Materials and Methods 72°C incubation for 10 minutes was performed at the end of the cycling.  2) Alu-T3 andAlu-T7PCK Alu-T3 and Alu-T7 PCR involves reactions using anAlu-specific primer and either of the sequencing primers T3 or T7. This type of PCR reaction was used to amplify the inter-Alu region from plasmids which contained a single Alu element. The Alu-specific primer used for these amplifications was either A1S or TC65A. The TC65A primer was made by deleting the Not I site (13 bp at 5’ end) from the TC-65 primer used by Nelson et al., 1989. The sequence of the TC65A primer is 5’ TFGCAGTGAGCCGAGAT 3’. The TC65A primer is homologous to positions 219 through 235 in the Alu consensus sequence (63bp to 46bp from the 3’ end of the Alu element). Standard PCR reaction conditions were used for Alu-T3 and Alu-T7 PCR, with 0.4 JM of either A1S or TC65A primer and 0.4 JM of either T3 or 17 primer added. Cycling conditions for Alu-T3 and Alu-T7 PCR were 25 cycles of 1 minute denaturation at 94°C, 1 minute annealing at 45°C and 3 minutes extension at 72°C, with an additional 10 seconds extension increase per cycle and a final 72°C incubation for 10 minutes.  3) Alu-LIC PCR and plasmid-LIC PCR PCR products suitable for LIC (section 2.2.9.3) were generated by amplification of 5PCR1 using the primer AlS-LIC and pUC19 using the primers PDJ8O and PDJ81 (Aslanidis and deJong, 1990). The sequence of the A1S-LIC primer is 5’ GATGGTAGTAGGCGAGACTCCATCTCAAA 3’. The sequence of the PDJ8O primer is 5’ CCTACTACCATCGGATCCCCGGGT 3’and the sequence  53  2. Materials and Methods of the PDJ81 primer is 5’ CCTACTACCATCGTCGACCTGCAG 3’. PCR conditions for the generation of LIC compatible products were the same as those -  used forA!u PCR.  4) D5S253 amplification of (GT) tract -  PCR primers flanking the (GT)n tract had the following sequences: D5S253-GT: 5’ GAAACCACCATGTAACTGATAG 3’ D5S253-CA: 5’ GGAAGGTI’ATAACAGAACACTG 3’ Standard reaction and cycling conditions were used with 0.4 j.LCi (0.8 pmoles) endlabeled D5S253-GT and 10 pmoles each of D5S253-GT and D5S253-CA primers.  5) D5S257 amplification of (GT) tract -  PCR primers flanking the (GT)n tract had the following sequences: D5S257-GT: 5’ ‘l’l’I’GAAAAGGGAAATACACTGTG 3’ D5S257-CA: 5’ CAACAAAGTAATFCCAGATACAC 3’ Standard reaction and cycling conditions were used with 0.8 ,.LCi (1.6 pmoles) endlabeled D5S257-GT primer and 10 pmoles each of D5S257-GT and D5S257-CA primers.  6) D5S260 amplification of (GT) tract -  PCR primers flanking the (GT)n tract had the following sequences: D5S260-GT: 5’AG’ITUI’CCTGAGAGTCATfAGC 3’ D5S260-CA: 5’AATfCAACTGTGACATATGCAAG 3’ Standard reaction and cycling conditions were used with 0.4 Ci (0.8 pmoles) end  54  2. Materials and Methods labeled D5S260-GT primer and 10 pmoles each of D5S260-CA and D5S260-GT primers.  7) D5S262 amplification ofAlu 3’ tail -  PCR primers flanking the Alu 3’ tail had the following sequences: D5S262-GT: 5’CCTCATAACACTGCTrGTAGAA 3’ D5S262-CA: 5’GTGGCAGTGAGCCGATCTCA 3’ Standard reaction and cycling conditions were used with 0.4 Ci (0.8 pmoles) endlabeled D5S262-GT primer and 10 pmoles each of D5S262-CA and D5S262-GT primers. The primer D5S262-CA has homology with the 3’ end of the Alu consensus sequence.  8) D5S265 amplification ofAlu 3’ tail -  PCR primers flanking the Alu 3’ tail had the following sequences: D5S265-A  =  TC65A primer  D5S265-T: 5’TrCAACCATITCCCCTGTrCG 3’ The TC65A primer has a 2 bp mismatch with the sequence of the Alu element at the D5S265 locus. Standard reaction conditions were used with 0.5 Ci (1 pmole) end labeled D5S265-T primer, 5 pmoles TC65A primer and 10 pmoles D5S265-T primer. Cycling conditions were 30 cycles of 1 minute denaturation at 94°C, 30 second annealing at 54°C and 1 minute extension at 72°C, with a final 72°C incubation for 10 minutes.  55  2. Materials and Methods 9) D5S266 amplification ofAlu 3’ tail -  PCR primers flanking the Alu 3’ tail had the following sequences: D5S266-A: 5’ CCTAGGTGATAGAGCAAGACC 3’ D5S266-T: 5’ CAAATATAACTACCACCTCCAG 3’ The primer D5S266-A has homology with the 3’ end of the Alu consensus sequence. PCR reactions were carried out using 1 mM MgCl with all other reaction components at standard values. Standard cycling conditions were used with 1 jCi (2 pmoles) end-labeled D5S266-T primer, and 10 pmoles of each of D5S266-A and D5S266-T primers.  10) D5S268 amplification of (GT) tract -  PCR primers flanking the (GT) tract had the following sequences: D5S268-GT: 5’AAGGTGAGGCAAAATGAGTGTA 3’ D5S268-CA: 5’CAATCAGGCCA’fllTl’AACTJ’CA 3’ Standard reaction and cycling conditions were used with 0.4 Ci (0.8 pmoles) end labeled D5S268-CA primer and 10 pmoles each of D5S268-CA and D5S268-GT primers.  56  2. Materials and Methods 2.2.12 Alu PCR differential hybridization Phage from the LAO5NSO1 or LAO5NLO3 libraries were plated at a density of 1000 to 1500 plaques per 10 cm plate and plaque lifts were hybridized with the labeled, preannealedAlu PCR product from the chromosome 5 somatic cell hybrid. Positive plaques were picked and stabbed multiple times onto a small area of a plate overlaid with bacteria to produce extremely large plaques. Duplicate plaque lifts were made of the secondary plates, one lift hybridized with labeled, preannealed Alu PCR product from the chromosome 5 hybrid and the other lift hybridized with the labeled, prearmealedAlu PCR product from the deletion 5 hybrid. Multiple exposures were made to compensate for overall differences in signal intensity between paired lifts. Phage which strongly hybridized with the chromosome S hybrid and failed to hybridize with the deletion 5 hybrid were picked and purified.  2.2.13 Statistical analysis  2.2.13.1 Radiation hybrid typing and analysis Radiation hybrid retention data were analyzed using the algorithms of Cox et al., 1990. Quattro-Pro compatible computer programs obtained from Dr. Cox were used for entering and analysis of marker retention data.  2.2.13.2 Polymorphism typing and analysis Polymorphisms associated with D5S205, D5S253, D5S257, D5S260, D5S262, A1u52A, AIu52B, D5S266 and D5S268 were typed in the CEPH panel of families (Dausset et al., 1990). CEPH typing data for D5S56, D5S59, D5S60, D5S6, DHFR,  57  2 Materials and Methods D5S37, D5S50 (Weiffenbach et a!., 1991), HPRTP2, D5S21, D5S76, D5S39, D5S78, D5S71 (Leppert et al., 1987), D2S51, D2S44, D2S54, D2S43 (O’Connell et al., 1989), D17S34, D17S30, D17S28, D17S31, D17S1, and MYH2 (Nakamura et al., 1988) were obtained from published data included in version 4 of the CEPH database (Dausset et al., 1990). The LINKAGE program package version 4.7 was used for data analysis (Lathrop et a!., 1984, 1985; Lathrop and Lalouel, 1988). Additional typing of the (GT) repeat polymorphism within D5S39 (Mankoo et a!., 1991) was performed for CEPH families informative for D5S262 in order to maximize the information available for linkage analysis with D5S262.  2.2.14 Somatic cell hybrid manipulations  2.2.14.1 Cell culture Somatic cell hybrids were grown in Dulbecco’s modified Eagle medium supplemented with 0.292 g/ml L-glutamine and 15% fetal bovine serum. Cells were subcultured in the following fashion. The media was removed from the cells, and the cells washed twice in 1X Hanks balanced salt solution. Cells were dislodged from the sides of the flask by the addition of 0.25% trypsin in Hanks solution and incubation with periodic gentle shaking for approximately 5 minutes at 37°C. An appropriate amount of cells was then transferred to a new flask, and fresh media added. Cells were trypsinized the day before harvesting for chromosomes.  58  2. Materials and Methods 2.2.1 4.2 Somatic cell hybrid DNA preparation DNA was extracted from somatic cell hybrids in the following fashion. Cells  were precipitated by spinning for 5 minutes at 2000 rpm, washed by resuspending in 0.85% (w/v) NaCJ, and reprecipitated by spinning for 5 minutes at 2000 rpm in a clinical centrifuge. One to 2 ml of 100 mM Tris; 40 mM EDTA pH 8.0 was added to the cells. Cells were lysed by the injection of 1 volume of lysis solution using a 5 ml syringe and a 16-18 gauge needle. An equal volume of TE saturated phenol was added to the lysed cell mixture, and the sample mixed vigorously for 10 minutes. The sample was centrifuged for 5 minutes at 2000 rpm in a clinical centrifuge and the upper aqueous layer removed with a large bore pipette and transferred to a fresh tube. The white interface from the phenol-cell lysis mixture was removed, mixed with 1 to 2 ml of 100 mM Tris; 40 mM EDTA pH 8.0 and re-extracted with phenol. The aqueous layers from two extractions were combined and extracted once with phenol and once with Sevags. The volume of the final aqueous layer was determined, 1/10 volume 4 M NH4OAc added and the sample mixed well. The DNA was precipitated by the addition of an equal volume of isopropanol. DNA was collected and recovered using a pasteur pipette with a curved end. The DNA on the end of the pipette tip was washed with 70% EtOH, then air dried. The DNA was dissolved in 0.5 to 1 mliX TE by slow mixing overnight on a rotator at 4°C. The yield of DNA was determined by taking an 0D 260 of a fraction of the sample. DNA samples were stored at 4°C.  59  2. Materials and Methods Lysis solution 100 mM Tris pH 8.0 40 mM EDTA 0.2% SDS  2.2.14.3 Preparation and staining of metaphase chromosomes Cells were harvested for metaphase chromosomes using the following procedure. Colcemid was added to 0.02 g/ml and the culture was incubated for 3 hours to arrest cells in metaphase. The cells were detached from the flask using trypsin as described previously. Trypsinized cells were transferred to a centrifuge tube and spun for 8 minutes at 1000 rpm in a clinical centrifuge. The majority of supernatant was removed and the cells resuspended in the residual supernatant by tapping gently. Cells were resuspended in approximately 5 ml 0.075 M KC1 by pipetting up and down with a pasteur pipette and incubated for 2 minutes at room temperature. Cells were precipitated by centriftigation at 1000 rpm for 8 minutes in a clinical centrifuge. The majority of supernatant was again removed and the cells resuspended in the residual supernatant by tapping gently. Cells were gradually resuspended in approximately 5 ml ice cold fix and left at room temperature for 10 minutes. Cells were precipitated by centrifugation at 1000 rpm for 8 minutes in a clinical centrifuge. Cells were washed by resuspending in ice cold fix, then precipitated by centrifugation at 1000 rpm for 8 minutes in a clinical centrifuge. If the suspension appeared to contain large amounts of debris, washing was repeated. Immediately prior to making slides, cells were resuspended in sufficient ice cold fix to create a milky colored solution. Slides were made by dropping the cell  60  2. Materials and Methods suspension onto a corner of a clean slide, then turning the slide so the solution moved down the slide. Amounts of cell suspension and slide angle were adjusted until chromosomes were well spread. Slides were made either immediately after harvesting cells or the day after harvesting. Slides were air dried and stored in covered containers for approximately 1 week before staining. Metaphase spreads were Giemsa banded (G-banding) using conventional techniques (Verma and Babu, 1989). Slides were treated as follows: 1) 0.1% trypsin; incubate for 10 to 15 seconds 2) 1% CaC12; incubate for 1 minute O; rinse twice 2 3) dH 4) Giemsa stain; incubate for approximately 1 minute O; rinse 2 5) dH Times for trypsinizing and staining were adjusted to obtain optimal G-banding.  Fix 1 volume glacial acetic acid 3 volumes methanol  Giemsa stain 2 ml Giemsa stain (Gurr’s improved R66) 20 ml 0.025 M phosphate buffer pH 6.8 (Fisher) O 2 30 ml dH  61  3. RESULTS  3.1 CLONE ISOLA TIONAND LOCALIZATION The initial objective of my thesis project was to isolate DNA fragments from within a region of human chromosome 5, qll.2-q13.3, defined by a segmental trisomy. This region was of interest due to the discovery of a Vancouver family in which the trisomic region co-segregated with schizophrenia and renal anomalies (Bassett et al., 1988; McGillivray et al., 1990). This region became of further interest due to reports of linkage between markers within this region and chronic spinal muscular atrophy (Brzustowicz et al., 1990; Melki et al., 1990; Gilliam et al., 1990). The technique of Alu PCR differential hybridization (Bernard et a!., 1991a) was developed to preferentially isolate DNA fragments from within the 5q11.2-q13.3 region. A schematic representation of the Alu PCR differential hybridization procedure is shown in Figure 3 (section 1.6). The localization of clones isolated by Alu PCR differential hybridization was confirmed by hybridization to the somatic cell hybrid GM1O114 (chromosome 5 hybrid) and absence of hybridization to the somatic cell hybrid HHW1O64 (deletion 5 hybrid).  3.1.1 Alu PCR differential hybridization Table 1 shows a summary of results obtained using Alu PCR differential hybridization in conjunction with the LAO5NSO1 and LAO5NLO3 phage libraries and the somatic cell hybrids GM1O114 (chromosome 5 hybrid) and HHW1O64 (deletion 5 hybrid). Two trials of the Alu PCR differential hybridization technique were performed with the LAO5NSO1 library and two trials were performed using the  62  3. Results LAO5NLO3 library. Approximately 5 X io phage from the LAO5NSO1 chromosome 5 phage library and approximately 1.3 X io phage from the LAO5NLO3 chromosome 5 phage library were screened with the Alu PCR product from the chromosome 5 hybrid. A total of 479 phage gave strong positive signals with the Alu PCR product from the chromosome 5 hybrid. The proportion of phage which hybridized with the Alu PCR product from the chromosome 5 hybrid was 0.4% for the LAO5NSO1 library and 2.2% for the LAO5NLO3 library. Hybridization of duplicate filters (Figure 5) revealed a total of 73 recombinant phage which hybridized to the chromosome 5 Alu PCR product and did not hybridize to the deletion 5 Alu PCR product. When these phage were replated and rescreened, 45 phage strongly hybridized to the chromosome 5 Alu PCR product and did not hybridize to the deletionS Alu PCR product. Phage which did not give differential signals when replated fell into two categories, those which no longer gave a strong signal with the Alu PCR product from either hybrid, and those which gave equivalently strong signals with both hybrids. No further experiments were performed with the phage which did not give differential signals upon rescreening. The 45 recombinant phage which gave strong differential signals upon rescreening were plaque purified and phage minipreps were made. Phage from trial #1 were isolated and purified as an initial group, while phage from trials #2 through #4 were isolated and purified as a second group. Localization to chromosome 5 and absence from HHW1O64 was later confirmed for 35/45 (78%) isolates (Table 1; section 3.1.3).  63  3. Results  Table 1. Clone isolation and localization  Trial  1 2 3 4  Library  #plaques screened  positivea  diffb  isolates  signal  rescreenC  d 10 confirmed  LAO5NSO1 LAO5NSO1 LAO5NLO3 LAO5NLO3  2.25X10 4 4 2.8X10 3 3.0X10 4 1.0X10  81 116 40 242  14 6 5 49  11 3 5 26  9 3 4 19  TOTAL  4 6.35X10  479  73  45  35  a clones positive with Alu PCR product from chromosome 5 hybrid b clones which exhibited differential signal on initial screen C  clones which exhibited differential signal when re-screened d clones for which localization to chromosome 5 and absence from HHW1O64 was confirmed by hybridization to Southern blots of the Alu PCR products from somatic cell hybrids HHW1O64 and GM1O114 (section 3.1.3)  64  3. Results Figure 5. Detection of clones derived from 5q11.2-q13.3 by differential hybridization  B  A  . •  •:•.• *e •  \b  ..  -  .  .  S.  .4  .  .  •• 4  ••••  S  O  Differential hybridization signals obtained when Alu PCR products from the chromosome 5 hybrid and deletion 5 hybrid were hybridized to duplicate plaque lifts. (A) Filter hybridized with the Alu PCR product of the chromosome 5 hybrid (GM1O114) and (B) duplicate filter hybridized with the Alu PCR product of the deletion 5 hybrid (HHW1O64). Arrows indicate three phage showing obvious differential hybridization which were picked for purification. Other apparent differences were less remarkable on the original autoradiographs.  65  3. Results 3.1.2 Alu PCR products from clones isolated by differential hybridization The 45 recombinant phage exhibiting strong differential signals were used as substrates for Alu PCR reactions with the A1S primer. Since these clones were isolated on the basis of hybridization to the Alu PCR product from the chromosome 5 hybrid, each clone necessarily contains the 3’ end of anAlu element. The second Alu element present in genomic DNA would also be present in each clone provided the inter-Alu region did not contain the restriction enzyme site used for cloning. The inter-Alu region could be amplified from 30 isolates (Table 2). These 30 isolates therefore contain two Alu elements oriented such that the 3’ ends of the elements point towards each other, allowing amplification of the inter-Alu region found in genomic DNA. The inter-Alu region could be amplified from 46% of isolates from the LAO5NSO1 library (Trials #1 and #2) and from 86% of isolates from the LAO5NLO3 library (Trials #3 and #4).  66  3. Results Table 2. Alu PCR products obtained from clones exhibiting differential signals  Trial  2  3  4  Lab name  Size inter-Alu elementa  5PCR1 5PCR2 5PCR3 5PCR4 5PCR5 5PCR6 5PCR7 5PCR8 5PCR9 5PCR1O 5PCR11 A1u16 A1u19 A1u20 A1u22 A1u24 A1u25 A1u26 A1u27 A1u28 A1u29 A1u30 A1u32 A1u36 A1u38  2.2kb  -  2.2kb  -  2.2kb 0.5kb nd 1.9kb (1.7kb, 1.6kb, 0.7kb) 2.2kb 1.0kb 4.4kb 2.8kb 1.0kb 4.4, 1.0 2.3kb 1.2kb 2.3kb 1.0kb (1.2kb, 1.4kb) 1.5kb  67  3. Results Table 2. (con’t) Trial  Lab name  Size inter-Alu elementa  4  A1u41 A1u43 A1u47 A1u48 A1u52 A1u54 A1u55 A1u56 A1u58 A1u59 Alu6O A1u61 A1u62 A1u64 A1u66 A1u67  4.4kb 2.8kb 3.0kb 1.0kb 2.5kb 2.8kb -  -  2.8kb nd 1.1kb 2.0kb 2.0kb nd 2.8kb -  A1u68 A1u71 A1u72 A1u73  3.0kb 2.0kb 3.8kb nd  a Size of product obtained byAlu PCR nd = no data = no Alu PCR product was detected Sizes in brackets are of minor amplification products. -  68  3. Results 3.1.2.1 Identification of multiple isolates and Alu-T3, Alu-T7 PCR for clones SPCR1 to 5PCR11 Clones 5PCR1 through 5PCR11 were the first group of phage isolated. Determination of clones isolated more than once (multiple isolates) for this group occurred at various steps during the purification and localization process. 5PCR4 and 5PCR9 were found to be analogous to 5PCR1 by hybridization of the PCR product from 5PCR1 to the PCR products from 5PCR4 and 5PCR9. 5PCR5 and 5PCR7 were initially postulated to be identical because they both contain a 3.8kb EcoRI fragment which hybridizes to a 2.9kbAlu PCR product from GM1O114. The identity of 5PCR5 and 5PCR7 was later confirmed by showing that the Alu-T7 PCR product from p5PCR5 (see below) hybridized to the 3.8kb insert from both phage. 5PCR2 and 5PCR6 were initially assumed to be equivalent because they both contain two EcoRI fragments of 2.2 and 0.8kb, and hybridize to a 3.4kb Alu PCR fragment from GM1O114. 5PCR2 and 5PCR6 were later demonstrated to be identical, since Alu-T7 PCR product from p5PCR2-1 hybridizes to the 2.2kb insert from each phage. The inserts from phage 5PCR1, 5PCR2, 5PCR3, 5PCR5, and 5PCR11 were subcloned into Bluescript II as EcoRI fragments. All phage with the exception of 5PCR2 contained a single EcoRI insert. Plasmid names from phage isolates with single EcoRI inserts are designated as phage names preceded by a “p” to denote the plasmid. As previously mentioned, phage 5PCR2 contained two EcoRI fragments of 2.2 and 0.8kb. Both of these fragments were subcloned into plasmids. The 2.2kb fragment was subcloned into plasmid p5PCR2-1 and the 0.8kb fragment subcloned into plasmid p5PCR2-2. Plasmid preps from p5PCR2-1, p5PCR2-2, p5PCR3,  69  3. Results p5PCR5, and p5PCR11 were then subjected toAlu-T7 and Alu-T3 amplifications. Results of amplifications are summarized in Table 3.  Table 3. Alu-T3 and Alu-T7 amplifications for clones 5PCR1 through 5PCR11  plasmid  insert size  Alu-T3  Alu-T7  p5PCR2-1  2.2kb  0.4kb  1.2kb  p5PCR2-2  0.8kb  -  -  p5PCR3  2.8kb  p5PCR5  3.8kb  p5PCR11  7kb  2.2kb -  -  -  1.6kb 1.4kb  3.1.2.2 Identification of multiple isolates for phage A1u19 to Alu 73 Isolates A1u16 through A1u73 were initially checked for identity with 5PCR1 through 5PCR11 by hybridization ofAlu,Alu-T3 orAlu-T7 fragments from 5PCR1 through 5PCR11 to phage plaque lifts. A1u20 was found to be equivalent to 5PCR1, A1u16 was equivalent to 5PCR5 and Alu24 and A1u59 were equivalent to 5PCR11. Clones A1u16 through A1u73 were then tested for multiple isolates. Clones were subdivided into two groups based on the size of their Alu PCR product. Those isolates which could not be Alu PCR amplified were included in both groups. For each group, phage were dotted onto a plate and plaque lifts made of the plate. Index clones which were potentially represented more than once were picked from  70  3. Results each group. Each index clone was hybridized to the plaque lift from the appropriate group. Multiple isolates were then determined on the basis of hybridization patterns. A summary of all multiple isolates obtained is shown in Table 4. For the majority of multiple isolates, all isolates in a group were from the same phage library and produced the same size Alu PCR product, indicating that the recombinant phage were likely identical in nature. However, the Alu PCR product from A1u28 contained an extra 4.4kb product in addition to the 1kb product observed for A1u22 and A1u48. A1u28 was therefore different from A1u22 and A1u48. Also, 5PCR11 was derived from the LAO5NSO1 library, while isolates A1u24 and A1u59 were from LAO5NLO3, indicating that recombinant phage within this group were not identical. To confirm that 5PCR11 and Alu24 contained overlapping sequences, the inter-Alu region from each of these isolates was hybridized to genomic DNA from GM1O114. Both 5PCR11 and A1u24 hybridized to a 4.4kbAlu PCR product from GM1O114, indicating that 5PCR11 and A1u24 contain inter-Alu element sequence in common. The identity of these two phage was further confirmed by hybridization of the Alu-T7 PCR product from p5PCR11 to restriction enzyme digested DNA from phage A1u24. The Alu-T7 PCR product from p5PCR11 hybridizes to a 7kb EcoRl fragment from phage A1u24, indicating that phage A1u24 likely contains the entirety of the 7kb EcoRl insert from phage 5PCR11. All sets of multiple isolates are referred to in subsequent sections using the  name of the lowest numerical isolate. A total of 30 distinct isolates were obtained which gave differential hybridization signals.  71  3. Results  Table 4. Multiple Isolates  Isolate  Library  Equivalent phage  Library  5PCR1  LAO5NSO1  5PCR4, 5PCR9, A1u20  LAO5NSO1  5PCR2  LAO5NSO1  5PCR6  LAO5NSO1  5PCR5  LAO5NSO1  5PCR7, A1u16  LAO5NSO1  5PCR11  L4O5NSO1  A1u24, A1u59  LAO5NLO3  A1u22  LAO5NLO3  A1u28, A1u48  LAO5NLO3  A1u25  LAO5NLO3  A1u43, A1u54, A1u58  LAO5NLO3  A1u62  LAO5NLO3  A1u71, A1u73  LAO5NLO3  72  3. Results 3.1.3 Confirmation of localization Alu PCR localization blots -  The localization of each of the 30 distinct isolates to the deletion region was confirmed by hybridization of DNA from each recombinant phage to Southern blots of the Alit PCR products from the chromosomeS and deletion 5 hybrids. Either the Alu PCR product, Alu-T7 PCR product, Alu-T3 PCR product or total phage DNA was used as a hybridization probe (Table 5). Repetitive probes were preannealed with a vast excess of unlabeled human DNA. Probes were classified as repetitive if they included the entire phage insert since each phage necessarily contained at least one Alu element due to their method of isolation. Probes were also classified as repetitive if initial hybridization of the Alu PCR product to an Alu PCR localization blot produced multiple strong bands. The results of this localization are described in Table 5. Examples of various probes hybridized to Alu PCR localization blots are shown in Figure 6. As is demonstrated in Figure 6, a single strongly hybridizing band was most prominent, with additional minor bands attributable to the hybridization of repetitive elements within the probe which were not completely blocked by preannealing. Twenty non-identical isolates were present in the chromosome 5 hybrid and absent from the deletionS hybrid. This corresponds to 35 of 45 phage isolated by Alu PCR differential hybridization if multiple isolates are included. Alu PCR differential hybridization therefore had success rate of 35/45 or 78%. Of the 10 isolates which could not be localized to the deletion region, three (5PCR1O, A1u56 and A1u64) were present elsewhere on chromosome 5, five (5PCR8, A1u27, A1u55, A1u67 and A1u72) failed to show any signal with either hybrid and two (A1u30, and A1u68) were highly repetitive and therefore could not be classified.  73  3. Results The 20 non-identical isolates localized to the deletion region were assigned locus names D5S205, and D5S251 through D5S269 (Table 5). Large scale phage preps were made for each of these isolates.  74  3. Results Table 5. Localization of clones by hybridization to somatic cell hybrids GM1O114 and HHW1O64  Locus  D5S205 D5S251 D5S252 D5S254 D5S253 D5S255 D5S256 D5S257 D5S258 D5S259 D5S260 D5S261 D5S262 D5S263 D5S264 D5S265 D5S266 D5S267 D5S268 D5S269  Phage/Plasmid name  Probe  Repetitive  Hybrid GM1O114  5PCR1 p5PCR2-1 p5PCR3 p5PCR5 p5PCR11 A1u19 A1u22 A1u25 Alu26 A1u29 A1u32 A1u36 A1u38 A1u41 A1u47 A1u52 Alu6O A1u61 A1u62 A1u66  Alu PCR Alu-T7 Alu-T3 Alu-T7 Alu-T7 Alu PCR Alu PCR Alu PCR Alu PCR Alu PCR Alit PCR Alu PCR Alu PCR Alu PCR Alu PCR Alu PCR Alu PCR Alu PCR Alu PCR Alit PCR  no yes no no yes no yes yes yes yes yes yes yes yes no  +  5PCR8 A1u27 A1u55 A1u67 A1u72  phage  yes yes no no yes yes yes yes yes no  phage phage phage Alu PCR  75  Hybrid HHW1O64 -  + +  -  +  -  +  -  + + + + + + + +  -  -  -  -  -  -  -  +  -  +  -  +  -  +  -  + + +  -  -  -  -  -  -  -  -  -  -  -  -  -  3. Results Table 5. (con’t)  Locus  Phage/Plasmid name  Probe  Repetitive  Hybrid GM1O114  Hybrid HHW1O64  A1u30 A1u68  Alu PCR Alu PCR  yes yes  ? ?  ? ?  5PCR1O A1u56 A1u64  Alu PCR phage phage  yes yes yes  +  +  +  +  +  +  76  3. Results Figure 6. Clone localization by hybridization to Southern blots of hybrid Alu PCR products  1  2  3  4  5  6  7  8  9 10 11 12  13 14  9—  3.8— I  — —  05—  Localization of clones by hybridization to Southern blots of the Alu PCR products of GM1O114 (chromosome 5 hybrid) and HHW1O64 (deletion 5 hybrid). Odd lanes (1, 3, 5, 7, 9, 11 and 13) contain the Alu PCR product from the chromosome 5 hybrid, and even lanes (2, 4, 6, 8, 10, 12, and 14) contain the Alu PCR product of the deletion 5 hybrid. Lanes were hybridized as follows: (1, 2) Alu PCR product of the chromosome 5 hybrid, (3, 4)Alu PCR product from 5PCR1, (5, 6) 5PCR2, (7, 8) 5PCR5, (9, 10) 5PCR11, (11, 12) 5PCR3, and (13, 14) 5PCR1O.  77  3. Results 3.1.4 Confirmation of localization Genomic localization blots -  The localization of clones from loci D5S205, D5S251, D5S252, D5S254, D5S253, D5S255, D5S264, and D5S267 to the deletion region of chromosome 5 was  further confirmed by hybridization to genomic DNA from GM1O114 (5 hybrid), HHW1O64 (deletion 5 hybrid), CHO (hamster) and a human lymphoblast sample. Probes from D5S205, D5S252 and D5S254 contained only unique sequences, hybridized to a single band in GM1O114 and human DNA with little background signal and did not hybridize HHW 1064 or hamster DNA (Figure 7). Similar results were obtained using probes from D5S255 and D5S264. Probes from D5S251, D5S253 and D5S267 produced discernable bands in the chromosome 5 hybrid DNA lane, no bands in the deletion 5 and hamster DNA lanes, and strong background signals in the human DNA lane. The localization to within the deletion region of chromosome 5 was therefore confirmed for all isolates tested.  3.1.5 Confirmation of localization PCR -  The localization of polymorphic systems associated with D55253, D5S257, D5S260, and D5S268 (section 3.3.1) was confirmed by amplification reactions using the HHW1O64 and GM1O114 hybrids as templates. Absence of amplification from HHW1O64 and amplification from GM1O114 was demonstrated for all systems.  78  3. Results Figure 7. Clone localization by hybridization to Southern blots of hybrid genomic DNA  A  B  C  1234  1234  1234  Kb 9.0—  3.8—  2.0—  Confirmation of localization of phage isolates by hybridization to Southern blots of EcoRI-digested genomic DNA. Lane 1, CHO (hamster); lane 2, GM1O114 (chromosome 5 hybrid); lane 3, HHW1O64 (deletion 5 hybrid); lane 4, human lymphoblast DNA. Panels were hybridized as follows: (A) Alu PCR product from D5S205, (B) Alu-T3 product from D5S252, and (C) Alu-17 product from D55254.  79  3. Results 3.1.6 Majority of clones do not correspond to visible differences between hybrid Alu PCR products The Alu PCR band to which each isolate corresponded was identified in order to determine if the band was noticeable as a difference between the Alu PCR products of the chromosome 5 and deletion 5 hybrids. The Alu PCR products of clones localized to the chromosome 5 deletion region were electrophoresed beside the Alu PCR products of the chromosome 5 and deletionS hybrids (Figure 8). The majority of isolates could not be detected as a difference between the Alu PCR product of the two hybrids. Several isolates, particularly those producing large inter-Alit fragments, corresponded to bands which were not visible on the ethidium bromide stained gel. Only the inter-Alu regions from loci D5S254 and D5S266 appear to correspond to a visually detectable difference between the Alu PCR products of the two hybrids.  80  3. Results Figure 8. Alu PCR products  1 2 3  4 5 6 7  8 9 101112 13 14 15 16 17 18 19 2  91  Ethidium bromide stained gel showing Alu PCR products from clones localized to the deletion region compared with the Alu PCR products from the HHW1O64 and GM1O114 hybrids. Lane 1, ) DNA digested with Hindill and SstII; Lanes 2 and 20, Alu PCR product from HHW1O64; Lanes 3 and 21, Alu PCR product from GM1O114. Lanes 4 to 19 contain the Alu PCR products from phage isolates. Lane 4, D5S266; Lane 5, D5S258; Lane 6, D5S261; Lane 7, D5S262; Lane 8, D5S255; Lane 9, D5S268; Lane 10, D5S267; Lane 11, D5S205; Lane 12, D5S259; Lane 13, D5S260; Lane 14, D5S265; Lane 15, D5S257; Lane 16, D5S269; Lane 17, D5S264; Lane 18, D5S263; Lane 19, D5S256 (A1u28).  81  3. Results 3.2 RADL4 TION HYBRID MAPPING The second goal of my thesis project was to physically map the 20 clones isolated from the 5q11.2-q13.3 region defined by the segmental trisomy. Radiation hybrid mapping (Cox et a!., 1990) was chosen to accomplish this goal. A radiation hybrid mapping panel is produced by subjecting a somatic cell hybrid containing a human chromosome of interest to a high dose of X-rays, followed by fusion of the irradiated cells with a rodent cell line. Fragments of the human chromosome are non-selectively retained in the radiation hybrids. Radiation hybrids can be analyzed for the presence or absence of markers, with coretention of two markers an indication of the physical distance between the two markers. A series of algorithms for the analysis of radiation hybrid retention data have been described by Cox et al., 1990. A radiation hybrid mapping panel consisting of 150 chromosome 5 hybrids was used to obtain order information on 18 clones isolated byAlu PCR differential  hybridization.  3.2.1 Radiation hybrid typing To determine the order of 18 of the 20 isolates which were localized to the deletion region of chromosome 5 (Table 5), isolates were typed in a chromosome 5 radiation hybrid panel consisting of 150 radiation hybrids. The Alu PCR products from D5S252 and D5S263 gave very weak signals when hybridized to Alu PCR localization blots and therefore were not scored in the radiation hybrid mapping panel. The inter-Alu region from the 18 remaining isolates was hybridized to Southern blots of the Alu PCR product from each of the 150 radiation hybrids. Visible Alu PCR amplification products were obtained for 92 of the 150 hybrids.  82  3. Results Figure 9 depicts the Alu PCR products from the amplification of hybrids 1 through 20 (17 radiation hybrids). Comparable results were obtained for the remainder of the hybrids. Isolates which had previously been determined to be repetitive when hybridized to Alu PCR localization blots were prearmealed with total human DNA. Isolates which gave multiple bands even after prearmealing due to their repetitive nature were hybridized to the Alu PCR hybrid blots singly. All other probes were hybridized in pairs. Figure 10 shows an example of hybridization signals obtained. Typing data indicating the presence or absence of each of the 18 probes in the 150 hybrids are included in Appendix 2. D5S39 typing results obtained from Dr. Ellen Solomon are also included in Appendix 2. Each of the 18 probes was nonselectively retained in 4.1% to 10.8% of the hybrids (Table 6).  83  3. Results Figure 9. Alu PCR products for radiation hybrids 1 through 20  1 2 3 4 5  6 7 8 9 10 1112 13 1415 16 1718 1920 2122  Ethidium bromide stained gel showing Alu PCR products obtained from radiation hybrids. Lanes 1 and 21,Alu PCR product from GM1O114; Lane 2,Alu PCR product from HHW1O64; Lane 10, Alu PCR product from PN/TS-1; Lane 22,) DNA digested with Hindlil and SstII. Lanes 3 through 9 and 11 through 20 contain the Alu PCR product obtained from radiation hybrids. Lane 3, hybrid 1; Lane 4, hybrid 2; Lane 5, hybrid 3; Lane 6, hybrid 4; Lane 7, hybrid 5; Lane 8, hybrid 6; Lane 9, hybrid 8; Lane 11 hybrid 10; Lanes 12 through 20, hybrids 12 through 20, respectively.  84  3. Results Figure 10. Probes from D5S260 and D5S262 hybridized to radiation hybrids 1 to 20  1 2  3 4  5  6  7 8 9  10 11 12 1314 15 16 171819 2021  I  Autoradiograph obtained by hybridization of probes from D5S260 and D5S262 to Southern blot ofAlu PCR products from radiation hybrids. Lanes 1 and 21,Alu PCR product from GM1O1 14; Lane 2, Alu PCR product from HHW1O64; Lane 10, Alu PCR product from PN/TS-1. Lanes 3 through 9 and 11 through 20 contain the Alu PCR product obtained from radiation hybrids. Lane 3, hybrid 1; Lane 4, hybrid 2; Lane 5, hybrid 3; Lane 6, hybrid 4; Lane 7, hybrid 5; Lane 8, hybrid 6; Lane 9, hybrid 8; Lane 11 hybrid 10; Lanes 12 through 20, hybrids 12 through 20, respectively.  85  3. Results Table 6. Radiation hybrid retention frequencies  Locus  D5S205 D5S251 D5S254 D5S253 D55255 D5S256 D5S257 D5S258 D5S259 D5S260 D5S261 D5S262 D5S264 D5S265 D5S266 D5S267 D5S268 D5S269 D5S39  Retention  Radiation Hybrids Scored  0.087 0.061 0.06 0.082 0.075 0.097 0.097 0.073 0.043 0.067 0.053 0.108 0.054 0.090 0.04 1 0.048 0.073 0.05 0.127  150 99 150 134 133 134 134 150 117 150 150 148 149 133 98 84 150 100 150  86  3. Results 3.2.2 Twopoint radiation hybrid analysis  To determine the most likely order of the isolates, markers were analyzed in each possible pairwise combination using the algorithm of Cox et a!., 1990. A lod score of 3 or more was taken as significant evidence for linkage (Table 7). The twopoint results were used to subdivide the markers into four groups, as indicated in Table 7.  87  3. Results Table 7. Twopoint radiation hybrid scores for markers linked at lod >3  Locus  Distance  Group #1 D5S253- D5S257 D5S257-D5S262 D5S268-D5S262 D5S260-D5S262 D5S262-D5S39 D5S39-D5S264  (cR5,üO)  LOD Score  80 75 95 89 97 58  3.56 3.98 3.13 3.42 3.36 6.35  63 65 81  3.58 4.20 3.50  42  3.37 3.66  Group #2 D5S259-D5S258 D5S258-D5S256 D5S256- D5S265 Group #3 Linked to 1 other marker D5S266-D5S261 D5S255-D5S254  77  Group #4 Linked to no other marker D5S205; D5S251; D5S267; D5S269 Group #5 Untyped in radiation hybrid panel D5S252; D5S263  88  3. Results 3.2.3 Fourpoint radiation hybrid analysis Cox et a!., 1990 devised algorithms for fourpoint radiation hybrid analysis which can be used to calculate the relative likelihoods of various marker orders and to determine radiation distances between markers. These fourpoint algorithms were used for group #1 and #2 markers. The odds against inversion of adjacent marker pairs was calculated by reversing the order of successive pairs of markers and calculating the likelihood of the order relative to the most likely order. Distances between markers in cR50,000 were calculated for the most likely marker orders. Group #1 consists of seven markers, five of which could be placed in a linear array from the results of the twopoint analysis (D5S253-D5S257-D5S262-D5S39D5S264). These 5 markers were analyzed in sets of 4 using the fourpoint algorithms of Cox et al., 1990. The results of the overlapping fourpoint analyses performed involving D5S253, D5S257, D5S262, D5S39 and D5S264 are shown in Figure 11. Radiation distances and odds against inversion of adjacent markers for the most likely marker order are also presented in Figure 11. Markers at either end of the group (D5S253 and D5S264) were each present in only one of the sets of four markers, so the odds of inversion of these markers was calculated using the pertinent fourpoint. Markers in the middle of the group (D5S257, D5S262 and D5S39) were present in both groups of four markers. Odds of inversion of these markers were calculated by inversion of the internal two markers of a set of four. The odds of inverting D5S257 and D5S262 was therefore calculated from the fourpoint involving D5S253, D5S257, D5S262 and D5S39, while the odds of inverting D5S262 and D5S39 was calculated from the fourpoint involving D5S257,  89  3. Results D5S262, D5S39 and D5S264. The other two markers in group #1 (D5S268 and D5S260) were linked by the twopoint data only to D5S262, one of the internal markers in the set. Fourpoint analyses involving both possible positions of D5S268 and D5S260 relative to D5S262 would not allow resolution of D5S268 and D5S260 from D5S262 (Figure 11). Each marker in group #2 was linked to a maximum of two other markers, indicating only one likely order. As depicted in Figure 12, the most likely order for group #2 markers is D5S259-D5S258-D5S256-D5S265. The odds of inversion of adjacent markers were calculated as described above for the group #1 markers. Radiation distances for this group and odds of inversion of adjacent markers for the most likely order of markers are shown in Figure 12.  90  3. Results Figure 11. Fourpoint Radiation hybrid analysis Group #1  marker order  -  odds  Dl  D2  D3  D5S253-D5S257-D5S262-D5S39 D5S257-D5S253-D5S262-D5S39 D5S253-D5S262-D5S257-D5S39 D5S253-D5S257-D5S39-D5S262  1 415 3.5X10 674  80  75  97  D5S257-D5S262-D5S39-D5S264 D5S262-D5S257-D5S39-D5S264 D5S257-D5S39-D5S262-D5S264 D5S257-D5S262-D5S264-D5S39  1 3 2.5X10 5 7X10 32  75  97  58  D5S257-D5S262-D5S260-D5S39 D5S257-D5S260-D5S262-D5S39  1 24  D5S257-D5S262-D5S268-D5S39 D5S257-D5S268-D5S262-D5S39  1 1.1  Odds against a given order were calculated relative to all other permutations of the set of four markers. Distances between markers in cR50,000 are indicated for the most likely marker orders. most likely order: odds cR50,000  415  3.5X105 32 7X105 D5S253 D5S257 D5S262 D5S39 D5S264 80 75 97 58  Odds against inversion of marker pairs are indicated above the map, while distances between markers in cR ,000 are indicated below. 50  91  3. Results Figure 12. Fourpoint Radiation hybrid analysis Group #2  marker order D5S259-D5S258-D5S256-D5S265 D5S258-D5S259-D5S256-D5S265 D5S259-D5S256-D5S258-D5S265 D5S259-D5S258-D5S265-D5S256  -  odds  Dl  D2  D3  1 35 383 294  63  65  81  Odds against a given order were calculated relative to all other permutations of the set of four markers. Distances between markers in cR50,000 are indicated for the most likely order of markers. most likely order: odds  35 D5S259  00 cR5O,0  383 D5S258  63  294 D5S256  65  D5S265 81  Odds against inversion of marker pairs are indicated above the map, while distances between markers in cR5O,000 are indicated below.  92  3. Results 3.3 POLYMORPHISMS A further aim of this thesis project was to place clones isolated from the 5q11.2-q13.3 region onto the genetic linkage map of human chromosome 5. The first step towards the accomplishment of this goal was to detect polymorphisms. Information obtained from radiation hybrid mapping was used to select clones of particular interest to use in the polymorphism screen. Screening was performed for a variety of types of DNA based polymorphisms. Polymorphism screening and typing is described in general in section 3.3.1. Polymorphisms detected are then described in further detail in sections 3.3.2. through 3.3.9.  3.3.1 Polymorphism screening and typing Polymorphism screening was done for three types of polymorphisms; conventional RFLPs, simple sequence repeat polymorphisms and polymorphisms in the polyA tails of Alit elements. A summary of polymorphisms identified is presented in Table 8. Polymorphisms detected were typed in the CEPH panel of families (Dausset et al., 1990). For each polymorphic system, hybridization to Southern blots or PCR reactions were performed on CEPH parents. CEPH families for which one or both parents were heterozygous were then typed. The heterozygosity and polymorphism information content (PlC) of each marker system was calculated from allele frequencies of the CEPH parents (Table 8).  93  3. Results Table 8. Polymorphisms  Locus  Polymorphism  #Alleles Het  Conventional RFLP D5S205 TaqI RFLP  D5S253 D5S257 D5S260 D5S268  (GT)n Tracts 23 (TG) 8 C(GT) 12 (TG) CG(TG) 6 (TG) 11 16 (GT)  (GT) Tracts at 3’ end of Alu elements D5S262 3 GA(TA) 5 (TA) (TA) (TG) 4 (TG) 5 (TA) 5 4 -TGGAA(TG) 5 T 3 ( T 6 TG) TA) A(TG) G D5S266 A 21 (TA) (TG) 10 6 3 CATACA(TA)  PlC  4  0.42  0.39  8 5 5 7  0.78 0.35 0.75 0.72  0.75 0.29 0.69 0.68  2  0.06  0.06  4  0.26  0.30  3 3  0.47 0.55  0.41 0.46  Alu polyA tract A1u52A A1u52B Het PlC  = =  heterozygosity polymorphism information content  94  3. Results Screening for conventional RFLPs was done for loci D5S205, D5S251, D5S252, D5S253, D5S254, D5S255 and D5S264. Probes were hybridized to Southern blots of 5 unrelated CEPH individuals digested with BamHI, BgllI, EcoRI, Hindill, MspI, PstI, PvuII and TaqI. A TaqI polymorphism was detected for D5S205. Codominant inheritance was observed for D5S205 in all CEPH families typed. During the course of the RFLP screen, it was reported (Weber and May, 1989) that simple sequence repeats of the (GT)n type were highly polymorphic in the human genome. Therefore, phage from all 20 loci within the deletion region were screened with poly(dC-dA)(dG-dT) to detect such tracts. Phage A1u24, A1u25, A1u32, A1u38, A1u60 and A1u62 (loci D5S253, D5S257, D5S260, D5S262, D5S266 and D5S268 respectively) hybridized poly (dC-dA)(dT-dG). The (GT)n tract from each of these phage was subcloned into Bluescriptil and sequenced. An autoradiograph of sequence obtained for the D5S257 (GT)n tract is shown in Figure 13. Comparable sequencing autoradiographs were obtained for the remainder of the (GT)n tracts. Sequences of the six (GT)n tracts are presented in Table 8. The SX  (GT)n tracts isolated fell into two categories, those which were present as  isolated tracts and those which were located at the 3’ ends of Alu elements. Primers flanking the (GT)n tracts were used to amplify the tracts to check for polymorphisms. All of the (GT) tracts amplified were polymorphic, with PlC values ranging from 0.06 to 0.75 (Table 8). Codominant Mendelian segregation was observed in CEPH families for all (GT)n tracts typed.  95  3. Results Figure 13. Sequence of D5S257 (GT) tract  TGCA  Sequence surrounding D5S257 (GT)n tract. Autoradiograph was obtained by denaturing polyacrylamide gel electrophoresis of a sequencing reaction on the plasmid p257RO.7 using the T7 sequencing primer.  96  3. Results (GT)n tracts were not present within loci D5S259, D5S258, D5S256 and D5S265, which were of interest since they comprised radiation hybrid mapping group #2. Similarly, D5S255 and D5S254 were linked by radiation hybrid mapping but did not contain (GT)n tracts. Alternate methods for generating polymorphisms for these loci were therefore explored. The polyA tail ofAlu elements had been reported to be polymorphic (Economou et al., 1990), and due to the method of isolation each of these clones contained at least one Alit 3’ end. Therefore, Alit elements from within these isolates were subcloned into Bluescriptil and sequence was obtained of the 3’ ends of the elements and flanking DNA. The insert from phage 5PCR5 (locus D5S254) had been subcloned into the plasmid p5PCR5. Portions of the phage inserts from A1u22, A1u26, A1u29 and A1u52 (D55259, D5S258, D5S256 and D5S265 loci respectively) were subcloned as EcoRI or Hindlil fragments. Plasmids containing single Alit elements were identified byAlu PCR, since plasmids with a single Alu element produce a single band in either a TC65AT3 PCR reaction or a TC65A-T7 PCR reaction. Plasmids containing a single Alit element were then sequenced using TC65A, T3 or T7 as primers. A total of l2Alu 3’ ends were sequenced, and 6 recognizable polyA tails were found. Sequences of the Alu elements with recognizable 3’ tails are shown in Table 9.  97  3. Results Table 9. Sequence ofAlu polyA tails  Locus  Plasmid  Alu polyA tail sequence  D55254 D55256 D5S258 D5S258 D5S259 D5S265  p5PCR5 p22E1A8 p26E2 p26H6 p29H2 p52H1  A 1 5 7 A T 3 G 5 2 A A AGA C 7 8 A GA A 8 7 A 1A A C 5 CA G 4 A 3 C 7 tj  Two of the Alu 3’ tails were felt to be of sufficient length to warrant polymorphism screening, namely the A 15 tract found in p5PCR5 (D5S254) and the 3 1 C 5 CA G 4 6 A A C tract from p52H1 (D55265). A primer 3’ of the polyA tail was 7 synthesized for both tracts. The primer for D5S254 was named D5S254-T, and had the sequence 5’ TTAAAATGTATGCATTATIICTGGA 3’. The primer for D5S265 was called D5S265-T and had the sequence 5’ TI’CAACCAIITCCCCTGTFCG 3’. The Alu element in p5PCR5 was then sequenced using the D5S264-T primer, and the Alu element in p52H1 sequenced using the D5S265-T primer. Both Alu elements were found to have significant homology to the TC65A primer. The Alu element in p5PCR5 had a 1 bp mismatch with TC65A at the 5’ end of the primer, and the Alu element in p52H1 had two 1 bp mismatches with TC65A inside the primer. The sequence of the p5PCR5 Alu element and surrounding region is shown in Figure 14, together with sequence alignment with the Alu consensus sequence. The sequence of the p52H1 Alu element is shown in Figure 24 (section 3.3.7.).  98  3. Results A PCR reaction using TC65A and each of the unique primers was carried out to amplify the Alu polyA tracts for each locus. The Alu polyA tract present within D5S254 could be amplified from the plasmid p5PCR5 and the phage 5PCR5, but could not be amplified from the somatic cell hybrid GM 10114 or from genomic DNA using a variety of PCR reaction conditions and cycling conditions. The polyA tract corresponding to D5S254 was therefore not investigated further. The polyA tract present within D5S265 was amplifiable from both plasmid and genomic DNA (section 3.3.7).  99  3. Results Figure 14. Sequence of D5S254 Alu polyA tract and flanking sequence and alignment with Alu consensus sequence alucon 5’ GGCTGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAG alucon 5’ GTGGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGG alucon 5’ TGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGC  liii liii 11111  D5S254  IllIllIllIllIll 11111111111  5’CCCCATCTC ACTAA TATACAAAAATTAGCCAGGCGTGGTGGCAT 3’ GGGGTAGAG TGATT ATATGTTTTTAATCGGTCCGCACCACCGTA  alucon 5’ GCGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCTGAGGCAGGAGAATCG D5S254  5 ‘GTGCCTGTAGTCCCAGCTAGTCAGGTGGCTGAGGC 3’ CACGGACATCAGGGTCGATCAGTCCACCGACTCCG  111111111  AGGAGAATCA TCCTCTTAGT  alucon 5’ CTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCAC  IIIIIItIIIIIIIIIIII,IIIIIIIIIIIIIIIIIIIIIIIIII  D5S254 5 ‘ATTGAACCCGGGAGGTGGAGGCTGCAGTGAGCCGAGATTGTGCCACTGTAC 3’ TAACTTGGGCCCTCCACCTCCGACGTCACTCGGCTCTAACACGGTGACATG alucon 5’ TGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCA D5S254 5 ‘T 3 ‘A  CCTGCGTGGGCAACAGAGCGAGACTCCATCTCAAAAAAAAAAAAA GGACGCACCCGTTGTCTCGCTCTGAGGTAGAGTTTTTTTTTTTTT  D5S254 5 ‘AATTCCAGAAATAATGCATACATTTTAAATAGCATGCCTTTCTGAGTTGTG 3’ TTAAGGTCTTTATTACGTATGTAAAATTTATCGTACGGAAAGACTCAACAC D5S254 5’ATGAAATCTTAT 3 ‘TACTTTAGAATA  TC65A primer sequence is underlined in the Alu consensus sequence and D5S254-T primer sequence is underlined in the D5S254 sequence. The Alu consensus sequence was obtained from the EMBL database version 27.  100  3. Results 3.3.2 D5S205 The polymorphism detected within locus D5S205 (Bernard and Wood, 1991) is a TaqI RFLP with a heterozygosity of 0.42 and a PlC of 0.39 calculated from allele frequencies obtained by typing 80 CEPH parents (Table 10). The TaqI polymorphism was detected by hybridization of the 2.2kb Alu PCR product from phage 5PCR1 to Southern blots of CEPH genomic DNA digested with TaqI. Table 10. D5S205 Allele frequencies  Allele  size (kb)  Al A2 A3 A4  9kb 6kb 3.2kb 11kb  frequency 0.12 0.74 0.12 0.02  D5S205 reference genotypes were as follows: 133101  =  A2,A2; 133102  =  A2,A2.  Segregation of the 9kb and 6kb alleles in family 1349 is shown in Figure 15.  101  3. Results Figure 15. D5S205 TaqI polymorphism  bEEb ‘666± 9 kb  6 kb  -  -  Segregation of the 9 kb and 6 kb alleles at the D5S205 locus in family 1349.  102  3. Results 3.3.3 D5S253 The polymorphism detected at locus D5S253 is a microsatellite repeat with a heterozygosity of 0.78 and a PlC of 0.75 calculated from allele frequencies obtained by typing 77 CEPH parents (Table 11). The (GT) tract within D5S253 was subcloned as a 3kb HindIll fragment into Hindill digested Bluescriptil to produce the plasmid p253H3.0. The region containing the D5S253-GT primer was subcloned within a 500bp RsaI fragment into EcoRV digested Bluescriptil to produce the plasmid p253R0.5. The sequence of the D5S253-GT primer was determined from the plasmid p253R0.5 using T3 and T7 primers. The sequence of the D5S253-CA primer was then determined from the p253H3.0 plasmid using the D5S253-GT primer. The sequence surrounding the (GT)n tract (Figure 16) was determined from the p253H3.0 plasmid using the D5S253-CA and D5S253-GT primers. Primer sequences were as follows: D5S253-GT: 5’ GAAACCACCATGTAACTGATAG 3’ D5S253-CA: 5’ GGAAGGTATAACAGAACACTG 3’ The sequence of the cloned product repeat unit was (GT) 23 which corresponds to an amplification product of 126bp. D5S253 reference genotypes were as follows: 133101  =  A4,A4; 133102  =  A1,A4. Segregation of the 124, 120 and 114 bp alleles  in family 1340 is shown in Figure 17.  103  3. Results Table 11. D5S253 Allele frequencies  Allele  size(bp)  frequency  Al A2 A3 A4 A5 A6 A7 A8  126 124 122 120 118 116 114 102  0.045 0.097 0.026 0.38 0.17 0.11 0.16 0.013  104  3. Results Figure 16. D5S253 (GT)n tract and flanking sequence 5’ AAAAATTAACTGGGTGTGGTAGTGTGCACCTGTGGTTCTAGCTACTCGGGAGGCTGAG 3’ TTTTTAATTGACCCACACCATCACACGTGGACACCAAGATCGATGAGCCCTCCGACTC GTAGGAGGCTTGCTTGACCCCAGGAGGTCAAGGCTGTGGTGAGCTGAGATTGTACCATTG CATCCTCCGAACGAACTGGGGTCCTCCAGTTCCGACACCACTCGACTCTAACATGGTAAC CACTCTAGCCTGGGCAACAGATCCAGACCTTGTCTCTAAATTAAAACAAAACAAAACCAA GTGAGATCGGACCCGTTGTCTAGGTCTGGAACAGAGATTTAATTTTGTTTTGTTTTGGTT ACAAAAAAACAGCTGNATAAGGAAGGTTATAACAGAACACTGTTCTCTCTTTACACACAC TGTTTTTTTGTCGACNTATTCCTTCCAATATTGTCTTGTGACAAGAGAGAAATGTGTGTG ACACACACACACACACACACACACACACACACACACACATCACACGTACAGGAATTATTT TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAGTGTGCATGTCCTTAATAAA TAACCTATCAGTTACATGGTGGTTTCACAGGTTTCAACTTCATCAACCCAGAACCACAAT ATTGGATAGTCAATGTACCACCAAAGTGTCCAAAGTTGAAGTAGTTGGGTCTTGGTGTTA CACAGATTTTGGCTAGACTCTGACTCTCATCTACTAGTGATAACAACAAGTTCCCTGTGG GTGTCTAAAACCGATCTGAGACTGAGAGTAGATGATCACTATTGTTGTTCAAGGGACACC AGTTTATAGCCCACAGATTATCA3’ TCAAATATCGGGTGTCTAATAGT5’  Sequence surrounding (GT)n tract. Primer sequences are underlined.  105  3. Results Figure 17. D5S253 (GT)n polymorphism  66EEEEr5 124 bp  -  l2Obp-  114 bp  -p  -  Segregation of the 124, 120 and 114 bp alleles at the D5S253 locus in family 1340. Allele sizes refer to predominant bands and are indicated on the left side of the figure. Minor bands present are shadow bands produced as an artifact of PCR.  106  3. Results 3.3.4 D5S257 The polymorphism detected at locus D5S257 (Bernard et a!., 1991b) is a microsatellite repeat with a heterozygosity of 0.35 and a PlC of 0.29 calculated from allele frequencies obtained by typing 79 CEPH parents (Table 12). The (GT) tract within D5S257 was subcloned as a 0.7kb RsaI fragment into EcoRV digested Bluescriptil to produce the plasmid p257R0.7. The sequence surrounding the D5S257 (GT)n tract (Figure 18) was determined from the plasmid p257R0.7 using T3 and V primers. Primer sequences were as follows: D5S257-GT: 5’ ‘ITI’GAAAAGGGAAATACACTGTG 3’ D5S257-CA: 5’ CAACAAAGTAATCCAGATACAC 3’  The sequence of the cloned product repeat unit was (TG) 8 which C(GT) 12 corresponds to an amplification product of 79bp. D5S257 reference genotypes were as follows: 133101  =  A2,A2; 133102  =  A2,A3. Segregation of the 79 and 77 bp  alleles in family 1332 is shown in Figure 19.  107  3. Results  Table 12. D5S257 Allele frequencies  Allele  size(bp)  frequency  Al A2 A3 A4 AS  81 79 77 75 69  0.006 0.79 0.19 0.013 0.006  Figure 18. D5S257 (GT) tract and flanking sequence 5’ ATAGCACTTTTAAATTGTTCTCATTCCCTTTGCTTAAAGGTAAGGTTTTTGAGCATTT 3’ TAT CGTGAAAATTTAACAAGAGTAAGGGAAACGAATTTCCATTCCAAAAACTCGTAAA  TAATACATCAAGTTAAAGTGTCAATTGTAGGCTTTGGTAAGATAATATAGATCAGTCATA ATTATGTAGTTCAATTTCACAGTTAACATCCGAAACCATTCTATTATATCTAGTCAGTAT TTTGAAAAGGGAAATACACTGTGTGTGTGTGTGTGTGTGTGTGCGTGTGTGTGTGTGTGT AAACTTTTCCCTTTATGTGACACACACACACACACACACACACGCACACACACACACACA ATCTGGAATTACTTTGTTGGAAT3’ TAGACCTTAATGAAACAACCTTA5’  Sequence surrounding (GT)n tract. Primer sequences are underlined.  108  3. Results Figure 19. D5S257 (GT) polymorphism  6EbEb6EE 79 bp 77 bp  -  -  Segregation of the 79 and 77 bp alleles at the D5S257 locus in family 1332. Allele sizes refer to predominant bands and are indicated on the left side of the figure. Minor bands present are shadow bands produced as an artifact of PCR.  109  3. Results 3.3.5 D5S260 The polymorphism detected at locus D5S260 (Bernard et a!., 1991c) is a microsatellite repeat with a heterozygosity of 0.75 and a PlC of 0.69 calculated from allele frequencies obtained by typing 69 CEPH parents (Table 13). The (GT) tract within D5S260 was present within the 2.3kb Alu PCR product of phage Alu32. The (GT) tract was subcloned from the Alu PCR product as a 0.5kb RsaI fragment into EcoRV digested Bluescriptil to produce the plasmid p260R0.5. The sequence surrounding the D5S260 (GT)n tract (Figure 20) was determined from the plasmid p260R0.5 using T3 and V primers. Primer sequences were as follows: GT strand primer: 5’AG’UITI’CCTGAGAGTCATfAGC 3’ CA strand primer: 5’AATI’CAACTGTGACATATGCAAG 3’ The sequence of the cloned product repeat unit was 11 CG(TG) which 6 (TG) corresponds to an amplification product of 146bp. D5S260 reference genotypes were as follows: 133101  =  A2,A4; 133102  =  A3,A4. Segregation of the 152, 148 and  146 bp alleles in family 1345 is shown in Figure 21  110  3. Results Table 13. D5S260 Allele frequencies  Allele  size(bp)  frequency  Al A2 A3 A4 A5  154 152 150 148 146  0.065 0.38 0.14 0.27 0.15  Figure 20. D5S260 (GT)n tract and flanking sequence 5’ CCAGTTTTCCTGAGAGTCATTAGCTCTGGCTTCTTGTGGATCCTTTCAGAAAGAATCT 3’ GGTCAAAAGGACTCTCAGTAATCGAGACCGAAGAACACCTAGGAAAGTCTTTCTTAGA GTGTGTATATTTGCATGTGTGTGTGTGCGTGTGTGTGTGTGTGTGTGTGTGAATGGGTAT CACACATATAAACGTACACACACACACGCACACACACACACACACACACACTTACCCATA  ATACACACTTGCATATGTCACAGTTGAATTTATTGTCTTGAATGGTAATAGTTAAGTCAT TATGTGTGAACGTATACAGTGTCAACTTAAATAACAGAACTTACCATTATCAATTCAGTA AAATATATAG3’ TTTATATATC5’  Sequence surrounding (GT)n tract. Primer sequences are underlined.  111  3. Results Figure 21. D5S260 (GT)n polymorphism  6EE6EE 152 bp 148 bp 146 bp  -  -  -  Segregation of the 152, 148 and 146 bp alleles at the D5S260 locus in family 1345. Allele sizes refer to predominant bands and are indicated on the left side of the figure. Minor bands present are shadow bands produced as an artifact of PCR.  112  3. Results 3.3.6 D5S262 The polymorphism detected at locus D5S262 is a microsatellite repeat located at the 3’ end of anAlu element with a heterozygosity of 0.06 and a PlC of 0.06 calculated from allele frequencies obtained by typing 78 CEPH parents (Table 14). The (GT) tract within D5S262 was subcloned as a 0.8kb SmaI-PstI fragment into SmaI-PstI digested Bluescriptil to produce the plasmid p262SP0.8. The sequence surrounding the D5S262 (GT) tract was determined from the plasmid p262SP0.8 using the T7 primer. The GT tract was present at the 3’ end of anAlu element (Figure 22). Primer sequences were as follows: D5S262-GT: 5’CCTCATAACACTGCTFGTAGAA 3’ D5S262-CA: 5’GTGGCAGTGAGCCGATCTCA 3’ The D5S262-CA primer has homology with the Alu consensus sequence. The sequence of the cloned product repeat unit was 3 GA(TA) (TA) 3 (TG) 4 (TA) 56 (TG) T 4 ( 5 T 3 GGAA(TG) TA) TG) A(TG) (TA) T 6 G which corresponds to an amplification product of 193bp. D5S262 reference genotypes were as follows: 133101  =  A1,A1; 133102  A1,A1.  Segregation of the 193 and 185 bp alleles in family 35 is shown in Figure 23.  Table 14. D5S262 Allele frequencies  Allele  size(bp)  Al A2  193 185  frequency 0.97 0.03  113  3. Results  Figure 22. D5S262 (GT) tract and flanking sequence aligned with Alu consensus sequence Sequence surrounding (GT)n tract and alignment with Alu consensus sequence. Primer sequences are underlined. The Alu consensus sequence was obtained from the EMBL database version 27.  114  3. Results alucon 5’ GGCTGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAG alucon 5’ GTGGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGG alucon 5’ TGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGC  III  D5S262  III  5’TGGCAGCGT 3 ‘ACCGTCGCA  alucon 5’ GCGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCTGAGGCAGGAGAATCG  1111111111  1111111111  1111111  D5S2 62 5’ GTGCCTGTAATGGCAGCTACTCGNN 3’ CACGGACATTACCGTCGATGAGCNN  GGNGAATTG CCNCTTAAC  alucon 5’ CTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCAC  11111111 111111  11111 IllIllIllIll  II  11111111  D5S262 5’ CTTGAACCTGGGAGGCAGAGGTGGCAGTGAGCCGATCTCATGCCACTGC 3’ GAACTTGGACCCTCCGTCTCCACCGTCACTCGGCTAGAGTACGGTGACG  alucon 5’ TGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCA  I  D5S262  II  5 ‘TTTCCATATATATATATA 3’ AAAGGTATATATATATAT  D5S 262 5’ CACACACACACATACACANATATATATATACACACACATTCCATATATATA 3’ GTGTGTGTGTGTATGTGTNTATATATATATGTGTGTGTAAGGTATATATAT D5S 262 5’ TACACACACACATATATATATACACACACATATATATCTATATACTGTGTT 3’ ATGTGTGTGTGTATATATATATGTGTGTGTATATATAGATATATGACACAA D5S 262 5’ AATTTAATGAGAGTTGGCAATTTTCTACAAGCAGTGTTATGAGGTATTG 3’ TTAAATTACTCTCAACCGTTAAAAGATGTTCGTCACAATACTCCATAAC  115  3. Results  Figure 23. D5S262 (GT)n polymorphism  0  EE6666Eb 193 bp  -  Segregation of the 193 and 185 bp alleles at the D55262 locus in family 35. Allele sizes refer to predominant bands and are indicated on the left side of the figure. Minor bands present are shadow bands produced as an artifact of PCR.  116  3. Results 3.3. 7D5S265 The subcloning and sequencing of the Alu polyA tract contained within D5S265 was described in section 3.3.1. The sequence surrounding the D5S265A1u polyA tract and alignment with the Alu consensus sequence is shown in Figure 24. Primer sequences were as follows: D5S265-A: TC65A D5S265-T: 5’ ‘fl’CAACCAUilCCCCTGTFCG 3’ The sequence of the cloned product repeat unit was 5 1A A C CA G 4 A 3 7 C , which corresponds to an amplification product of 172bp. The D5S265 primers were used for a PCR reaction involving the plasmid p52H1 and an amplification product of 172bp was obtained. An amplification product of 172 bp was also obtained for GM 10114, and no amplification product was observed when the HHW1O64 hybrid was used as a template. However, when DNA samples from CEPH individuals were used as templates, two polymorphic systems were amplified (Figure 25). The smaller system is referred to as AIu52A and the larger system as AIu52B. When the PCR annealing temperature was increased, the intensity of the bands in each of the systems decreased by the same proportion. The presence of the second system was therefore not due to detection of a sequence with lower homology to the primers used. No amplification product was observed using plasmid DNA or CEPH DNA as a template when TC65A was not added to the PCR reaction. Therefore, products observed were not due to amplification between two D5S265-T primers. The TC65A primer was unlabelled, indicating that amplification products observed were not due to priming between  117  3. Results adjacent TC65A sequences. The products observed for D5S265 were therefore due to amplification between the D5S265-T and TC65A primers.  118  3. Results  Figure 24. D5S265 Alu polyA tract and flanking sequence compared with Alu consensus sequence alucon 5’ GGCTGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAG alucon 5’ GTGGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGG alucon 5 ‘TGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGC  1111111  D5S265  5’CGGTGGCGC 3’ GCCACCGCG  alucon 5’ GCGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCTGAGGCAGGAGAATCG D5S265 5’ATGCCTGTAA CTCAGCT CTCAGGAGGCTGAGGC 3’TACGGACATT GAGTCGA GAGTCCTCCGACTCCG  AGGAGAATCG TCCTCTTAGC  alucon 5’ CTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCAC D5S265 5 ‘CTTGA CCTGGGAGGCAGAGGTTGC GTGAGCTGAGATAGCGCCATTGCAC 3’ GAACT GGACCCTCCGTCTCCAACG CACTCGACTCTATCGCGGTAACGTG alucon 5’ TGCACTCCAGCCTGGGCGACAGAGCGAGACTC CGTCTCA  I  D5S265 5’T 3 ‘A  11111111  11111111 1111111  111111  CCAGCCTGA CGACAGAGTGAGACTCTTGTCTCAAAAAAAAAAAA GGTCGGACT GCTGTCTCACTCTGAGAACAGAGTTTTTTTTTTTT  D5S2 65 5’ AAAAACAAAAACAAAAGCCCCAAAAAACAGTATTATCAACAAAATTGTAGG 3’ TTTTTGTTTTTGTTTTCGGGGTTTTTTGTCATAATAGTTGTTTTAACATCC D5S265 5 ‘GGGCAAAAAGGAAAAAGGCAATCTGAACGAACAGGGGAAATGGTTGAATCC 3’ CCCGTTTTTCCTTTTTCCGTTAGACTTGCTTGTCCCCTTTACCAACTTAGG D5S265 5’ATTAGTCA3’ 3 ‘TAATCAGT3’  Sequence surrounding Alu polyA tail and alignment with Alit consensus sequence. TC65A primer sequence is underlined in the Alu consensus sequence and D5S265-T primer sequence is underlined in the D5S265 sequence. The Alit consensus sequence was obtained from the EMBL database version 27.  119  3. Results Figure 25. D5S265 Alu polyA tract polymorphisms  12345678  ——  186177172  Amplification obtained using D5S265-T and TC65A primers on CEPH parents. Lane 1, 202; Lane 2, 1201; Lane 3, 1202; Lane 4, 1701; Lane 5, 1702; Lane 6, 2101; Lane 7, 2102; Lane 8, 88401. Allele sizes refer to predominant bands and are indicated on the left side of the figure. Minor bands present are shadow bands produced as an artifact of PCR.  120  3. Results 3.3. Zi System A1uS2A System A1u52A consisted of 3 fragments of 172, 177 and 186 bp. CEPH parents observed had phenotypes of 172, 177/172, 186/172 and 186/177/172. Table 15 lists offspring phenotypes observed for all informative matings. Various segregation models for the AIu52A system were tested to obtain the best fit to the data. A single locus model with allele sizes of 186, 177 and 172bp is not possible due to the presence of individuals with a 186, 177, 172 phenotype. A total of 5 families containing at least one 186, 177, 172bp individual were observed (2 parents and 15 children Table 15). -  121  3. Results Table 15. Observed matings AIu52A -  parental phenotypes  family  offspring phenotypes 172  172,177  172,186 172,177,186  172 X 172,177 2 104 1332 1344 1349 1362 1375 1418 1420 1333 1340 13291 13292 13293 Total  3 6 1 29  5 77  17 1423 Total  1 5 6  1346 1350 Total  0 0 0  0 0 0 6 4 1 3 3 0 0 2  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0  3 4  0 0 0  5 3 8  3 4  6 10 10 2 4 9 4 5 7 9 3 2 1  172 X 172,186  7  172 X 172,177,186  122  7  0 0 0  3. Results Table 15 (con’t)  parental phenotypes  family  offspring phenotypes 172  172,177  172,186 172,177,186  172,177 X 172,177 102 1377 1408 1345 Total  0 3 2 2 7  13 5 3 5 26  0 0 0 0 0  0 0 0 0 0  884 1421 1341 Total  0 0 0 0  4 0 3 7  0 3 4 7  8 5 1 14  21 23  0 0  Total  0  3 1 4  1 3 4  0 1 1  1347 1424  3 0  6 8  0 0  0 0  172,177 X 172,186  172,177 X 172,177,186  ? X 177,172  123  3. Results A slightly more complex model for segregation involving two loci, one of which was polymorphic and the other of which was monomorphic for the 172 bp band was then tested for fit to the A1u52A data. Due to the presence of the postulated constant 172bp band, a distinction could not be made as to whether the polymorphic system involved three alleles of 186, 177 and 172 bp or three alleles of 186, 177 and no amplification. The 172bp band is therefore indicated in brackets when referred to as part of the A1uS2A polymorphic system. The test for fit to this model was carried out as follows. Parental phenotypes were converted to the most likely genotypes using family segregation data. Allele frequencies for CEPH parents were calculated based on genotypes inferred by the model (Table 16). Allele frequencies were then used to predict expected CEPH parent phenotype values based on Hardy-Weinberg equilibrium (Hedrick, 1983). 2 AX test (Freedman et a!., 1980) was then used to compare observed and expected values, and a p value calculated. As seen in Table 17, the expected and observed values were closely correlated, with a 0.10< p <0.30 indicating that the data fit the single polymorphism model.  124  3. Results Table 16. Allele frequencies for polymorphic locus within A1u52A system assuming a single polymorphism  Allele  size(bp)  frequency  Al A2 A3  (172) 177 186  0.675 0.260 0.065  Table 17. Expected versus observed genotypes for polymorphic loci within Alu52A system assuming a single polymorphism  genotype  expected  observed  (172),(172) (172),177  35 27 7  39 20 4 8 4 1  (172),186 177,177 177,186 186,186  X ( 2 4)  =  5.69  5 3 0  0.1< p <0.3  125  3. Results To determine if CEPH offspring phenotypes followed Mendelian segregation assuming the single polymorphism model, a comparison was made between expected and observed numbers of offspring for each parental mating (Table 18). A test was used to compare expected and observed offspring numbers for each mating class. A p value of greater than 0.10 was observed for all classes, indicating that the data fit the model well. Assuming the single polymorphism model, the polymorphic system within the D5S265 locus has a heterozygosity of 0.47 and a PlC of 0.41, using allele frequencies observed for 77 CEPH parents. A1u52A reference genotypes were as follows: 133101  =  A1,A1; 133102  =  A1,A1. Segregation of the 177 and 172 bp alleles at the  A1u52A locus in family 1349 is shown in Figure 26.  126  3. Results Table 18. Expected versus observed offspring phenotypes assuming a single polymorphism model for A1u52A  predicted parental genotypes at the polymorphic locus  offspring phenotypes  Obs Exp  (i72)XL12Z) (172) 177 (1) 2 x  =  0.56 0.30< p <0.50  Ohs Exp  UIZ)XJ17 (172) 177  Ohs Exp  U2Z)Xf.ii) (172) 186 (1) 2 X  =  0.077 0.70< p <0.90  Z)Xj77 (172) 186 (1) 2 x  =  177 (1) 2 x  Ohs Exp  0.067 0.70< p <0.90 Ohs Exp  177 =  1.07 p  0.30  172  172,177  172,186  172,177,186  29 32  35 32  0 0  0 0  families:  1344, 1349, 1362, 1375, 1418, 1340, 13291, 13292, 13293  0 0  42 all  families:  2, 104, 1332, 1420, 1333  6 6.5  0 0  families: 0 0  0 0  7 6.5  0 0  7  0  7.5  0  0  0 0  17, 1423 8 7.5  families:  1346, 1350  7 5  13 15  families:  1377, 1408, 1345  127  0 0  0  3. Results Table 18. (con’t)  predicted parental genotypes at the polymorphic locus  offspring phenotypes  Obs Exp  U22)X177 (172) 177  172  172,177  172,186  172,177,186  0 0  13 all  0 0  0 0  3 2  4 2  1 2  3 4  5 4  8 6  1  family 102 U7)X(172) 177 186  (3) 2 x  =  5  0.10< p  Ui)X186 177 186 (1) 2 x  =  0.5  mXU2z) 177 (1) 2 x  186 =  1.33  U7)X177 177 186 (2) 2 x  =  2.11  p  <  Obs Exp  0 2  0.30  family 1341  Obs Exp  0 0  0 0  family 1421  0.50 Obs Exp  0.10< p <0.30 Obs Exp 0.30< p <0.50  0  4  0  6  0 0  0  4 4.5  4 2.25  families:  21, 23  family 884 0  Parental genotypes were predicted assuming a single polymorphism model. Expected values for offspring phenotypes were calculated assuming simple Mendelian segregation involving the predicted parental genotypes.  128  2.25  3. Results 3.3. Z2 System A1u52B System A1u52B consisted of 3 fragments of 308, 310 and 312 bp. System A1u52B exhibited codominant Mendelian segregation for all CEPH families typed. A heterozygosity of 0.55 and a PlC of 0.46 was obtained from allele frequencies observed for 69 CEPH parents (Table 19). Segregation of the 308 and 310 bp alleles at the AIu52B locus in family 1349 is shown in Figure 26.  Table 19. A1u52B Allele frequencies  Allele  size(bp)  Al A2 A3  308 310 312  frequency 0.36 0.56 0.08  129  3. Results Figure 26. A1u52A and AIu52B polymorphisms  310 bp 308 bp  -  -  Segregation of the 172 and 177 bp alleles at the A1u52A locus and 308 and 310 bp alleles at the Alu52B locus in family 1349. Allele sizes refer to predominant bands and are indicated on the left side of the figure. Minor bands present are shadow bands produced as an artifact of PCR.  130  3. Results 3.3.8 D5S266 The polymorphism detected at locus D5S266 is a microsatellite repeat located at the 3’ end of anAlu element with a heterozygosity of 0.26 and a PlC of 0.30 calculated from allele frequencies obtained by typing 53 CEPH parents (Table 20). The (GT)n tract within D5S266 was subcloned as a 1.5kb Hindifi fragment into Hindifi digested Bluescriptil to form the plasmid p266H1.5. A portion of the DNA surrounding the (GT) tract was then subcloned as a 0.8kb XhoI-HindIII fragment to produce the plasmid p266XHO.8 by digestion of p266H1.5 with XhoI and religating. A different portion of the DNA surrounding the (GT)n tract was subcloned as a 0.4 kb HpaII fragment into Clal digested Bluescriptil to produce the plasniid p266H0.4. The sequence surrounding the D5S266 (GT)n tract (Figure 27) was determined from the plasmids p266H0.4 and p266XH0.8 using T3 and T7 primers. As shown in Figure 27, the (GT) tract was at the 3’ end of anAlu element. The GT strand primer has homology with the Alit consensus sequence. The CA strand primer did not have significant homology with any sequences in the EMBL database. An additional Alu element was present in reverse orientation immediately adjacent to the region to be amplified (Figure 27). The area surrounding the D5S266 (GT)n polymorphism was therefore extremely rich inAlu elements. Primer sequences were as follows: D5S266-GT: 5’CCTAGGTGATAGAGCAAGACC 3’ D5S266-CA: 5’CAAATATAATACCACCI’CCAG 3’ The sequence of the cloned product was 10 3 2 A ( 6 (TA) CATACA(T TG) 1 A) which corresponds to an amplification product of 123bp. D5S266 reference genotypes  131  3. Results were as follows: 133101  =  A4,A4; 133102  =  A4,A4. Segregation of the 123 and 129  bp alleles in family 1375 is shown in Figure 28. The D5S266 polymorphism was somewhat difficult to amplify, and difficult to score due to the presence of multiple shadow bands. Results obtained from the CEPH panel were therefore rather limited for this system.  Table 20. D5S266 Allele frequencies  Allele  size(bp)  frequency  Al A2 A3 A4  129 127 125 123  0.085 0.057 0.009 0.82  132  3. Results  Figure 27. D5S266 (GT)n tract and flanking sequence compared with Alu consensus sequence  Sequence surrounding (GT)n tract and alignment with Alu consensus sequence. Primer sequences are underlined. The Alu consensus sequence was obtained from the EMBL database version 27.  133  3. Results alucon 5’ GGCTGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAG alucon 5’ GTGGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGG alucon 5’ TGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGC alucon 5’ GCGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCTGAGGCAGGAGAATCG alucon 5’ CTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCAC  I I  D5S266  III  5’TCGATGCCAC 3 ‘AGCTACGGTG  alucon 5’ TGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCA D5S266 5 ‘TGCACTCCAGCCTAGGTGATAGAGCAAGACCCTGTCTCAAAAAAAAAAAAA 3’ ACGTGAGGTCGGATCCACTATCTCGTTCTGGGACAGAGTTTTTTTTTTTTT  D5S 266 5’ AAAAAAAATATATATATATATGTGTGTGTGTGTGTGTGTGCATACATATAT 3’ TTTTTTTTATATATATATATACACACACACACACACACACGTATGTATATA D5S266 5 ‘ACACCGAAGCTGGAGGTGGTAGTTATATTTGTCAGCAAATTTTGCAACAAA 3’ TGTGGCTTCGACCTCCACCATCAATATAAACAGTCGTTTAAAACGTTGTTT D5S266 5 ‘TTTAGGTTTTGCTTCAGAGTTGTCATTCCTTTTATGTTTCTTTTTTTTTTT 3’ AAATCCAAAACGAAGTCTCAACAGTAAGGAAAATACAAAGAAAAAAAAAAA D5S266 5 ‘TGAGACAGAGTCTCAATCTGTCACCCAGGCTGGAGTGCAGTGGCACC  3’ ACTCTGTCTCAGAGTTAGACAGTGGGTCCGACCTCACGTCACCGTGG  111111 1111111  111111  alucon 3 ‘ACTCTGCCTCAGAGCGAGACAGCGGGTCCGACCTCACGTCACGTCACCGCG D5S266 5’GTTGTCAGCTCACTGCAACCT3’ 3’ CAACAGTCGAGTGACGTTGGA5’ alucon 3’ CTAGAGCCGAGTGACGTTGGAGGTGGAGGGCCCAAGTTCGCTAAGAGGACG  alucon 3 ‘GAGTCGGAGTCGGAGGGCTCATCGACCCTAATGTCCGCGCGCGGTGGTGCG alucon 3 ‘GGCCGATTAAAAACATAAAAATCATCTCTGCCCCAAAGTGGTACAACCGGT alucon 3’ CCGACCAGAACTTGAGGACTGGAGTCCACTAGGTGGGTGGAGCCGGAGGGT alucon 3’ TTCACGACCCTAATGTCCGCACTCGGTGGTGCGGGTCGG’ 5  134  3. Results  Figure 28. D5S266 (GT)n polymorphism  bEEbbbbE  Segregation of the 123 and 129 bp alleles at the D5S266 locus in family 1375. Allele sizes refer to predominant bands and are indicated on the left side of the figure. Minor bands present are shadow bands produced as an artifact of PCR.  135  3. Results 3.3.9 D5S268 The polymorphism detected at locus D5S268 (Bernard et al., 1991b) is a microsatellite repeat with a heterozygosity of 0.72 and a PlC of 0.68 calculated from allele frequencies obtained by typing 80 CEPH parents (Table 21). The (GT)n tract within D5S268 was subcloned as a 0.4kb Alul fragment into EcoRV digested Bluescriptil to produce the plasmid p268A0.4. The sequence surrounding the D5S268 (GT)n tract (Figure 29) was determined from the plasmid p268A0.4 using T3 and V primers. Primer sequences were as follows: D5S268-GT: 5’AAGGTGAGGCAAAATGAGTGTA 3’ D5S268-CA: 5’CAATCAGGCCA’UITITAACTrCA 3’ Due to a typing error when the primer D5S268-GT was ordered, the primer is deleted for one base relative to the correct sequence. The absence of this base did not appear to affect the amplification of this polymorphic (GT) tract. The sequence of the cloned product repeat unit was (GT) 16 which corresponds to an amplification product of ll4bp. D5S268 reference genotypes were as follows: 133101  =  A4,A6; 133102  =  A4,A6. Segregation of the 114, 118, 120 and 122 bp  alleles in family 1332 is shown in Figure 30.  136  3. Results Table 21. D5S268 Allele frequencies  Allele  size(bp)  frequency  Al A2 A3 A4 A5 A6 A7  124 122 120 118 116 114 112  0.019 0.038 0.038 0.33 0.31 0.25 0.012  Figure 29. D5S268 (GT) tract and flanking sequence 5’ TGCTCCTCAAAATGGCCCAATTATCAGGGGGACCAATCAGGCCATTTTTAACTTCATT 3’ ACGAGGAGTTTTACCGGGTTAATAGTCCCCCTGGTTAGTCCGGTAAAAATTGAAGTAA  TTGATTAACATTTTAGTAATGTAACTAAATGCACACACACACACACACACACACACACAC AACTAATTGTAAAATCATTACATTGATTTACGTGTGTGTGTGTGTGTGTGTGTGTGTGTG ACATACATACACTCATTTTCGCCTCACCTTTTGCAGGAAAAAAACAGAATT3’ TGTATGTATGTGAGTAAAAGCGGAGTGGAAAACGTCCTTTTTTTGTCTTAA5’  Sequence surrounding (GT) 11 tract. Primer sequences are underlined.  137  3. Results Figure 30. D5S268 (GT) polymorphism  6±6± 666±±  Segregation of the 114, 118, 120 and 122 bp alleles at the D5S268 locus in family 1332. Allele sizes refer to predominant bands and are indicated on the left side of the figure. Minor bands present are shadow bands produced as an artifact of PCR.  138  3. Results 3.4 LINKAGE ANALYSIS A goal of this thesis project was to place clones isolated from the 5q11.2q13.3 region onto the genetic linkage map of human chromosome 5. To achieve this objective, linkage analyses using likelihood tests (Morton, 1955) were performed with each new polymorphic system in conjunction with previously positioned markers. New polymorphic markers were also positioned relative to each other. Linkage calculations were performed using the computer program package LINKAGE version 4.7 (Lathrop et a!., 1984, 1985; Lathrop and Lalouel, 1988).  3.4.1 Twopoint linkage analysis New polymorphic markers were positioned relative to previously reported chromosome 5 polymorphic markers (index markers). The index markers used for analysis, D5S56, D5S59, D5S60, HPRTP2, D5S21, D5S76, D5S6, D5S39, D5S78, D5S71, D5S37 and D5S50, have been positioned on a chromosome 5 linkage map by Weiffenbach et al., 1991. Twopoint linkage analyses were performed using the MLINK option of the LINKAGE program package version 4.7 (Lathrop et al., 1984, 1985; Lathrop and Lalouel, 1988). New markers were initially subjected to a twopoint analysis with the index markers D5S76, D5S6, D5S39, D5S78 and D5S71. These index markers were chosen since they span the region of the 5q11.2-q13.3 deletion (Gilliam et al., 1989) and form a contiguous linkage group (Weiffenbach et al., 1991). New markers were also tested in a twopoint analysis with DHFR, which is located within the approximately 29cM region between D5S78 and D5S71, to ensure that linkage in this interval was detected. Further twopoints were then carried out until each new  139  3. Results marker was linked to at least two flanking index markers on each side of the new marker or significant linkage (lod >2) could no longer be obtained. Linkage analysis placed the new markers into three groups, those which were linked to markers within the 5q11.2-13.3 region (D5S201, D5S253, D5S260, D5S262 and D5S266), those which were unexpectedly linked to markers on 5p (D5S257 and D5S268), and those which were unlinked to any chromosome 5 marker (AIu52A and AIu52B). For each group of chromosome 5 markers, new markers within each group were tested versus each other in a twopoint analysis to determine their relative positioning. A1u52A and A1u52B were tested versus highly informative markers from other chromosomes. Linkage was detected between A1u52A and markers on chromosome 2. A1u52B was found to be linked to markers on chromosome 17. Twopoint analyses for A1u52A and A1u52B were carried out using chromosome 2 and 17 index markers until significant linkage (lod >2) could no longer be detected. Chromosome 2 index markers used (D2S51, D2S44, D2S54 and D2S43) have been placed on a linkage map by O’Connell et al. (1989). A linkage map of chromosome 17, including index markers D17S34, D17S30, D17S31, D17S1 and MYH2, was published by Nakamura et al. (1988). For all loci pairs with a lod score of 2 or greater, the effective number of recombinants and effective number of informative meioses was calculated by the method of Edwards (1976). The derivation of Edward’s formula is presented in Appendix 1. Table 22 contains lod score data for various recombination fractions for all twopoints with a lod of 2 or greater. Maximal likelihood values for recombination fractions, maximal lod scores, effective numbers of recombinants and  140  3. Results effective numbers of informative meioses are also shown in Table 22. An estimate for the number of informative meioses scored for a polymorphic system can be obtained by a twopoint analysis involving the marker versus itself at a recombination fraction of 0. The method of Edwards (1976) can then be used to convert the lod score obtained to the effective number of informative meioses. This calculation was performed for all new polymorphic systems (Table 23). The effective number of informative meioses varied between a low of 24 for the relatively uninformative D5S262 locus to a high of 447 for the highly informative D5S260 locus.  141  D5S257-D5S76 D5S257-D5S6 D5S268-D5S257  D5S257-D5S56 D5S257-D5S59 D5S257-D5S60 D5S257-HPRTP2  D5S268-D5S76 D5S268-D5S6  13.74 4.42 21.82  NEW 5P MARKERS D5S268-D5S56 D5S268-D5S59 D5S268-D5S60 D5S268-HPRTP2  6.03 -40.88 -24.01 5.53 7.36 11.49 2.86 -11.92 -13.16 25.17  0.05  Marker loci  23.65  6.70 8.99 11.59 5.03 -3.27 -5.24  18.76 9.05 23.67 7.64 -19.60 -6.54  0.10  19.13 10.46 21.05  7.39 -3.14 5.68 6.43 8.69 9.72 5.60 2.91 0.67 18.98 7.83 -9.11 1.56 6.81 9.17 10.85 5.65 0.80 -1.41 21.49  0.20  19.82 10.43 22.95  0.15  e  5.76 7.80 8.32 5.16 3.87 1.74 16.19  17.35 9.61 18.39 6.60 0.28 7.48  0.25  13.16  7.69 4.86 6.61 6.74 4.43 4.05 2.12  14.79 8.25 15.14 5.56 2.02  0.30  Table 22. Twopoint meiotic linkage analysis  0.13 0.13 0.08 0.17 0.28 0.31 0.03  0.15 0.17 0.10 0.14 0.35 0.28  2.13 25.35  19.82 10.56 23.67 7.85 2.55 7.76 6.84 9.20 11.70 5.69 4.05  9 45 50 63 128 178 51 69 65 55 93 66 105  3  7 9 5 9 26 20  25 17 15  R  169 102 148  N  NEW 5Q MARKERS D5S205-D5S76 D5S205-D5S6 D5S205-D5S39 D5S205-D5S78 D5S205-DHFR D5S205-D5S71 D5S205-D5S37 D5S205-D5S50 D5S253-D5S76 D5S253.-D5S6 D5S253-D5S39 D55253-D5S78 D5S253-DHFR D5S253-D5S71 D5S253-D5S37 D5S253-D5S50  Marker loci  -8.10 13.99 8.12 3.97 6.34 2.71 -3.21 -1.07 -23.05 -2.99 1.47 8.64 11.55 -1.69 7.23 16.22  0.05  10.90 1.90 12.29 22.03  -6.24 7.89 7.84 11.45  -0.15 16.02 10.95 5.51 5.74 4.26 1.70 4.20  0.10  3.39 13.57 23.01  9.87 12.02 9.94  3.31 15.80 11.34 5.87 5.09 4.65 3.65 6.11 1.49 12.06  0.15  8  9.32 10.58 7.59 3.98 11.91 19.59 10.09 11.60 8.82 3.96 13.22 21.91  4.53 4.35 6.59 5.38 13.34  5.31 12.77 9.36 5.24 3.71 4.08 4.31 6.23 7.06 12.91  0.25  4.87 14.59 10.67 5.72 4.42  0.20  Table 22. (con’t)  3.79 5.32 7.27 11.35 7.97 9.12 6.24 3.60 9.96 16.43  5.00 10.54 7.62 4.52 2.96 3.40  0.30  0 0.15 0.22 0.20 0.28 0.21 0.18 0.15 0.04 0.21 0.16 0.14  0.25 0.12 0.14 0.15  6.59 7.34 13.37 10.15 12.02 11.61 4.00 13.60 23.03  5.32 16.10 11.37 5.88 6.91 4.64 4.41  105 102 51 51 124 184  79 169 172  61  94 114 91 50 23 40  N  0 6 13 16 47 36 19 15 2 11 20 26  24 14 13 8  R  0.05  0.10  NEW 5Q MARKERS -CONTINUED 8.28 5.08 D5S260-HPRTP2 13.01 15.50 D5S260-D5S21 7.61 17.52 D5S260-D5S76 34.20 36.50 D5S260-D5S6 4.94 12.34 D5S260-D5S39 6.08 1.82 D5S260-D5S78 2.87 2.89 D5S262-D5S39 4.12 3.84 D5S266-D5S76 28.50 28.07 D5S205-D55253 -0.44 -9.86 D55205-D5S260 3.54 3.69 D5S205-D5S262 -31.91 -10.59 D5S253-D5S260 2.34 2.14 D5S260-D5S262 0.64 -2.06 D5S260-D55266  Marker loci  2.28  -0.63 2.27 1.79  3.60 23.37  20.82 32.09 14.55 7.77 2.45  8.94 14.29  0.20  5.60 2.87 4.54 2.07  3.97 26.09 3.71 3.25  9.13 15.44 20.65 35.15 14.56 7.52 2.69  0.15  0  5.78 1.96 7.64 1.45 2.19 6.14 2.44 6.99 1.79 2.36  6.96 10.21 16.42 22.96 11.12 6.36 1.82 2.44 16.54  0.30  8.17 12.49 19.23 27.94 13.25 7.30 2.16 3.08 20.16  0.25  Table 22. (con’t)  0.16 0.12 0.18 0.10 0.17 0.19 0.07 0.08 0.06 0.25 0.05 0.30 0.11 0.24  15.66 21.01 36.51 14.76 7.79 2.91 4.08 28.65 6.15 3.69 7.64 2.34 2.37  9.16  9 38  16  108 17 214  39 23 24 17 1 2 218 228 143 87 15 23 142  9 27 1 64 2  13 13  R  83 111  N  UI  0.05  4.78 17.68 8.28 6.64  0.10  15.18 27.60 8.34 13.95 4.09 5.03  4.49 16.13 7.38 6.30  0.15  25.58 7.75 12.67 3.82 4.73  14.42  5.68  4.06 14.30 6.41  0.20  12.89 22.60 6.84 11.04 3.40 4.25  3.53 12.27 5.39 4.89  0.25  =  maximum likelihood value for recombination fraction 2 = maximum lod score R = effective number of recombinants (Edwards, 1976) N = effective number of informative meioses (Edwards, 1976)  CHROMOSOME 17 MARKERS 11.31 14.69 AIu52B-D17S34 25.22 28.09 A1u52B-D17S30 8.39 7.25 AIu52B-D17S28 14.16 14.67 A1u52B-D17S31 4.14 3.68 AIu52B-D17S1 5.02 4.34 AIu52B-MYH2  CHROMOSOME 2 MARKERS 4.79 AIu52A-D2S51 18.75 A1u52A-D2S44 9.01 A1u52A-D2S54 6.32 A1u52A-D2S43  Marker loci  e  Table 22. (con’t)  10.84 18.88 5.70 9.17 2.86 3.61  2.93 10.04 4.33 4.01  0.30  0.14 0.11 0.12 0.09 0.10 0.13  0.07 0.03 0.02 0.09  e  122 187 60 87 26 38 5.07  25 78 36 39  N  15.21 28.17 8.46 14.71 4.14  4.84 18.86 9.22 6.65  z  7 8 3 5  17 21  2 2 1 4  R  3. Results Table 23. Effective number of informative meioses (N) for new polymorphic markers  Locus  Marker  lod  N  D5S205  5PCR1  75.14  250  D5S253 D5S257 D5S260 D5S262  A1u24 A1u25  131.85 57.97 134.63 7.22 44.07 69.54 17.11 136.23  438 193  D5S266 D5S268  A1u32 A1u38 A1u52A A1u52B A1u60 A1u62  447 24 146 231 57 453  Lod = lod score obtained at e = 0 for each marker versus itself N = effective number of informative meioses for each system calculated using the method of Edwards, 1976.  146  3. Results 3.4.2 Multipoint linkage analysis  3.4.2 1 ChromosomeS markers A general position for each of the new chromosome 5 markers was obtained from twopoint linkage data. D5S266 and D5S262 could not be positioned on the chromosome 5 index map since they were each linked to only one index marker. Multipoint linkage analyses to position D5S253, D5S205, D5S260, D5S257 and D55S268 on the index map were performed using the CILINK option of the LINKAGE program package version 4.7 (Lathrop et al., 1984, 1985; Lathrop and Lalouel, 1988). Index markers were included in the analysis if they gave a twopoint lod score of 3 or greater with the new marker in question. Each new marker was varied across a region encompassing at least two index markers on either side of the new marker and odds obtained for each order (Table 24). Recombination fractions were obtained for the most likely marker order (Table 24). D5S268 could be unambiguously placed between index markers D5S60 and HPRTP2, since the odds against alternate locations were all greater than io6. D5S257 could be placed between D5S59 and HPRTP2, but could not be positioned relative to D5S60 on the index map. However, when D5S268 was added to the index map, D5S257 could be placed between D5S60 and D5S268 with odds of greater than  io against alternate positions.  D5S253 could be positioned between D5S78 and D5S71, with odds of greater than io against all other positions. D5S205 could not be positioned on the index map. However, when D5S253 was added to the index map, D5S205 could be positioned between D5S78 and D5S253, with odds against alternate positions of  147  3. Results greater than 102. D5S260 could be unambiguously placed on the chromosome 5 index map between D5S21 and D5S76, with odds of greater than i0 6 against all other positions. D5S266 was significantly linked to D5S260 and index marker D5S76. D5S266 could be positioned distal of D5S260 by a multipoint analysis, but could not be positioned relative to D5S76. D5S262 was linked to D5S205, D5S39 and D5S260. The order D5S260D5S39-D5S205 could be determined from the multipoint data. The position of D5S262 was therefore varied across the D5S260-D5S39-D5S205 region (Table 24). The position of D5S262 could not be determined by multipoint analysis. A summary of published index marker map distances (Weiffenbach et a!., 1990) together with a new linkage map incorporating markers D5S257, D5S268, D5S260, D5S205 and D5S253 is shown in Figure 31. Recombination distances shown in Figure 31 were calculated using Kosambi’s mapping function (Kosambi, 1944).  3.4.2.2 A!uS2A and A1u52B The position of A1u52A with respect to chromosome 2 index markers and Alu52B with respect to chromosome 17 index markers was determined using CILINK (Table 24). AIu52A could be positioned between markers D2S44 and D2S43, but could not be positioned relative to marker D5S254. A1u52B could be positioned between index markers D17S28 and D17S1, but could not be positioned relative to D17S31.  148  D5S59-D5S60-D5S257-D5S268-HPRTP2 D5S257-D5S59-D5S60-D5S268-HPRTP2 D5S59-D5S257-D5S60-D5S268-HPRTP2 D5S59-D5S60-D5S268-D5S257-HPRTP2 D5S59-D5S60-D5S268-HPRTP2-D5S257  D5S59-D5S60-HPRTP2-D5S76-D5S257  D5S59-D5S60-D5S257-HPRTP2-D5S76 D5S257-D5S59-D5S60-HPRTP2-D5S76 D5S59-D5S257-D5S60-HPRTP2-D5S76 D5S59-D5S60-HPRTP2-D5S257-D5S76  0.08  0.08  0.07  0.07  0.09  0.07  D5S59-D5S60-D5S268-HPRTP2-D5S6 D5S268-D5S59-D5S60-HPRTP2-D5S6 D5S59-D5S268-D5S60-HPRTP2-D5S6  D5S59-D5S60-HPRTP2-D5S268-D5S6 D5S59-D5S60-HPRTP2-D5S6-D5S268  2 e  01  Marker order  0.04  0.21  0.16  Table 24. Multipoint linkage analysis  0.14  1 12 1.7X10 8 1.0X10 1.9X io 11 2. 1X10  1.8X10 9 13 7.6X10  1 3 8.9X10 11 0.16  odds  1 6 6.4X10 9 8. 1X10 1 1.3X10’ 26 1.6X10  05  0.16  04  3 e 0.18  0.09  0.06  e2 0.12  0.10  0.10  el 0.11  0.09  0.10  Marker order  D5S39-D5S78-D5S253-D5S71-D5S37 D5S253-D5S39-D5S78-D5S71-D5S37 D5S39-D5S253-D5S78-D5S7 1-D5S37 D5S39-D5S78-D5S71-D5S253-D5S37 D5S39-D5S78-D5S71-D5S37-D5S253  D5S6-D5S39-D5S205-D5S78-D5S71 D5S205-D5S6-D5S39-D5S78-D5S71 D5S6-D5S205-D5S39-D5S78-D5S71 D5S6-D5S39-D5S78-D5S205-D5S71 D5S6-D5S39-D5S78-D5S71-D5S205  D5S39-D5S78-D5S205-D5S253-D5S71 D5S205-D5S39-D5S78-D5S253-D5S71 D5S39-D5S205-D5S78-D5S253-D5S7 1 D5S39-D5S78-D5S253-D5S205-D5S71 D5S39-D5S78-D5S253-D5S71-D5S205  Table 24. (con’t)  0.17  1 11 2.2X10 2 3.0X10 3 1.2X10 13 2. 1X10  36 9 2.3X10  1 4.8 4 2.8X10 0.21  odds 1 ° 1 1.4X10 9 1.6X10 18 1.0X10 11 2.5X10  e  0.02  e4  I’  IS  Ui  0.09  0.12  HPRTP2-D5S21-D5S260-D5S76-D5S6-D5S39 D5S260-HPRTP2-D5S21-D5S76-D5S6-D5S39 HPRTP2-D5S260-D5S21-D5S76-D5S6-D5S39 HPRTP2-D5S21-D5S76-D5S260-D5S6-D5S39 HPRTP2-D5S2 l-D5S76-D5S6-D5S260-D5S39  0.11  0.12  1 4 1.3X10 2.6  1 1.03 1.18 10 0.14 0.17  D5S260-D5S39-D5S205-D5S262 D5S260-D5S39-D5S262-D5S205 D5S260-D5S262-D5S39-D5S205 D5S262-D5S260-D5S39-D5S205  1 12 4.8X10 9 1.6X10 6 4.3X10 9 2.7X10 9 9.3X10 0.09  0.09  odds  85  0.18  0.05  e4  03  D5S260-D5S76-D5S266 D5S266-D5S260-D5S76 D5S260-D5S266-D5S76  HPRTP2-D5S21-D5S76-D5S6-D5S39-D5S260  e2  01  Marker order  Table 24. (con’t)  odds  odds against an order relative to the most likely order  0.06  0.08  6 2X10  537  1 4 6X10 1.1  0.11  0.10  D 17S28-AIu52B-D 17S3 1-D 17S 1-MYH2 A1u52B-D 17S28-D 17S3 1-D 17S 1-MYH2 D 17S28-D 17S3 1-A1u52B-D 17S 1-MYH2 D 17S28-D 17S3 1-D 17S 1-AIu52B-MYH2 D 17S28-D 17S3 1-D 17S 1-MYH2-AIu52B  D2S5 1-D2S44-D2S54-D2S43-A1u52A  1 6 6X10 229 21 9 6X10  0.13  0.02  0.03  0.10  odds  D2S51-D2S44-AIu52A-D2S54-D2S43 AIu52A-D2S5 1-D2S44-D2S54-D2S43 D2S5 1-AIu52A-D2S44-D2S54-D2S43 D2S5 1-D2S44-D2S54-AIu52A-D2S43  05  04  03  02  01  Marker order  Table 24. (con’t)  3. Results Figure 31. Chromosome 5 multipoint linkage analysis Summary  published index map (Weiffenbach, 1991)  -  New map  5pter  5pter  D5S59  D5S59 7  8  D5S60  D5S60 7 D5S257 20  4 D5S268 14  HPRTP2  HPRTP2 10  12  D5S21  D5S21 9 16  D5S260 12  D5S76  D5S76 11  11  D5S6  D5S6 8  9  D5S39  D5S39 12  10  D5S78  D5S78 10 D5S205 29  6 D5S253 18  D5S71  D5S71  5qter  5qter  Map distances in cM calculated using the Kosambi mapping function are indicated to the right of each map. New markers placed on the linkage map are underlined.  153  3. Results 3.5 HHW1O64 CHARACTERIZATION -  Clones isolated byAlu PCR differential hybridization were assigned to the 5q11.2-q13.3 region based on their absence of hybridization with the somatic cell hybrid HHW1O64. Genetic linkage results obtained with new polymorphic markers led to an examination of the DNA content of the HHW1O64 hybrid. The polymorphic systems isolated as a part of this thesis necessitated an inspection of regions within both the p arm and the q arm of the human chromosome 5 present in the HHW1O64 hybrid.  3.5.1 HHJV1O64 Sp deletion Two of the new polymorphic markers analyzed by meiotic linkage analysis mapped to chromosome 5p (D5S257 and D5S268). Both of these markers had been isolated on the basis of their absence of hybridization to the somatic cell hybrid HHW1O64, which was believed to contain a chromosome 5 deleted only for 5q11.2q13.3. The localization of D5S257 and D5S268 to Sp was therefore completely unexpected. This localization was confirmed and the remainder of the isolates localized by hybridization ofAlu, Alu-T3 or Alu-T7 PCR products to Southern blots of Alu PCR products from the somatic cell hybrids HHW213, GM1O114 and HHW1O64. GM1O114 contains a human chromosome 5 and HHW213 contains a human chromosome 5 consisting almost entirely of 5p material (Overhauser et a!., 1986a). Markers were classified as being derived from 5q11.2-q13.3 if they hybridized GM1O114 and failed to hybridize to both HHW1O64 and HHW213. Markers were classified as located on 5p if they hybridized to GM1O114 and HHW213 and failed to hybridize to HHW1O64. Four of the twenty markers isolated  154  3. Results byAlu PCR differential hybridization were located on 5p based on this hybridization scheme (Table 25). The remaining 16 were located within the expected 5q11.2-q13.3 region. These results demonstrate that the somatic cell hybrid HHW1O64 contains a deletion on 5p in addition to the expected 5q11.2-q13.3 deletion. D5S257 was physically localized using a somatic cell deletion mapping panel to 5p15.l, within interval I as defined by Overhauser et a!., 1987 (John McPherson, pers. comm.). This physical localization agrees with the linkage mapping of D5S257 between HPRTP2 and D5S60, since HPRTP2 has been physically positioned to 5pl4. (Overhauser et al., 1986b) and D5S60 has been cytogenetically mapped to 5pl5.l (Weiffenbach et a!., 1991). The Sp deletion in the HHW1O64 hybrid therefore involves at least a portion of the 5pl5.l band.  155  3. Results Table 25. Marker localization to 5p or 5q11.2-q13.3  Locus  D5S205 D5S251 D5S252 D5S254 D5S253 D5S255 D5S256 D5S257 D5S258 D5S259 D5S260 D5S261 D5S262 D5S263 D5S264 D5S265 D5S266 D5S267 D5S268 D5S269  Probe name  Location  5PCR1 5PCR2 5PCR3 5PCR5 5PCR11 A1u19 A1u22 A1u25 A1u26 A1u29 A1u32 A1u36 Alu38 A1u41 A1u47 A1u52 A1u60 A1u61 A1u62 A1u66  5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5p15.1 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3 5q11.2-13.3  156  3. Results 3.5.2 HHW1O64 Sq deletion Linkage maps of human chromosome 5 report the following marker order: D5S21-D5S76-D5S6-D5S39 (Weiffenbach et a!., 1991; Westbrook et a!., 1991). The 5q11.2-q13.3 deletion within HHW1O64 encompasses markers D5S6 and D5S39 but does not include markers D5S21 and D5S76 (Gilliam et al., 1989). The position of the proximal deletion breakpoint can therefore be assigned to between D5S76 and D5S6. The new marker D5S260 is not present in the HHW1O64 hybrid but maps to the D5S21-D5S76 region based on linkage studies. The linkage results for D5S260 are therefore inconsistent with the somatic cell hybrid data. Linkage and somatic cell hybrid data for markers D5S21, D5S76, D5S6, D5S39, and D5S260 were therefore investigated. The somatic cell hybrid data obtained for hybrid HHW1O64 with markers D5S76 and D5S260 were re-examined. The first possibility addressed was that D5S260 had been misappropriately assigned as absent from the HHW1O64. This possibility was excluded, since absence of D5S260 from the HHW1O64 hybrid was conclusively demonstrated by both hybridization and PCR testing (sections 3.1.3, 3.1.5 and 3.5.1). The possibility that D5S76 had been erroneously positioned outside the deleted region was therefore investigated. D5S76 hybridized to a 10 kb TaqI fragment in HHW1O64, indicating that D5S76 maps outside the deleted region. The inconsistency observed between the D5S260 linkage data and somatic cell hybrid positioning was therefore not due to errors in marker typing in HHW1O64. Another potential explanation for the mapping inconsistency observed for D5S260 is that D5S260 was incorrectly positioned by linkage mapping. This seems unlikely, given the multipoint linkage analysis data placing D5S260 between D5S21  157  3. Results and D5S76, with odds against all other orders of greater than i0 6 (Table 24). However, the position obtained for D5S260 is dependent on the order of the index markers used for analysis. An incorrect position for D5S260 could be obtained if the order of index markers was incorrect. The odds for all possible orders of the index markers D5S21, D5S76, D5S6 and D5S39 were therefore calculated using a multipoint analysis. As seen in Table 26, the most likely order for markers was D5S21-D5S76-D5S6-D5S39, with odds against all other orders of greater than . These linkage results are compatible with the published order of index 2 7X10 markers. An investigation of the linkage and somatic cell hybrid data involving markers D5S21, D5S76, D5S6, D5S39, and D5S260 did not reveal any obvious errors. The inconsistency between the positioning of D5S260 by linkage analysis and the absence of D5S260 from the somatic cell hybrid HHW1O64 may therefore result from unanticipated alterations in the HHW1O64 hybrid. The mapping inconsistency could be explained by the presence of a complex 5q deletion involving  two non-contiguous regions in the somatic cell hybrid HHW1O64. A deletion of the region of DNA proximal to D5S76 in the HHW1O64 hybrid would explain the absence of D5S260, while the deletion noticed on karyotyping the hybrid would include the markers distal to D5S76. Such a complex deletion would likely be missed by cytogenetic analysis if the deleted region proximal to D5S76 was small. Such a complex rearrangement is the most likely explanation for the inconsistencies in the D5S260 mapping data.  158  3. Results Table 26. Multipoint linkage analysis involving index markers D5S21, D5S76, D5S6, and D5S39  Marker order  1 e  e2  D5S21-D5S76-D5S6-D5S39 D5S21-D5S76-D5S39-D5S6 D5S21-D5S6-D5S39-D5S76 D5S21-D5S6-D5S76-D5S39 D5S21-D5S39-D5S6-D5S76 all other orders  0.15  0.13  oddsa 0.09  1 2 7.0X10 3 5. 1X10 9. 1X10 5 2.0X10 > i0 9  a odds against an order relative to the most likely order  3.5.3 Cytogenetic analysis of HHW1064 An aliquot of the HHW1O64 hybrid cells used to prepare the hybrid DNA was cytologically examined. The HHW1O64 hybrid cells contained a single human chromosome 5 and the only cytologically detectable deletion was the expected constitutional deletion of 5q11.2-q13.3 (Figure 32). Approximately 1/3 of all metaphases examined exhibited a partial or complete breakage of the human chromosome 5 near the centromere on the long arm of the chromosome, in a region consistent with the Sq deletion breakpoints.  159  3. Results Figure 32. Metaphase chromosomes from somatic cell hybrid HHW1O64  cc LI  (I  Arrow indicates human chromosome 5 with constitutional deletion for 5q11.2-q13.3.  160  3. Results 3.6 CHARACTERIZATION OF CHROMOSOMAL REARRANGEMENT SEGREGATING IN TRISOMY FAMILY The somatic cell hybrid HHW1O64 carries a deleted chromosome 5 derived from a carrier female of the family in which schizophrenia and renal anomalies co segregate with trisomy for the 5q11.2-q13.3 region (Bassett et al., 1988; McGillivray  et al., 1990). The chromosome 5 present in HHW1O64 was demonstrated to contain a 5p deletion and likely contains a complex rearrangement on 5q. The derivative chromosome 5 present within the carrier female could contain rearrangements identical to that found in HHW1O64, or could contain the expected 5q11.2-q13.3 deletion as the sole rearrangement. An investigation of the segregation of the new polymorphic markers in the trisomic family was therefore carried out to determine the DNA content of the rearrangement segregating in this family.  .3.6.lSp The somatic cell hybrid HHW1O64 contains a deletion on the p arm of chromosome 5. The 5p deletion could have arisen during the generation of the HHW1O64 hybrid, or could be part of a complex constitutional rearrangement in the carrier individual from whom HHW1O64 was derived. To determine if there was a constitutional deletion of 5p in addition to the 5q11.2-q13.3 deletion in the carrier individual who was the source of the chromosome 5 in HHW1O64, D5S257 and D5S268 were analyzed in the carrier’s family. D5S257 was partially informative and  D5S268 was fully informative in this family (Figure 33). Segregation data indicate that the carrier individual does not have a constitutional deletion on Sp. The HHW1O64 hybrid therefore contains a secondary, non-cytologically visible deletion  161  3. Results of the p arm of chromosome 5 in addition to the expected constitutional 5q11.2q13.3 deletion.  3.6.1 Sq The HHW1O64 hybrid may contain a complex rearrangement involving the 5q11.2-q13.3 region, with deletions of the regions on both sides of the marker D5S76. The new marker D5S260 maps proximal of D5S76 and the new marker D5S253 maps distal to D5S76. The family segregating for the segmental trisomy was therefore typed for the polymorphic loci associated with D5S260, D5S76 and D5S253 (Figure 33). D5S76 and D5S260 were partially informative in this family and D5S253 was uninformative as all individuals were homozygous for the l2Obp allele. Segregation data indicate that the rearrangement in the trisomic family involves D5S260. D5S76 segregates in a normal Mendelian fashion, indicating that this locus is not involved in the rearrangement.  162  3. Results Figure 33. Family segregating for segmental trisomy typed for D5S268, D5S257, D5S76 and D5S260  D5S268  120,114  124,120  126,114  126,124  124,124  D53257  77,77  77,77  79,77  79,77  79,77  D5S260  154,150  154,152,150  150,150  152,150  152,150  D5S76  io,io  14,10  14,10  14,10  no data  •  46, XY, der(1) 46, XX,  El  nv Ins  nv ins(1;5)(qe23q13.3q11.2)  46, XV  Sizes of alleles at D5S268, D55257, D5S76 and D5S260 loci are indicated under symbols for individuals  163  4. DISCUSSION  4.1 CLONE ISOLATION A variety of techniques have been developed for the isolation of human DNA fragments from chromosomal subregions. Prior to the invention of PCR, recombinant libraries containing regions of interest were produced by manipulations of genomic DNA, metaphase chromosomes or somatic cell hybrids. Recombinant libraries produced by subtractive DNA cloning (Kunkel et a!., 1985; Nussbaum et a!., 1987) have the disadvantage that they typically involve large quantities of genomic DNA, while libraries produced by physical microdissection of metaphase chromosomes (Kaiser et a!., 1987) require complex manipulations. Libraries produced from somatic cell hybrids containing all or part of a single human chromosome (for example; Scambler et a!., 1987) must be screened in order to isolate human DNA sequences from the rodent background. Region-specific human fragments were therefore difficult to obtain prior to the use of PCR.  Alu mediated PCR has certain advantages over conventional DNA fragment isolation. Human-specific fragments can be directly isolated from somatic cell hybrids using Alu PCR. Conventional selection procedures for distinguishing human and rodent isolates are therefore eliminated. Alu PCR can be performed with small quantities of starting DNA. Regional localization ofAlu PCR derived clones can be accomplished by hybridization to the Alu PCR products from somatic cell hybrids. Since the complexity of DNA in anA!u PCR reaction is reduced relative to the somatic cell source, localization ofAlu PCR derived clones can be accomplished in a shorter length of time than for conventional Southern blot  164  4. Discussion analysis. Four techniques for the isolation ofAlu PCR fragments from a specific chromosomal region were reported prior to my report onAlu PCR differential hybridization. The first technique reported, Alu PCR on YACs or phage followed by regional localization to DNA from somatic cell hybrids (Nelson et a!., 1989), requires YACs or phage derived from the region of interest. Alu PCR followed by cloning of the Alu PCR material (Brooks-Wilson et a!., 1990), requires a somatic cell hybrid containing only the region of interest. The two other techniques reported (Ledbetter et a!., 1990; Patel et a!., 1990) require the detection of visible differences between PCR products from various hybrids. The Ledbetter et a! (1990) technique involves the isolation of differences between the Alu-L1 PCR products of two somatic cell hybrids which are visible on an ethidium bromide stained gel. The procedure reported by Patel et a! (1990) is rather complex and involves the following steps: (1) A!u PCR on a monochromosomal hybrid, (2) separation of the fragments generated by agarose gel electrophoresis, (3) division into subfractions by cutting out agarose plugs from various sizes of Alu PCR product, and (4) hybridization of the subfractions to the A!u PCR product of various hybrids in order to detect differences by hybridization. This procedure therefore also relies on the presence of visually detectable differences between the PCR products of various hybrids. Alu PCR differential hybridization requires the segregation of a chromosome containing a deletion of the region of interest into a somatic cell hybrid. Such deletion chromosomes are often readily available from patients with genetic diseases. The other components used for this procedure, a chromosome specific  165  4. Discussion phage library and a somatic cell hybrid containing an intact copy of the chromosome of interest, are readily available for all human chromosomes. Alu PCR differential hybridization therefore complements and extends the previously existing methods for isolation of region-specific Alu PCR fragments. The derivation of the Alu PCR differential hybridization technique depended in a large part on the materials which were available at the time the technique was developed. When research was initiated, two chromosome 5 hybrids were available, one (GM1O114) which contained an intact human chromosome 5 and the other (HHW1O64) which contained a human chromosome 5 with an interstitial deletion for 5q11.2-q13.3. Two chromosome 5 phage libraries, LAO5NSO1 and LAO5NLO3 were also available. The technique ofAlu mediated PCR (Nelson et a!., 1989, Brooks-Wilson et al., 1990) was reported soon after my thesis research started. A strategy for the isolation of clones within 5q11.2-q13.3 was therefore developed which made use of all of these components. This technique was called Alu PCR differential hybridization to highlight the use of Alu PCR products derived from different sources as hybridization probes. Alu PCR differential hybridization is applicable to two somatic cell hybrids which differ only in that one contains a deletion of the region of interest. When this technique was used in conjunction with chromosome 5 materials, isolate localization could be confirmed by hybridization to Alu PCR localization blots for 35/45 (78%) of clones isolated byAlu PCR differential hybridization (Table 1). Alu PCR differential hybridization is therefore highly effective for the isolation of region specific clones. Alu PCR differential hybridization should be generally applicable to any  166  4. Discussion hybrid deletion hybrid pair. However, the proportion of isolates localized to the -  deleted region will depend on the size of the region which is deleted. Alu elements appear to be deficient in Giemsa (G) positive bands and enriched in G negative bands (Korenberg and Rykowski, 1988). Therefore, the number ofAlu PCR isolates localized to the deleted region will also depend on the chromosomal composition of the hybrids involved. The cytologically visible deletion within HHW1O64 encompasses approximately 10% of chromosome 5, and contains both G positive and G negative bands (Gilliam et a!., 1989). A total of 479 chromosome 5 recombinant clones were screened for differential hybridization, and 35 were absent from the HHW1O64 hybrid (Table 1). Twenty-five of these 35 isolates were from 5q and 10 were from 5p. The cytologically visible deletion therefore accounted for 25/479 (5%) of isolates and 10/479 (2%) were from a non-cytologically detectable p arm deletion. The fraction of isolates from Sq was close to that expected based on the cytologically determined size of the deletion. The large fraction of p arm isolates is likely due to an abundance ofAlu elements within this region. Flow sorted chromosome 5 libraries were used rather than genomic phage libraries to minimize the number of phage screened, and to reduce the number of falsely positive phage in the initial screen. False positives could arise due to incomplete preannealing of repetitive elements present in the Alu PCR product of the chromosome 5 hybrid. These false positives should be eliminated by the secondary screen, since the Alu PCR product from the deletion 5 somatic cell hybrid would be expected to contain the same unblocked repetitive elements. Conversely, some isolates located within the deletion region could be missed if they contained repetitive elements which were not preannealed.  167  4. Discussion The large insert phage library LAO5NLO3 was of greater value for Alu PCR differential hybridization than the LAO5NSO1 library for several reasons. A larger proportion of phage from the LAO5NLO3 library were positive when hybridized with the Alu PCR product of the chromosome 5 hybrid (2.2% compared with 0.4% for LAO5NSO1), thereby decreasing the number of phage in the initial screen. The proportion of clones containing the 3’ ends of both flanking Alu elements was also higher in the LAO5NLO3 library than the LAO5NSO1 library. The inter-Alu region could be amplified from 15/15 clones obtained from the LAO5NLO3 library but from only 1/5 clones from the LAO5NSO1 library. The use of a large insert phage library therefore reduces the effort required for clone isolation and localization. Isolate location was confirmed by hybridization to cell hybrid genomic DNA for 8/8 clones tested (section 3.1.4). Absence of amplification from the somatic cell hybrid HHW1O64 was confirmed by PCR for 4/4 systems tested (section 3.1.5). These confirmations were done to determine the proportion of clones which were inappropriately assigned to the deletion region using Alu PCR localization blots. Such isolates could arise due to polymorphic differences between chromosomes in the placement or structure ofAlu elements. Differences in the degree of Alu PCR amplification between the two hybrids could also lead to falsely assigned clones. No isolates were found which had been falsely assigned by the Alu PCR localization blots. Therefore, differences between GM1O114 and HHW1O64 inAlu placement orAlu PCR amplification do not appear to be frequent. The AlS primer used for Alu PCR was from the extreme 3’ end of the Alu consensus sequence, and therefore generates a minimum ofAlu-homologous material (Brooks-Wilson et al., 1990). The use of the A1S primer therefore allows  168  4. Discussion the production of unique Alu PCR products. However, only 5/l6Alu PCR products isolated byAlu PCR differential hybridization were unique (Table 5). This repetitive nature of the Alu PCR products suggests a potential clustering of repetitive elements in the inter-Alu region. The nature of the repetitive elements present within the Alu PCR products was not pursued further, since their presence did not hinder the isolation of clones byAlu PCR differential hybridization. Only 2/20 isolates located within the deletion region were discernible as a difference between the Alu PCR product of the chromosome 5 hybrid and the deletion 5 hybrid on an ethidium bromide stained gel (section 3.1.6). The method of differential hybridization therefore can be used for isolation of non-visually discernible differences between two related somatic cell hybrids. In summary, Alu PCR differential hybridization is a highly specific method for the isolation of region-specific DNA fragments which complements and extends previously existing methods. Localization of clones was rapidly confirmed by hybridization to the Alu PCR products of the somatic cell hybrids. Alu PCR differential hybridization should be generally applicable to any somatic cell hybriddeletion hybrid pair.  4.2 RADIATION HYBRID MAPPING The recently developed procedure of radiation hybrid mapping allows the ordering of widely spaced markers in the human genome (Cox et a!., 1990). Radiation hybrid mapping complements and extends conventional mapping techniques, such as meiotic linkage mapping, PFGE with rare-cutting enzymes, and in situ hybridization. Radiation hybrid mapping also has certain advantages since it  169  4. Discussion can be performed with small, moderately repetitive non-polymorphic markers, and does not depend on recombination frequencies or restriction enzyme site positioning. Radiation hybrid mapping was therefore selected for physically ordering clones isolated byAlu PCR differential hybridization. To type the chromosome 5 radiation hybrid mapping panel each of the hybrids was amplified using Alu PCR, and then Southern blotted. The Alu PCR products from the clones isolated byAlu PCR differential hybridization were then hybridized to the Southern blots of the hybrid Alu PCR products. The use of Alu PCR allows rapid typing of markers, since the decreased complexity of Alu PCR products relative to hybrid genomic DNA allows a reduction in exposure time. Since radiation hybrids can be somewhat unstable, it is recommended that a single batch of hybrid DNA be used to screen all probes (Cox et a!., 1990). If multiple investigators wish to screen the same batch of hybrid DNA, it is useful if each screen can be accomplished with a minimal amount of DNA. The use ofAlu PCR enables screening of a large number of probes with a very small amount of radiation hybrid DNA. Alu PCR also offers the advantage that probes which are highly repetitive on hybridization to genomic DNA may appear non-repetitive or only slightly repetitive when used to screenAlu PCR products, due to the reduced complexity of the Alu PCR product relative to the genomic DNA source. Time spent isolating unique probes can therefore be reduced. Obviously, care must be taken that the same primer(s) used to isolate the Alu PCR probe are used to amplify the radiation hybrids. D5S39 was typed by Dr. Solomon by hybridization to Southern blots of genomic DNA from the radiation hybrids. AnAlu PCR isolate was not available for  170  4. Discussion D5S39, so the genomic typing results were used for this probe. The sensitivity of typing using hybrid genomic DNA may be somewhat different from that obtained by using hybrid Alu PCR products. However, these differences were not large, since comparable retention frequencies were obtained for D5S39 and the Alu PCR isolates. As will be discussed later, the positioning of D5S39 by radiation mapping was comparable to that obtained by meiotic linkage analysis. Therefore, inclusion of this probe in the radiation hybrid analysis did not appear to cause any discrepancies. Radiation hybrid mapping in conjunction with Alu PCR was used to rapidly obtain radiation hybrid retention data for clones isolated byAlu PCR differential hybridization. The algorithms of Cox et a!., 1990 (Appendix 1) were used for analysis of the radiation hybrid retention data. Markers were initially analyzed in pairs using twopoint algorithms. A lod score of 3 was considered evidence of significant linkage between markers. Using this criteria, markers could be placed into four groups. Group #1 consists of 7 linked markers, group #2 of 4 linked markers, group #3 of 2 pairs of linked markers, and group #4 of 4 markers unlinked to any other marker (Table 7). Each marker was significantly linked to a maximum of two other markers. The order of markers suggested by the twopoint analysis was therefore assumed to be the most likely. The relative likelihoods of inversion of pairs of markers was then calculated for groups #1 and #2 using fourpoint algorithms. A linear order was obtained for 5/7 markers in group #1 (Figure 11) and for the 4 markers in group #2 (Figure 12). The remaining two markers in group #1 could not be positioned. Radiation hybrid mapping was therefore useful for grouping markers and obtaining a rough order of certain groups of markers.  171  4. Discussion The fact that not all markers could be ordered by radiation hybrid mapping reflects in part the density of markers used. The cytologically visible deletion in the somatic cell hybrid HHW1O64 spans approximately 10% of chromosome 5, or roughly 20Mb. The 18 markers typed in the chromosome 5 radiation panel were expected to be located within this 20Mb region. As will be discussed later, the 18 markers actually span a much larger region, since 3 of the markers were located on 5p. The density of markers used to screen the chromosome 5 radiation panel was therefore relatively low. The amount of radiation used to create the chromosome 5 radiation hybrid mapping panel was another reason why linkage was not obtained between all markers typed in the radiation panel. Marker retention frequencies vary according to the amount of radiation used to make a panel. Cox et a!. (1990) used 8,000 rads to create their radiation hybrid panel and obtained marker retention frequencies of 32% to 59%. The chromosome 5 radiation panel was made using 50,000 rads and marker retention frequencies of 4% to 13% were obtained for this panel (Table 6). Markers typed in the chromosome 5 panel must therefore be very close together to obtain overlapping retentions and therefore significant linkage. Radiation hybrid distances indicate the frequency of breakage between markers after exposure to a given amount of x-rays. Radiation distances between significantly linked markers in my data set ranged from 42 cR50000 to 97 cR50000, which corresponds to a breakage frequency between markers of 42% to 97%. Significant linkage between markers was therefore obtained at very close to the 100 50000 limit (100% frequency of breakage between markers). cR Radiation hybrid mapping was used to position 18 new chromosomeS  172  4. Discussion markers and D5S39 into 4 groups. This information was then used to determine which markers to investigate further. Radiation hybrid mapping therefore serves as a valuable tool for the rapid ordering of non-polymorphic loci.  4.3 POLYMORPHISM SCREENING AND LINKAGE ANALYSIS The loci used for polymorphism screening were determined largely by the results of the radiation hybrid mapping. Radiation hybrid group #1 consisted of 7 markers, 5 of which could be placed in a linear order by radiation hybrid mapping. The development of polymorphisms from within this group was therefore of interest to allow the comparison of radiation hybrid mapping and linkage mapping. Group #1 was of further interest because it included D5S39, which had been shown to map close to the SMA disease gene. Polymorphisms from radiation hybrid groups #2 and #3 were of interest to allow comparison of radiation and linkage maps, and to position these groups within the 5q11.2-q13.3 region. During the course of this thesis, screening was done for three types of polymorphisms; conventional RFLPs, polymorphic (GT)n microsatellite repeats and polymorphisms inAlu polyA tails. Screening for conventional RFLPs was done prior to radiation hybrid mapping and before much information was available on (GT)n polymorphisms. RFLP screening was therefore done with the isolates from the first Alu PCR differential hybridization trial (D5S205, D5S251, D5S252, D5S253, D5S254 and D5S255) and with two non-repetitive isolates from later trials (D5S255 and D5S264). A TaqI polymorphism with a PlC of 0.39 was detected for D5S205 (section 3.3.2). D5S205 was mapped by linkage analysis between D5S78 and D5S71.  173  4. Discussion Subsequent to the screen for conventional RFLPs, the radiation hybrid mapping was finished and extensive information was available on the informativeness of (GT)n tract polymorphisms (Weber and May, 1989; Weber, 1990). All isolates were therefore screened for the presence of (GT)n tracts. Hybridization conditions were used such that tracts containing approximately 10 or more repeats would be detected (Weber and May, 1989), since tracts with more than 10 repeats had been reported to often be polymorphic (Weber, 1990). A total of 6 (GT) tracts were detected, 5 of which were from radiation hybrid group #1 and one which was from radiation hybrid group #3. All of these tracts were therefore of interest, and were tested to determine if they were polymorphic. All of the tracts were polymorphic, with PlC values ranging between 0.06 and 0.75 (Table 8). (GT)n tracts isolated fell into two categories, four tracts which were present as isolated tracts and two which were at the 3’ ends of Alu elements. The four isolated (GT)n tracts amplified well and alleles were easy to score. D5S253 and D5S268 had uninterrupted GT runs of 16 and 23 and were highly informative, with PlC values of 0.75 and 0.68, respectively. D5S260 and D5S257 had 11 and 12 uninterrupted GT residues, and had PlC values of 0.69 and 0.29. These four tracts therefore followed the general rules for informativeness determined by Weber (1990), namely that tracts of 16 or more uninterrupted GT repeats would be highly informative, while tracts with repeat numbers of between 11 and 15 would be highly variable in terms of their PlC values. Linkage mapping positioned D5S253 between index markers D5S78 and D5S71 (Figure 31). D5S253 is currently the closest marker to the distal 5q11.2-  174  4. Discussion q13.3 deletion breakpoint, and provides a highly informative marker within the 29 cM gap between D5S78 and D5S71 (Weiffenbach et a!., 1991). D5S260 linkage maps between index markers D5S21 and D5S76 (Figure 31). This positioning was unexpected, since both D5S21 and D5S76 map outside the 5q11.2-q13.3 deletion region (Gilliam et a!., 1989). The positioning of the (GT) polymorphisms associated with D5S257 and D5S268 was also unexpected, since both D5S257 and D5S268 were found to linkage map to 5p (Figure 31). As will be discussed later, the discovery of these three markers led to an analysis of the HHW1O64 hybrid and the deletion chromosome 5 from which the hybrid was derived. The two (GT) 11 tracts at the ends ofAlu elements were of two types. The D5S262 tract was a cluster of short repeats of TG and TA, and the longest uninterrupted repeat was 6 units long (Figure 22). Although this system was predicted to be uninformative using Weber’s (1990) rules, the tract was investigated because of its position in radiation hybrid group #1. The D5S262 polymorphism was, as expected, relatively uninformative, with the least common allele just above the 0.01 frequency level normally used to define a system as polymorphic (Hedrick, 1983). The probability that D5S266 would be polymorphic was much harder to assess, since variation could occur in both the (GT) tract and in the Alu polyA tail (Figure 27). The PlC observed for D5S266 was 0.30, which is more than expected for a (GT) tract of 10 repeats (Weber, 1990). The presence of the Alu polyA tail may therefore have contributed to the level of informativeness. Neither D5S262 nor D5S266 could be accurately positioned on the chromosomeS linkage map. D5S262  was insufficiently polymorphic to allow placement. Amplification of the D5S266 (GT) tract and 3’ Alu region resulted in the production of a large amount of  175  4. Discussion shadow bands, positioned at 1 bp intervals from approximately 2 bp above to 10 bp below the major band amplified (Figure 28). Similar shadow bands are generally observed at 2 bp intervals for most isolated (GT)n tracts. The shadow bands are an in vitro artifact, since they are also seen for amplifications involving plasmid templates. For the majority of (GT)n systems, these shadow bands do not interfere with typing. However, the presence of excessive shadow bands made typing extremely difficult for the D5S266 system. (GT)n tracts were not present for loci D5S259, D5S258, D5S256, D5S265, D55255 and D5S254, which were of interest because they formed radiation hybrid group #2 and a portion of group #3. Alternate methods for detection of polymorphism were therefore investigated for these loci. Since clones corresponding to these loci were isolated byAlu PCR differential hybridization, each of the loci contained at least one Alu 3’ end. A variety of PCR-based assays for the detection of polymorphisms inAlu elements have been reported (Orita et a!., 1989, 1990; Economou et a!., 1990; Epstein et a!., 1990; Xu et al., 1991; Zuliani and Hobbs, 1990). The technique described by Economou et a! (1990) involved detection of polymorphisms inAlu polyA tails by amplification using a primer specific to the Alu element in question along with a primer from the non-repetitive region flanking the Alu element. The hypothesis was tested that this procedure could be modified slightly, such that the Alu primer was derived from the Alu consensus sequence, rather than the sequence of the specific Alit element. To isolate Alu elements, portions of the recombinant phage from loci of interest were subcloned into plasmids, and the plasmids screened for the presence of a single Alu element. Sequence for the Alu polyA tail was then obtained using  176  4. Discussion the TC65A primer, which is homologous to the Alu consensus sequence. Since only a few polymorphisms involving Alu polyA tails have been detected thus far, guidelines have not been established for estimating the probability that a tract will be polymorphic. Taking (GT) tracts as an example, the polymorphism level ofAlu polyA tails is likely proportional to the number of repeat units. Tracts from D5S254 and D55265, with 15 and 17 residues respectively, were therefore investigated for potential polymorphisms. A specific amplification product for the D5S254 Alu polyA tail was not obtained under a variety of amplification conditions. This was somewhat unexpected, since the TC65A primer had only one mismatch at the 5’ end with the sequence of the D55254 Alu element. An appropriately sized amplification product could be obtained using plasmid or phage template, which implies that the problem did not involve the primers or PCR system. One possible explanation is that the phage could have been rearranged during cloning, potentially due to recombination between Alu elements. The sequence obtained could therefore be different from that present in genomic DNA. Although each unique primer used for PCR was carefully screened for secondary structure and for sequence homologies, the primer flanking the Alu element could have been the problem if it either would not bind to the target sequence due to secondary structure constraints, or was somewhat repetitive, and bound to numerous places elsewhere in the genome. The D5S254 polyA tail tract was not investigated further. The Alu polyA tract from D5S265 was amplifiable with the expected 172 bp size from both plasmid and genomic DNA. Additional amplification products were observed in genomic samples at 177 and 186 bp, which were initially assumed to represent alleles at the D5S265 locus. However, CEPH individuals were observed  177  4. Discussion with a phenotype of 186,177,172, (Table 15) which is not possible under a single locus model. A variety of segregation models were therefore examined for fit to the data. The model which best fit the data was found to be two loci, one of which was polymorphic and the other which was monomorphic for the 172 bp band (single polymorphism model). The polymorphic locus was found to be in Hardy-Weinberg equilibrium assuming a single polymorphism model, which would be highly unlikely if the model was incorrect. Also, phenotype frequencies for all CEPH offspring fit those expected for a single polymorphism model for all parental mating classes. Linkage analysis demonstrated that the polymorphic locus was located on chromosome 2. An additional polymorphic system with fragment sizes of 308, 310 and 312bp was observed using the TC65A and D5S265-T primers (Figure 25). The larger system was not amplifiable from the GM1O114 hybrid, indicating that the amplification products observed were not from chromosome 5. Linkage analysis demonstrated that this system was located on chromosome 17. Three systems were amplified using the TC65A and D5S265-T primers, one from each of chromosomes 2, 5 and 17. No amplification product was observed using D5S265-T primer alone and the TC65A primer was unlabelled. The systems observed for D5S265 were therefore due to amplification between the D5S265-T primer and the TC65A primer. As the annealing temperature for PCR was increased, the amount of product obtained decreased in an identical fashion for each of the three systems. Therefore, the primer binding sites for these three systems were very similar or identical. The three systems amplifiable using the D5S265-T and TC65A primers likely  178  4. DLccussion arose due to duplication by an unknown mechanism. One possible duplication mechanism would involve Alu element transposition. This mechanism is unlikely, however, since Alu elements are thought to transpose utilizing an RNA intermediate (Ulla and Tschudi, 1984; Chen et a!., 1985) and the transpositional event postulated here would necessarily include flanking sequences. The formal possibility exists that the D5S265-T primer sequence serves a functional role on chromosomes 2, 5 and 17. This seems somewhat unlikely, however, due to the fact that the primer was derived from anonymous sequence. A more likely explanation is that the D5S265-T primer is located within a low copy repetitive element which had not yet been sequenced when the database search was made. The juxtaposition of the D5S265-T primer sequence with the 3’ end of anAlu element could have arisen due to transposition of the Alu element into the low copy repetitive element. The fact that Alu elements have a tendency to transpose into the A-rich tails of repetitive elements (Weiner et al., 1986) provides support for this type of mechanism. The presence of sequences homologous to D5S256-T on chromosome 2 together with an identical size amplification product to that obtained for chromosome 5 could then be explained by a duplication event of a low copy repetitive element containing the Alu element. The chromosome 17 locus could be the result of an independent Alit insertional event, or could be due an additional transposition event of the low copy repeat containing the Alu element. Further evidence for the common origin of the chromosome 2 and 5 systems, with a more distant origin for the chromosome 17 system is the appearance of the amplification products for the various systems. The chromosomes 2 and 5 systems exhibited a large number of shadow bands, similar to that observed for the D5S266 system,  179  4. Discussion while the chromosome 17 system was relatively devoid of shadow banding. While the difference in the proportion of shadow bands could be a technical artifact, it could also be due to sequence differences within the amplified products from the various loci, with fewer repeats present in the polyA tail from chromosome 17. For the D5S265 Alu polyA tail amplification, difficulties experienced were directly related to the primer flanking the Alu element. The presence of two mismatches between the TC65A primer and the Alu sequence did not appear to have any detrimental effect. The causes of the amplification problems for the D5S254 system are more difficult to ascertain, but are unlikely to solely involve the mismatch present between the TC65A primer and the D5S254A1u sequence. Alu polyA tail polymorphism detection using a primer specific to the Alu consensus sequence together with a unique flanking primer is therefore a useful technique. However, extreme care must be taken in the choice of the unique flanking primer. Various types of PCR based polymorphic systems were investigated during the course of this thesis. As was expected from the guidelines devised by Weber (1990), (GT)n tracts with a repeat number of 16 or more were found to be the most informative, followed by (GT)n tracts with between 10 and 15 repeats. (GT) tracts associated with Alu polyA tails were more difficult to type due to the presence of excessive shadow banding. Alu polyA tracts can serve as sources of polymorphisms when (GT)n tracts are unavailable, but do not seem generally attractive due to potential problems with shadow bands and difficulties with amplification of the tract of interest.  180  4. Discussion 4.4 COMPARISON OF MAPPING METHODS Positional information on markers isolated byAlu PCR differential hybridization was initially obtained using radiation hybrid mapping. Linkage mapping was then performed for markers of interest to place them relative to other polymorphic markers. Figure 34 depicts results obtained for markers which were mapped using both methods, which included index marker D5S39 and new markers D5S257, D5S268, D5S260, and D5S253. All markers mapped using both methods were present within radiation hybrid group #1. The orientation of radiation hybrid group #1 as indicated in Figure 34 was based on the somatic cell hybrid localization of markers D5S39 to 5q11.2-q13.3 (Gilliam et a!., 1989) and D5S257 to 5pl5.1 (John McPherson, pers. comm.). Although D5S262 and D5S264 were not positioned on the linkage map, they are shown in the radiation map in Figure 34 to prevent discontinuities. D5S260 and D5S268 could not be positioned relative to D5S262 by radiation hybrid mapping, and are therefore indicated in brackets immediately adjacent to D5S262 on the radiation map. The order of markers obtained by radiation hybrid mapping was analogous to that obtained by linkage mapping for D5S257, D5S260, D5S268 and D5S39. The sole discrepancy between the two maps was the position of D5S253, which was placed between D5S78 and D5S71 by linkage mapping, but was positioned next to D5S257 by radiation hybrid mapping. Somatic cell hybrid localization has positioned D5S78, D5S71 and D55253 onto 5q (Tables 25 and 27), while D5S257 has been placed on 5p (Table 25). The linkage placement of D5S253 between D5S78 and D5S71 is therefore confirmed by the somatic cell hybrid localization data, indicating that D5S253 was incorrectly positioned by radiation hybrid mapping.  181  4. Discussion  While the map order obtained by radiation hybrid mapping is very similar to that obtained using linkage analysis, inconsistencies exist when distances between markers are considered. D5S257 maps to 5pl5.l and D5S262 maps to 5q11.2-q13.3 by somatic cell hybrid mapping (Table 25). The distance between D5S257 and D5S262 therefore encompasses approximately 1/4 of chromosome 5, and yet these markers were linked by radiation hybrid mapping. It seems highly unlikely that D5S257 and D5S262 would be linked by radiation hybrid analysis given that no radiation hybrid linkage was detected for the marker pairs D5S257-D5S268 and D5S205-D5S253, which are located close to each other on the linkage map. This difference in detection of radiation hybrid linkage can be partially attributed to differences between regions in the ratio of physical distance to linkage distance. However, the physical distance between D5S257 and D5S268 cannot be very large, since they are both present within a non-cytologically detectable 5p deletion. Therefore, it is highly unlikely that physical: linkage ratio differences could completely explain the radiation hybrid linkages obtained.  182  4. Discussion  Figure 34. Comparison of radiation hybrid map and meiotic linkage map  Published Order  Linkage map  Radiation hybrid map  5pter D5S59 D5S253 D5S60 D5S257 D5S268  D5S257  D5S260  D5S262 (D5S260; D5S268)  HPRTP2 D5S21 D5S76 D5S6 D5S39  D5S39 D5S264  D5S78 D5S205 D5S253 D5S71 qter  183  4. Discussion 4.5 CHARACTERIZATION OF HHWJO64 AND SEGMENTAL TRISOMY The somatic cell hybrid HHW1O64 was demonstrated to contain a deletion for 5q11.2-q13.3 by the absence of hybridization of markers known to be present within this interval (Gilliam et a!., 1989). Lack of hybridization with HHW1O64 was therefore used as a basis for the isolation of probes from 5q11.2-q13.3 byAlu PCR differential hybridization. As a result of genetic linkage analysis with new polymorphic markers, the DNA content of the HHW1O64 hybrid was examined. The polymorphic systems isolated as a part of this thesis necessitated an inspection of regions within both the p arm and the q arm of the human chromosome 5 present in the HHW1064 hybrid. An examination was also made of the derivative chromosome 5 present in the carrier female from whom HHW1O64 was derived. Linkage data on two of the clones isolated byAlu PCR differential hybridization (D5S257 and D5S268) unequivocally positioned them on the p arm of chromosome 5. HHW1O64 therefore carries a non-cytologically visible deletion on the p arm of chromosome 5 in addition to the expected constitutional 5q11.2-q13.3 deletion. The position of the 5p secondary microdeletion was partially delineated by physical mapping of D5S257 using a chromosome 5 somatic cell hybrid mapping panel (John McPherson, pers comm). D5S257 maps to a tightly defined region of 5p15.l within region I as defined by Overhauser et a!., 1987. The HHW1O64 5p microdeletion therefore encompasses at least part of the 5pl5.l band. To confirm the 5p localization of D5S257 and D5S268 markers and to localize the remainder of the new markers, each isolate was hybridized to HHW213, a hybrid which contains predominantly 5p material. Four out of twenty distinct isolates were found to be located on the p arm of chromosome 5 (Table 25). When  184  4. Discussion multiple isolates are included, 10/35 (29%) of isolates were from the noncytologically detectable deletion within 5p. The large number of p arm isolates compared to the number of isolates obtained from the cytologically detectable 5q deletion is likely due to the relative Alu-richness of the two regions, with the area of the p arm deletion being particularly rich in Ala elements. The 5q deletion within the HHW1O64 hybrid involves markers D5S6 and D5S39 but does not include markers D5S76 and D5S21 (Gilliam et al., 1989). The marker D5S260 was positioned between D5S21 and D5S76 by multipoint linkage mapping, but is not present within the somatic cell hybrid HHW 1064. An examination of the somatic cell hybrid and linkage data was performed in an attempt to resolve this inconsistency. D5S260 was clearly demonstrated to be absent from the HHW1O64 hybrid and D5S76 was found to be present within this hybrid. Therefore, no errors in marker typing of the somatic cell hybrid HHW1O64 were detected. Linkage results for the D5S21-D5S39 region were closely examined to determine any potential errors. The consensus order obtained from linkage analysis was D5S21-D5S76-D5S6-D5S39 (Weiffenbach, et a!., 1991; Westbrook et a!., 1991; this thesis). However, a careful examination of these data revealed certain problems with the linkage map in this region. Weiffenbach et a!., 1991 used the BUILD option of CR1-MAP to produce a multipoint linkage map of markers positioned with greater than 1000:1 odds against all other positions. However, this paper also reports data using the FLIPS option of CR1-MAP, which gives the odds of inversion of 1:1.6 for D5S76 and D5S6 and odds of “better than 1:1000” for inversion of the entire D5S76-D5S6-D5S39 segment (Weiffenbach et a!., 1991). The  185  4. Discussion linkage map reported by Westbrook et al, 1991 was made by the compilation of data from both CEPH families and other families. While the order of markers was reported to be the same for all sources of data, the relative odds of inversion of markers in each study was not indicated (Westbrook et a!., 1991). Finally, twopoint linkage data obtained for D5S260 placed D5S260 closer to both D5S21 and D5S6 than to D5S76 (Table 22). These results suggest a problem with the linkage data for D5S76. A careful examination of the linkage data therefore suggests that the order of markers in the vicinity of D5S76 is somewhat questionable. As the number of polymorphic markers identified in humans increases, it has become evident that data errors are a limiting factor in the production of multipoint linkage maps (Keats et al., 1991; Sheilds et a!., 1991). Data errors can include errors in reading or entering data, interchange or mislabelling of samples or misinterpretation of bands or reactions (Keats et al., 1991). Data errors can alter both distances between markers and marker order. Duplicate typing of all loci will control for data error, but is not feasible for the majority of systems due to time and cost constraints. Retesting of all apparent recombinants over small distances and all double recombinants over moderate distances is helpful but is not done routinely due to logistic constraints (Keats et a!., 1991; Shields et a!., 1991). Alternate mapping methods can often provide information regarding potential errors in linkage maps. Physical localization data for chromosome 5 index markers from published reports were therefore examined (Table 27). D5S21 was positioned within 5pl3-pll using a somatic cell hybrid localization panel (Overhauser et a!., 1987). D5S6 and D5S39 were mapped by in situ hybridization to 5q12-q13.1 and 5q13, respectively (Mattei et al., 1991). The order of these markers  186  4. Discussion Obtained by physical localization therefore agrees with the consensus linkage order (D5S21-D5S6-D5S39; Weiffenbach et al., 1991, Westbrook et a!., 1991; this thesis). The assignment of D5S76 to 5cen-qll.2 (Bishop and Westbrook, 1990) is based on a combination of linkage data reported by Leppert et a!., 1987 and the presence of D5S76 in the HHW1O64 somatic cell hybrid observed by Gilliam et a!., 1989. Physical localization data for D5S76 which are clearly distinguishable from linkage data are therefore not available. While the physical localization results are clearly incomplete without data on D5S76, no evidence was observed against the linkage order D5S21-D5S76-D5S6-D5S39. Also worthy of mention is the HHW1O64 data placing D5S21 and D5S76 outside the deletion while D5S6 and D5S39 are located within the deleted region (Gilliam et a!., 1989). Any inversion of marker orders including D5S76 such as D5S21-D5S6-D5S76-D5S39 or D5S21-D5S39-D5S6-D5S76 implies the presence of two noncontiguous deletions in the HHW1O64 hybrid. The HHW1O64 hybrid data therefore lend support to the D5S21-D5S76-D5S6-D5S39 order. In the absence of evidence to the contrary, the somatic cell hybrid HHW1O64 contains a complex deletion in the 5q11.2-q13.3 region, encompassing at least two non-contiguous segments on either side of D5S76. A diagrammatic representation of the derivative 5 chromosome postulated to be present in the HHW1O64 hybrid shown in Figure 35. Cytological locations for index markers were used to determine approximate breakpoint positions (Table 27).  187  4. Discussion Table 27. Physical location of index markers  locus  cytological location  reference  D5S59 D5S60 HPRTP2 D5S21 D5S76 D5S6 D5S39  5pl5.2-5p15.l 5pl5.l 5pl4 5pl3-pll 5cen-qll.2 5q12-q13.1 5q13 5q11.2-q13.3 5q14-q21 5q21  Weiffenbach et a!., 1991 Weiffenbach et a!., 1991 Overhauser et a!., 1986b Overhauser et a!., 1987 Bishop and Westbrook., 1990 Mattei et al., 1991 Mattei et a!., 1991 Gilliam et al., 1989 Bishop and Westbrook., 1990 Stewart et a!., 1987  D5S78 D5S71 D5S37  A breakage of the human derivative chromosome 5 present within HHW1O64 was detected at or near the 5q11.2-q13.3 deletion breakpoint in approximately 1/3 of metaphases examined. Since a common fragile site is not present at or near this region of chromosome 5 (McAlpine et al., 1990), this fragility appears to be a specific to the derivative chromosome 5. Fragility at translocation breakpoints has been documented in the lymphocytes of various translocation carriers (for example, Juberg et al., 1983; Drets and Therman, 1983), and is generally attributed to the juxtaposition of regions of DNA not normally found next to each other. The fragility at the 5q11.2-q13.3 deletion breakpoints in the HHW1064 hybrid may have been due to such DNA interactions or may merely have been an artifact of culture. Fragility at this site was not examined in the carrier female from whom HHW1O64 was derived. However, the carrier female was  188  4. Discussion phenotypically normal, indicating that the postulated fragile site had no clinical significance. The cytogenetically balanced carrier from whom HHW1O64 was derived has a deleted chromosome 5 and a chromosome 1 with an insertion of chromosome 5 material. This individual is a member of a family in which segmental trisomy for 5q11.2-q13.3 segregates with schizophrenia and other anomalies (Bassett et a!., 1988; McGillivray et a!., 1990). Segregation for the (CA) polymorphisms associated with D5S268 and D5S257 was studied in this family to determine if the 5q11.2-q13.3 deletion chromosome in the carrier individual was also deleted for 5p. Segregation at the D5S76, D5S253 and D5S260 loci was also investigated to determine if the 5q deletion was the result of a complex rearrangement. The D5S268 locus was fully informative in this family (Figure 33). The polymorphisms associated with D5S257, D5S76 and D5S260 were partially informative and D5S253  was uninformative (Figure 33). The carrier individual has two alleles at the D5S268 locus, 126 and 124bp. Her affected son inherits the derivative 1 chromosome and a normal chromosome 5 from his mother and a normal chromosome 5 from his father (Figure 33). The affected son is disomic at the D5S268 locus, inheriting the maternal 124 allele. If the D5S268 locus were translocated to the der(1) chromosome then the affected son would have been trisomic for D5S268. It is unlikely that the D55268 locus is deleted from the der(5) chromosome and inserted elsewhere in the genome since the intact chromosome 5 transmitted to each of the sons carries a different maternal D5S268 allele necessitating at least one recombination event. Therefore, the chromosomal rearrangement segregating in this family does not involve 5p.  189  4. Discussion The deletion 5 chromosome present within the carrier female contains the 10 kb D5S76 allele, since this allele is present in the HHW1O64 hybrid. The normal chromosome 5 present within the carrier female therefore carries the 14 kb D5S76 allele, which is passed to both sons. Normal Mendelian segregation is therefore observed at the D5S76 locus (Figure 33). The carrier female has 150 and 152 bp alleles at the D5S260 locus, one of which is located on her normal chromosome 5 and the other which is on her derivative 1 chromosome. She passes both alleles at the D55260 locus to her affected son, since the affected son is trisomic at the D5S260 locus (Figure 33). The trisomic region therefore includes the D55260 locus but does not contain the D5S76 locus. The deletions on either side of D5S76 in the derivative chromosome 5 present in the somatic cell hybrid HHW1O64 were therefore derived from the carrier female. The chromosomeS rearranged as a part of the segmental trisomy may have had a marker order of D5S21-D5S260-D5S76-D5S6, which is identical to that obtained in the CEPH panel. Given this marker order, the deletion of D55260 and D5S6 without the deletion of D5S76 requires a complex rearrangement. Alternatively, if the chromosome 5 carried an inverted marker order of D5S21D5S76-D5S260-D5S6, a single deletion event would explain the removal of D5S260 and D5S6 without deletion of D5S76. This inversion event could have arisen when the complex rearrangement occurred in the family segregating for the segmental trisomy, or could be present in an ancestral chromosome. A distinction between the various origins of the rearrangement could be made by determining the orientation of D5S76 on the deleted chromosome. The results presented as a part of this thesis indicate that a de novo  190  4. Discussion deletion is present in the HHW1O64 somatic cell hybrid. Results were also obtained supporting the hypothesis that a complex rearrangement involving 5q11.2-q13.3 is present in HHW1O64 and is segregating in the trisomic family from which HHW1O64 was derived. The presence of a complex rearrangement segregating in the trisomic family has important implications regarding the position of the putative genes for schizophrenia susceptibility and renal development. The region involved in the segmental trisomy, and thus the region to which these genes would be assigned, can now be expanded to include regions both proximal and distal to D5S76. Linkage studies involving schizophrenia and hereditary renal adysplasia must therefore include markers both proximal and distal to D5S76. Diagrammatic representations of the derivative chromosome 5 postulated to be present within HHW1O64 and the carrier female member of the trisomic family are shown in Figure 35.  191  4. Discussion Figure 35. Derivative chromosome 5 present in HHW1O64 and carrier female from whom HHW1O64 was derived  5  marker  HHW1O64  15.3 15.2 15.1  I I  D5S59  H-  D5S60 D5S257 D5S268  14 13.3  13.2 13.1  HPRTP2  12 11 11.1  D5S21  11.2 12 13.1 13.2 13.3  D5S260  I  D5S76 D5S6 D5S39 D5S78 D5S205 D5S253  14  15  D5S71 21  carrier female  D5S37  ‘I.,  23.1 23.2 23.3 31.1 31.2 31.3 32 33.1  9  32.3 34 35.1 35.2 35.3  I present in derivative chromosome 5 • absent from derivative chromosome 5  192  4. DLccussion 4.6 CONCLUSIONS The research described in this thesis generated a variety of results relevant to the field of human molecular genetics. A new technique, Alu PCR differential hybridization, was developed for the isolation of region-specific human DNA fragments from mixed DNA sources. This technique is applicable to any hybriddeletion hybrid pair and complements and extends previously reported techniques. Alu PCR differential hybridization was used to isolate clones from a region of the human genome, 5q11.2-q13.3, defined by a segmental trisomy. This region was of interest due to co-segregation of the trisomic region with schizophrenia and renal anomalies in a Vancouver family (Bassett et a!., 1988; McGillivray et a!., 1990). Linkage between markers in the 5q11.2-q13.3 region and chronic spinal muscular atrophy made this region of additional interest (Brzustowicz et a!., 1990; Melki et a!., 1990; Gilliam et a!., 1990). A rough order of clones isolated byAlu PCR differential hybridization was determined using radiation hybrid mapping. While a linear order could not be determined for all clones using radiation hybrid mapping, insights were made regarding parameters required to observe significant linkage in the radiation hybrid mapping panel. Order information obtained from radiation hybrid mapping was used to select markers to screen for polymorphisms. Polymorphic systems detected allowed the characterization of the HHW 1064 somatic cell hybrid, which is of value to other investigators utilizing this hybrid. The new polymorphic systems provided information regarding the segmental trisomy segregating in the Vancouver family. These results have important implications regarding the positioning of genes for schizophrenia and hereditary renal adysplasia postulated to be present within the  193  4. Discussion trisomic region. Polymorphic markers isolated during the course of this thesis also provide useful markers for linkage studies involving disease genes in the vicinity of the new markers.  4.7 SUMMARY 1. Alu PCR differential hybridization, a novel technique for the isolation of regionspecific human DNA fragments was devised. This technique should be generally applicable to any somatic cell hybrid-deletion hybrid pair.  2. Alu PCR differential hybridization was used in conjunction with two chromosome 5 somatic cell hybrids (GM1O114 and HHW1O64) to isolate 20 clones absent from the HHW1O64 hybrid.  3. Radiation hybrid mapping together with Alu PCR was used to rapidly obtain positional information on 18 of the new isolates.  4. Clones of interest were screened for the presence of three types of polymorphisms; conventional RFLPs, variations in the number of (GT) repeats, and variations inAlu polyA tails. Nine polymorphisms were detected.  5. A highly informative (GT) tract within chromosome 5q11.2-q13.3 was detected at the D5S253 locus. D5S253 is currently the closest marker to the distal breakpoint within the 5q deletion region. D5S253 is located in an approximately 29 cM gap between D5S78 and D5S71.  194  4. Discussion  6. A highly informative (GT)n tract was detected in locus D5S260. D5S260 is not present in the somatic cell hybrid HHW1O64, but linkage maps between the index markers D5S21 and D5S76, which are located outside the region deleted in the HHW1O64 hybrid. Examination of the HHW1O64 typing data and linkage data for markers in this region of chromosome 5 suggests that the HHW1O64 hybrid contains a complex deletion encompassing two non-contiguous fractions on either side of D5S76.  7. A moderately informative RFLP marker (D5S205) and two moderately informative (GT)n tracts (D5S262 and D5S266) were isolated from 5q11.2-q13.3.  8. Amplification of the Alu polyA tract from D5S265 using D5S265-T and TC65A primers was found to detect three systems, one on each of chromosomes 2, 5 and 17. The chromosome 5 system was monomorphic, while the chromosome 2 and 17 systems were polymorphic and moderately informative. One possible explanation for the observed amplifications is that D5S265-T primer was within a low copy repetitive element into which anAlu element had transposed.  9. The highly informative (GT)n tract polymorphism associated with D5S268 and the moderately informative (GT)n tract polymorphism associated with D5S257 were unexpectedly found to linkage map to chromosome 5p.  10. The regional localization of the remainder of isolates was performed using the  195  4. Discussion somatic cell hybrid HHW213, which contains mainly 5p material. Four out of twenty of clones isolated byAlu PCR differential hybridization were located on  chromosome 5p. The HHW1O64 hybrid therefore contains a non-cytologically detectable deletion on chromosome 5p.  11. The carrier individual from whom HHW1O64 was derived does not have a 5p deletion, based on segregation analysis of D5S268 and D5S257 in her family. Segregation analysis for D5S76 and D5S260 in this family was used to demonstrate that the complex rearrangement on 5q postulated for HHW1O64 was derived from the balanced carrier.  4.8 PROPOSALS FOR FURTHER RESEARCH Listed below are several experiments which could be performed to extend the results obtained in this thesis.  1. Isolate more clones from the 5q11.2-q13.3 region using Alu PCR differential hybridization, prescreen these isolates for the presence of (GT) tracts and develop more polymorphisms for this region.  2. Perform Alu PCR differential hybridization using different somatic cell hybrids to delineate the limits of resolution of this technique, and/or to isolate clones useful for the mapping of different disease genes.  196  4. Discussion 3. Perform Alu PCR differential hybridization using cosmids as the source of cloned DNA in an attempt to increase the amount of DNA present for each isolate. Alu PCR differential hybridization would presumably work with gridded cosmid arrays, which will soon be generally available for all human chromosomes.  4. Obtain more information on radiation hybrid typing of index markers such as D5S6 in the mapping panel which was used for this thesis. The presence of additional markers would increase the density and presumably increase the chance of detecting significant linkages.  5. Map isolates in a different radiation hybrid mapping panel. A panel which was made using a lower dose of radiation would presumably allow the linkage of all markers within the 5q11.2-q13.3 region.  6. Perform linkage analysis using informative markers in the 5q11.2-q13.3 region (both previously reported and as a result of this thesis) and Asian families segregating for schizophrenia. This study would investigate the formal possibility that the postulated locus for schizophrenia within 5q11.2-q13.3 was race-specific.  7. Perform linkage analysis in hereditary renal adysplasia pedigrees to look for linkage to the 5q11.2-q13.3 region.  8. Isolate more Alu polyA tracts, and develop PCR systems for amplification of polyA tails. Primers from the Alu consensus sequence with varying amounts of  197  4. Discussion homology to the Alu element in question could then be tested to determine the amount of homology necessary for tract amplification. General rules for the informativeness of Alu polyA tails could also be formulated.  9. Sequence more of the region around Alu element in p52H1 (D55265) to determine if any evidence exists for the postulated low copy repetitive element. Evidence for such an element could also be demonstrated by the hybridization of various regions to Southern blots of genomic DNA.  10. Isolate the 5q constitutional deletion breakpoints and the 5p deletion breakpoints from the HHW1O64 hybrid. 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This method is based on the assumption that the further apart two markers are on a chromosome, the higher the probability of a radiation-induced breakage between them. The frequency of breakage between two markers can be estimated in the following fashion (Cox et a!., 1990). For two markers, A and B, the observed segregation can be defined in terms of four unknowns: e  frequency of breakage = probability of retention of a fragment containing A and not B = probability of retention of a fragment containing B and not A J3 = probability of retention of a fragment containing both A and B 1 P =  The fraction of hybrids retaining marker A but not B is therefore equivalent to breakage between A and B plus retention of the fragment containing A and nonretention of the fragment containing B, ie. (A+B-) T  8PA(l-PB)  =  Similarly,  (A-B +) T  (A+B+) T  (A-B-) T  ePB(l-PA)  =  =  =  ePAPB +(1-e)P  e(l-Pj(l-PB) + (1-e)(1-P)  209  Appendix 1. Algorithms These equations can then be used to solve for e in terms of A and B to obtain: 8=  (A+B-) + (A-B+) T(PA + B 2 AB) -  A and B can be estimated from the observed marker segregation data as RA and RB where RA is equal to the fraction of all hybrids analyzed for marker A that retain marker A and RB is equal to the fraction of all hybrids analyzed for marker B that retain marker B. e can then be calculated from the observed data, and varies between 1 (markers are always broken apart) and 0 (markers are never broken apart). A mapping function D -ln(1-e), which is analogous to the Haldane mapping function for meiotic linkage mapping, is then used to convert the frequency of breakage into an estimate of the distance between markers (D). D is expressed in centiRays (cR) with the amount of radiation used to make the radiation hybrid panel indicated as a subscript ie 000 0, means 50,000 rads were used to make the 5 cR panel. A distance of 1 cR50000 is therefore equivalent to a breakage frequency between two markers of 1% when 50,000 rads are used. The odds of obtaining a given distance between two markers relative to the that the markers are unlinked (e =1) can be calculated in a similar fashion to odds meiotic linkage mapping (Cox et al., 1990). A lod score can then be obtained from this calculation. Likelihood determinations for various orders of four markers (fourpoint analyses) can be calculated in a similar fashion to twopoint likelihoods (Cox et a!., 1990). It is therefore possible to obtain an estimation of the most possible order of four markers, and the odds for inversion of marker pairs.  210  AppendL 1. Algorithms 2. Mapping Functions - Haldane and Kosambi Mapping functions are used to convert observed recombination fractions (e) into recombination distances (d), such that for any contiguous map region + d. It has been observed that recombination fractions follow 1 + d 2 + dli = d the general formulae: ...  (1) The corresponding equation for map distance can be written: (2)  12 d + 1 =d 2 d  If 0 is then thought of as a function of recombination distance ie e =f(x), where x is equated with map distance, equation (1) becomes, (3)  1 +x2) f(x  ) 1 f(x  =  +  ) pf(x)f(x) 2 f(x -  When (3) is rearranged, divided by x2, and the limit taken as x2 -> 0, the left hand of side of the equation can be written as the standard definition for a derivative. (4)  lim X2->0  )-f(x 2 ± f( ) 1 = X2  f(,{1-pf(x)} X2  solving the differential using the fact that f(x)/x -> 1 as x -> 0 leads to: (5)  f’(xl)  =  ) 1 1-pf(x  or, (6)  .=l-pe dx  Haldane (1919) then integrates with respect to e, to obtain: (7)  x  =  -  1. ln(1 p0) p -  or  0  =  .1 (1 eP9 -  p  211  AppendL 1. Algorithms Haldane then makes the observation that when x is very large, e approaches 0.5 asymptotically. He therefore replaces p with 2 to obtain: x  (8)  =  -  1 ln(1 28) -  or  e  =  2  1 (1  -  9 2 e  2  which is the Haldane mapping function  Kosambi (1944) makes the hypothesis that p can be replaced by 4e, which when substituted into (6), leads to the differential equation de=1-49 2 dx  (9)  or  dx  =  de 2 1-4e  This differential can be easily solved using standard tables to give  (10)  x  =  j. 4  ln  [  1 + 2e 1-28  or  e =1 tanh (2x) 2  which is the Kosambi mapping function More complex mapping functions have since been derived in an attempt to obtain a better fit to the observed data (Ott, 1985). For the majority of human data, however, either the Haldane or Kosambi mapping function will provide a good fit to the data.  212  Appendix 1. Algorithms 3. Polymorphism Information Content (PlC) The PlC of a marker indicates the probability that any meiosis will be informative, based on observed allele frequencies (Botstein et al., 1980). If the system is in Hardy-Weinberg equilibrium, the expected proportions of each parental genotype can be calculated from observed allele frequencies. For each mating class, the frequency of mating can expressed as the product of the two parental genotypic frequencies. The probability that an offspring will be informative can be determined for each mating class by inspection of the parental genotypes. The percentage of informative meioses for each mating class is the product of the mating frequency and the probability that an offspring will be informative. The PlC is then calculated by summing this product for all mating classes. Mathematically, the simplest expression of PlC is { 1 (the proportion of uninformative matings)} or, -  n-i  n PlC  =  1  -  E  i=1  2 Pi  -  n  E  E  i=1  j=i+1  1 p 2 2p  Where j represents the allele frequency of the th allele.  213  Appendix 1. Algorithms 4. Effective number of Informative Recombinants and Meioses  For organisms in which genetic analysis is carried out by planned crosses, the numbers of recombinants and non-recombinants can be directly counted. For human genetic analysis, however, linkage analysis is carried out using likelihood analysis with both phase known and phase unknown families (Morton, 1955). A method was therefore developed for determining the numbers of recombinants and non-recombinants in phase known families which would give the same lod score as that observed (Edwards, 1976). These values were termed effective numbers of recombinants and non-recombinants, the sum of which is necessarily the number of effective number of informative meioses (Edwards, 1976). Based upon the sequential likelihood analysis of Morton (1955), Edwards (1976) derived the following equations for calculation of effective numbers:  {e  (1)  1  (2)  r=se  (3)  n=s-r  s  [log e log 0.5] -  +  (1- e) [log (1-e) log (0.5)] -  Where,  e  =  1 = s = r = n  most likely recombination fraction lod score at most likely value of e effective number of meioses effective number of recombinants effective number of non-recombinants  214  }  APPENDIX 2. RADIATION HYBRID DATA 1 0  =  probe present in hybrid probe not present in hybrid ND = no data  =  HYBRID D5S205 D5S251 D5S254 D5S253 D5S255 D5S256 D5S257 1 2 3 4 5 6 8 10 12 13 14 15 16 17 18 19 20 21 22 23 25 26 27 28 29 30 31 32 33 34 35 37 38 39 40  0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0  0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0  215  0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0  0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 1 1 1 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0  Appendix 2. Radiation hybrid data HYBRID D5S205 D5S251 D5S254 D5S253 D5S255 D5S256 D5S257 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 63 64 65 66 67 68 69 71 72 73 74 75 76 77 78 81 82 83 84 85 86 87 88 89 90  0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 0 0 0 0 0 0 0 0 0 1  0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 1  216  0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 1 0 1 0 0 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0  Appendix 2. Radiation hybrid data HYBRID D5S205 D5S251 D5S254 D5S253 92 93 94 95 96 97 98 99 100 101 104 105 106 107 109 110 111 112 113 114 115 117 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 139 140 141 143 144 145  0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0  0 0 0 0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 1 0 0 0 0  0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 0 0 0 0 0 0  217  D5S255 D5S256 D5S257 0 0 0 0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0  1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 0 1 0 0 0 0  0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1 0 0 0 0 0 0 0 0  AppendL 2. Radiation hybrid data HYBRID D5S205 D5S251 D5S254 D5S253 146 149 150 151 152 153 154 155 156 157 158 159 160 162 163 164 165 166 167 168 172 y z  0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0  0 0 0 1 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0  218  D5S255 D5S256 D5S257 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0  1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0  Appendb 2. Radiation hybrid data HYBRID D5S258 D5S259 D5S260 D5S261 D5S262 D5S264 D5S265 1 2 3 4 5 6 8 10 12 13 14 15 16 17 18 19 20 21 22 23 25 26 27 28 29 30 31 32 33 34 35 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51  0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0  219  0 0 0 0 0 0 0 1 1 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 1 1 0 0 0 0 1 1 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  Appendix 2. Radiation hybrid data HYBRID D5S258 D5S259 D5S260 D5S261 D5S262 D5S264 D5S265  52 53 54 55 56 57 58 59 60 61 63 64 65 66 67 68 69 71 72 73 74 75 76 77 78 81 82 83 84 85 86 87 88 89 90 92 93 94 95 96 97 98 99 100 101 104  1 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 ND ND ND ND ND ND ND  0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 1 0 0 1  1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1  220  0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 1 0 0 1  0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0  0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 ND ND ND ND ND ND ND  Appendix 2. Radiation hybrid data HYBRID D5S258 D5S259 D5S260 D5S261 D5S262 D5S264 D5S265 105 106 107 109 110 111 112 113 114 115 117 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 139 140 141 143 144 145 146 149 150 151 152 153 154 155 156 157 158  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0  ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0  221  0 0 0 0 0 0 0 0 0 0 0 0 0 0 ND ND 1 0 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 ND 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  ND ND ND ND ND ND ND ND ND ND 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0  Appendk 2. Radiation hybrid data HYBRID D5S258 D5S259 D5S260 D5S261 159 160 162 163 164 165 166 167 168 172 y z  0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0  222  D5S262 D5S264 D5S265 1 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0  1 0 0 0 0 0 0 0 0 0 0 0  Appendix 2. Radiation hybrid data HYBRID D5S266 D5S267 D5S268 D5S269 1 2 3 4 5 6 8 10 12 13 14 15 16 17 18 19 20 21 22 23 25 26 27 28 29 30 31 32 33 34 35 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51  0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0  0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0  223  D5S39 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  Appendix 2. Radiation hybrid data HYBRID D5S266 D5S267 D5S268 D5S269 52 53 54 55 56 57 58 59 60 61 63 64 65 66 67 68 69 71 72 73 74 75 76 77 78 81 82 83 84 85 86 87 88 89 90 92 93 94 95 96 97 98 99 100 101 104  1 0 0 0 0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND  0 1 0 0 0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 ND ND ND ND ND ND ND  0 1 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0  0 1 0 0 0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 ND ND ND ND ND ND ND  224  D5S39 0 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 1  Appendix 2. Radiation hybrid data HYBRID D5S266 D5S267 D5S268 D5S269 105 106 107 109 110 111 112 113 114 115 117 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 139 140 141 143 144 145 146 149 150 151 152 153 154 155 156 157 158  ND ND ND ND ND ND ND ND ND ND 0 0 0 0 0 0 ND 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0  ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  225  D5S39 0 0 0 0 0 1 0 1 0 0 0 0 1 0 0 0 0 1 0 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  Appendix 2. Radiation hybrid data HYBRID D5S266 D5S267 D5S268 D5S269 159 160 162 163 164 165 166 167 168 172 y z  0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0  1 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0  226  D5S39 0 0 1 0 0 0 0 0 0 0 0 0  

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