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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 CLONESFROM HUMAN CHROMOSOME 5byLYNN ELIZABETH BERNARDB.Sc., Simon Fraser University, 1987A THESIS SUBMITI’ED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESGENETICS PROGRAMMEWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril, 19920 Lynn Elizabeth Bernard, 1992Signature(s) removed to protect privacyIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at The University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission forextensive copying of this thesis for scholarly purposes may be granted by the Headof my Department or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.DEPARTMENT OF MEDICAL GENETICSThe University of British Columbia2075 Wesbrook PlaceVancouver, CanadaV6T 1W5April 22, 1992Signature(s) removed to protect privacyABSTRACTThis thesis describes the isolation and mapping of DNA clones from humanchromosome 5, with emphasis on the 5q11.2-q13.3 region. This region is of interestbecause of a Vancouver family in which trisomy for the 5q11.2-q13.3 region cosegregates with schizophrenia and renal anomalies. The 5q11.2-q13.3 region is ofadditional interest since chronic spinal muscular atrophy has been linked to markerswithin this region.A novel technique, Alu PCR differential hybridization, was developed toisolate clones from the 5q11.2-q13.3 region. The somatic cell hybrid HHW1O64contains a chromosome 5 with an interstitial deletion of 5q11.2-q13.3, derived froma carrier member of the family segregating for the segmental trisomy. Alu PCRdifferential hybridization was used to isolate twenty chromosome 5 clones absentfrom the HHW1O64 hybrid. Radiation hybrid mapping was used to determine arough order of clones isolated byAlu PCR differential hybridization. Orderinformation was used to select clones to screen for polymorphisms. Ninepolymorphic systems were detected.Multipoint linkage mapping of two of the new polymorphisms (D5S257 andD5S268) 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 HHW1O64hybrid. The HHW1O64 somatic cell hybrid therefore contains a deletion within thep arm of chromosome 5 in addition to the expected interstitial deletion of 5q11.2-q13.3. Analysis of D5S257 and D5S268 in the family segregating for the segmentaltrisomy indicated that the rearrangement in this family does not involve Sp.Multipoint linkage analysis of the polymorphism associated with D5S26011Abstractplaced 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 ofabsence from the HHW1O64 hybrid. The most likely explanation for theinconsistency between the linkage and somatic cell hybrid data is that thechromosome 5 present in the HHW1O64 hybrid is deleted for DNA both proximaland distal to D5S76. This complex 5q rearrangement is present in the familysegregating for the segmental trisomy, based on the analysis of D5S260 and D5S76in this family.111TABLE OF CONTENTSABSTRACT iiTABLE OF CONTENTS ivLIST OF TABLES viiiLIST OF FIGURES xDEDICATION xiiACKNOWLEDGEMENTS xiii1. INTRODUCTION 11.1 THE 5q11.2-q13.3 REGION OF THE HUMAN GENOME 11.2 CHROMOSOMAL REARRANGEMENTS AND SOMATIC CELLHYBRIDS 41.3 ISOLATION OF DNA MARKERS 61.4PCR 61.SALUELEMENTS 71.6ALUPCR 91.7 RADIATION HYBRID MAPPING 131.8 POLYMORPHISM SCREENING 141.9 LINKAGE MAPPING 172. MATERIALS AND METhODS 222.1 MATERIALS 222.1.1 Somatic cell hybrids 222.1.2 Phage libraries 232.2 METHODS 242.2.1 Restriction enzyme digestions 242.2.2 Gel electrophoresis 252.2.3 Southern blotting 252.2.4 Oligolabeling 272.2.5 Preannealing with human DNA 292.2.6 Prehybridization, hybridization and washing 292.2.7 (GT) tract detection and isolation 302.2.8 Phage maniulations 312.2.8.1 Phage plating 312.2.8.2 Plaque lifts and plaque purification 32ivTable of Contents2.2.8.3 Small scale phageprep 332.2.8.4 Large scale phage prep 342.2.9 Plasmid manjpulations 362.2.9.1 Ligation 362.2.9.2 Production ofcompetent cells and transformation 372.2.9.3 Ligation independent cloning (LIC) 382.2.9.4 Colony screens 402.2.9.5 Plasmid minprep 412.2.9.6 Plasmid large scale prep 422.2.10 Sequencing 442.2 10.1 Denaturation of template 442.2.1 0.2 Sequencing reactions 452.2.1 0.3 Sequencingprimers 462.2.10.4 Denaturingpolyaciylamide gel electrophoresis 462.2.1 0.5 Manipulation ofsequence data 482.2.11 Polymerase chain reaction (PCR) 492.2.11.1 Standard PCR reaction conditions 492.2.11.2 Standard cycling conditions 492.2.11.3 PCR primers - synthesis and purification 502.2.11.4 Primer end-labeling 512.2.11.5 Electrophoresis of radiolabeled PCR products 522.2.11.6 Specific PCR reactions and primer sequences 522.2.l2Alu PCR differential hybridization 572.2.13 Statistical analysis 572.2.13.1 Radiation hybrid typing and analysis 572.2.13.2 Polymorphism typing and analysis 572.2.14 Somatic cell hybrid maniulations 582.2.14.1 Cell culture 582.2.14.2 Somatic cell hybrid DNA preparation 592.2.14.3 Preparation and staining of metaphase chromosomes 603. RESULTS 623.1 CLONE ISOLA TIONAND LOCALIZATION 623.1.1 Alu PCR differential hybridization 623.1.2 Alu PCR products from clones isolated by differential hybridization.... 663.1.2.1 Identification of multiple isolates and Alu- T3, Alu-T7PCR forclones 5PCR1 to 5PCRJ1 693.1.2.2 Identification of multiple isolates forphage A1u19 to A1u73 .. 70VTable of Contents3.1.3 Confirmation of localization - Alu PCR localization blots 733.1.4 Confinnation of localization - Genomic localization blots 783.1.5 Confirmation of localization - PCR 783.1.6 Majority of clones do not correspond to visible differences betweenhybrid Alu PCR products 803.2 RADL4 TION HYBRID MAPPING 823.2.1 Radiation hybrid typing 823.2.2 Twopoint radiation hybrid analysis 873.2.3 Fouipoint radiation hybrid analysis 893.3 POLYMORPHISMS 933.3.1 Polymorphism screening and typing 933.3.2D5S205 1013.3.3 D5S253 1033.3.4 D5S257 1073.3.5D5S260 1103.3.6D5S262 1133.3.7D5S265 1173.3. Zi System AluS2A 1213.3. Z2 System A1uS2B 1293.3.8D5S266 1313.3.9D5S268 1363.4 LINKAGE ANALYSIS 1393.4.1 Twopoint linkage analysis 1393.4.2 Multipoint linkage analysis 1473.4.2.1 ChromosomeS markers 1473.4.2.2A1u52A andAluS2B 1483.5 H13W1064 - CHARACTERIZATION 1543.5.1 HHWi064 Sp deletion 1543.5.2 HHW1O64 Sq deletion 1573.5.3 Cytogenetic analysis ofHRW1064 1593.6 CHARACTERIZATION OF CHROMOSOMAL REARRANGEMENTSEGREGATING IN TRISOMYFAMILY 161.3.t5.lsp 1613.6.15q 1624. DISCUSSION 1644.1 CLONE ISOLATION 1644.2 RADIATION HYBRID MAPPING 169viTable of Contents4.3 POLYMORPHISM SCREENING AND LINKAGE ANALYSIS 1734.4 COMPARISON OF MAPPING METHODS 1814.5 CHARACTERIZATION OFHHW1O64AND SEGMENTAL TRISOMY..1844.6 CONCLUSIONS 1934.7SUMMARY 1944.8 PROPOSALS FOR FURTHER RESEARCH 196REFERENCES 199APPENDIX 1. ALGORITHMS 2091. Radiation Hybrid Mapping 2092. Mapping Functions - Haldane And Kosambi 2113. Polymorphism Information Content (PlC) 2134. Effective Number OfInformative Recombinants And Meioses 214APPENDIX 2. RADIATION HYBRID DATA 215viiLIST OF TABLESTable 1. Clone isolation and localization 64Table 2. Alu PCR products obtained from clones exhibiting differential signals 67Table 3.Alu-T3 and Alu-T7 amplifications for clones 5PCR1 through 5PCR11 70Table 4. Multiple Isolates 72Table 5. Localization of clones by hybridization to somatic cell hybrids GM1O114and HHW1O64 75Table 6. Radiation hybrid retention frequencies 86Table 7. Twopoint radiation hybrid scores for markers linked at lod >3 88Table 8. Polymorphisms 94Table 9. Sequence ofAlu polyA tails 98Table 10. D5S205 Allele frequencies 101Table 11. D5S253 Allele frequencies 104Table 12. D5S257 Allele frequencies 108Table 13. D5S260 Allele frequencies 111Table 14. D5S262 Allele frequencies 113Table 15. Observed matings - Alu52A 122Table 16. Allele frequencies for polymorphic locus within A1u52A system assuminga single polymorphism 125Table 17. Expected versus observed genotypes for polymorphic loci within A1u52Asystem assuming a single polymorphism 125Table 18. Expected versus observed offspring phenotypes assuming a singlepolymorphism model for Alu52A 127Table 19. Alu52B Allele frequencies 129Table 20. D55266 Allele frequencies 132Table 21. D5S268 Allele frequencies 137viiiList of TablesTable 22. Twopoint meiotic linkage analysis 142Table 23. Effective number of informative meioses (N) for new polymorphicmarkers 146Table 24. Multipoint linkage analysis 149Table 25. Marker localization to 5p or 5q11.2-q13.3 156Table 26. Multipoint linkage analysis involving index markers D5S21, D5S76, D5S6,and D5S39 159Table 27. Physical location of index markers 188ixLIST OF FIGURESFigure 1. Family segregating for segmental trisomy 3Figure 2. Polymerase chain reaction (PCR) 8Figure 3. Schematic representation ofAlu PCR differential hybridization procedure11Figure 4. Representation of human chromosome present in somatic cell hybridsHHW1O64 and GM1O114 12Figure 5. Detection of clones derived from 5q11.2-q13.3 by differential hybridizationFigure 6. Clone localization by hybridization to Southern blots of hybrid Alu PCRproducts 77Figure 7. Clone localization by hybridization to Southern blots of hybrid genomicDNA 79Figure 8. Alu PCR products 81Figure 9. Alu PCR products for radiation hybrids 1 through 20 84Figure 10. Probes from D5S260 and D5S262 hybridized to radiation hybrids 1 to 20Figure 11. Fourpoint Radiation hybrid analysis - Group #1 91Figure 12. Fourpoint Radiation hybrid analysis - Group #2 92Figure 13. Sequence of D5S257 (GT)11 tract 96Figure 14. Sequence of D5S254 Alu polyA tract and flanking sequence andalignment with Alu consensus sequence 100Figure 15. D5S205 TaqI polymorphism 102Figure 16. D5S253 (GT) tract and flanking sequence 105Figure 17. D5S253 (GT) polymorphism 106Figure 18. D5S257 (GT)n tract and flanking sequence 108Figure 19. D5S257 (GT)n polymorphism 109xList ofFiguresFigure 20. D5S260 (GT)n tract and flanking sequence 111Figure 21. D5S260 (GT)n polymorphism 112Figure 22. D5S262 (GT)n tract and flanking sequence aligned with Alu consensussequence 114Figure 23. D5S262 (GT)n polymorphism 116Figure 24. D5S265 Alu polyA tract and flanking sequence compared with Aluconsensus sequence 119Figure 25. D5S265 Alu polyA tract polymorphisms 120Figure 26. AIu52A and A1u52B polymorphisms 130Figure 27. D5S266 (GT)n tract and flanking sequence compared with Alu consensussequence 133Figure 28. D5S266 (GT)n polymorphism 135Figure 29. D5S268 (GT)n tract and flanking sequence 137Figure 30. D5S268 (GT)n polymorphism 138Figure 31. Chromosome 5 multipoint linkage analysis - Summary 153Figure 32. Metaphase chromosomes from somatic cell hybrid HHW1O64 160Figure 33. Family segregating for segmental trisomy typed for D5S268, D5S257,D5S76 and D5S260 163Figure 34. Comparison of radiation hybrid map and meiotic linkage map 183Figure 35. Derivative chromosome 5 present in HHW1O64 and carrier female fromwhom HHW1O64 was derived 192xiDEDICATIONTo the memory of Paul Douglas Leigh Bernard.xliACKNOWLEDGEMENTSI would like to thank my supervisor, Dr. Stephen Wood, for making thisthesis possible through his encouragement, advice and support. I would also like tothank the members of my supervisory committee, Dr. Ann Rose, Dr. Fred Dill, Dr.Robert McMaster and Dr. Paul Goodfellow, for helpful discussions and adviceduring the course of my research.I would like to thank the members of my lab, Mike Schertzer, CraigKreklywich, Heather Mitchell and Karen Henderson for advice and support, withspecial gratitude to Craig Kreklywich for assistance with sequencing and typing ofmicrosatellite repeats. I am also grateful for the technical advice provided by themembers of the Rose and Goodfellow labs, especially from Dr. Terry Starr andAngela Brooks-Wilson. I would also like to thank Dr. Fred Dill for assistance withmetaphase chromosome preparation and karyotyping.I wish to express my gratitude to my parents and my brother, Gord, for theirlove and their belief in my abilities. I would like to thank my husband, Bruce, for hislove, patience and interest in my research, and the members of his family for theirunfailing support. I would also like to thank my friends for their patience andencouragement, with special thanks to Naazlin Devji.This thesis was supported in part by a studentship from the MedicalResearch Council of Canada.xlii1. INTRODUCTIONA long-term goal in the field of human genetics is the development ofmethods for the localization and isolation of genes causing human disease. Thisgoal has been complicated by the large size of the human genome, 3X109base pairs(bp), the complexity of the human body, and by our long generation times withrelatively few offspring. To counter these difficulties, geneticists studying humanshave made various adaptations to conventional genetic methods. Since the humangenome is so large, it has been subdivided into more manageable pieces throughvarious cloning technologies. Variability at the DNA level has been utilized for thestudy 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 regionof human chromosome 5, qll.2-q13.3, and to provide information on the physicaland genetic location of these new markers. These new markers would then provideadditional tools for the investigation of disease loci which lie within this region.1.1 THE 5q11.2-q13.3 REGION OF THE HUMAN GENOMEA Vancouver family of Asian descent with a chromosomal rearrangementinvolving 5q11.2-q13.3 presented the first clue that genes within this region may playa role in the etiology of certain cases of schizophrenia, and may also be involved inkidney development (Bassett et al, 1988, McGillivray et a!, 1990). Schizophreniaand renal anomalies were observed in the two members of this family who aretrisomic for the 5q11.2-q13.3 region. An unaffected carrier family member has a11. Introductionbalanced direct insertion of chromosome 5 material into chromosome 1 (46, XX, mvins (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 wasoriginally supported by a linkage study involving 7 families of British and Icelandicdescent (Sherrington et al, 1988). This study demonstrated linkage between 5qmarkers and schizophrenia, with maximal lod scores for the disease locus betweenthe markers D5S39 and D5S76. A variety of modes of inheritance and criteria forschizophrenia were investigated, along with various levels of penetrance. Themaximal lod scores were obtained if individuals with schizophrenia, schizophreniaspectrum disorders and various fringe disorders were classified as affected, and thedisease was considered dominantly inherited with 86% penetrance. This studypublished by Sherrington et a! (1988) has thus far been the sole report of linkagebetween chromosome 5q markers and schizophrenia. Four studies demonstratingstrong evidence against linkage between this region of chromosome 5 andschizophrenia have been published (Kennedy et a!, 1988; St. Clair et a!, 1989;Detera-Wadleigh et a!, 1989; McGuffin et a!, 1990). Taken together, the publishedlinkage data seem to indicate that a major gene predisposing to schizophrenia doesnot lie within the 5q11.2-q13.3 region. However, the possibility still exists that asmall proportion of cases of familial schizophrenia involve gene(s) within 5q11.2-q13.3.The members of the Vancouver family who carried the segmental trisomy for5q11.2-q13.3 had renal abnormalities. The proband has an abnormal left kidney,while his maternal uncle lacks a left kidney. These abnormalities are consistent witha diagnosis of hereditary renal adysplasia, an autosomal dominant condition21. Introduction48, XV, der(1) mv ins (1;5)(q323;q13Sqll2)48, XX, mv ins(1:5)(q32.3;qlS.3q11.2)48, XVFigure 1. Family segregating for segmental trisomyD31. Introduction(MIM #191830; McKusick, 1990). Genetic linkage studies involving markers withinthe 5q11.2-q13.3 region and hereditary renal adysplasia have not yet beenperformed.The 5q11.2-q13.3 region became of additional interest due to reports oflinkage between markers within this region and childhood-onset proximal spinalmuscular atrophy (SMA; Brzustowicz et a!, 1990; Melki et at, 1990; Gilliam et a!,1990). Proximal SMA is characterized by the progressive degeneration of the lowermotor neurons in the spinal cord and brain stem motor nuclei, leading to paralysisof the limbs and trunk (Wessel, 1989). SMA is the second most common fatalrecessive disorder after cystic fibrosis (Pearn, 1980). SMA has been subdivided intothree types on the basis of the age of onset and severity of the disease: acuteWerdnig-Hoffmann disease (type I), intermediate Werdnig-Hoffmann disease (typeII) and Wohlfart-Kugelberg-Welander disease (type III). All three types areiuherited in an autosomal recessive fashion, and together account for over 90% ofchildhood SMA. Genetic evidence currently suggests that all three forms map tothe 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 TIYBRJDSCytologically visible chromosomal rearrangements often provide the firstclues for positioning human diseases. Confirmation of gene localization can then beperformed by the isolation of additional rearrangements involving the samechromosomal region or by genetic linkage studies. Detection of chromosomal41. Introductionrearrangements has served as a starting point for the localization of many diseasegenes, 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 withimmortalized rodent cells. These cell lines randomly lose human chromosomesuntil 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 ofselection. Somatic cell hybrids therefore allow the separation of the human genomeinto the naturally occurring division of the chromosome. The segregation ofchromosomes from an individual with a chromosomal rearrangement often affords amethod for further subdividing large amounts of DNA. Somatic cell hybridscontaining a single rearranged human chromosome can often be useful not only indelineating the extent of the rearrangement, but also for positioning DNA markers.This positioning can be confirmed using somatic cell hybrid mapping panels, whichconsist of a number of somatic cell hybrids containing a variety of chromosomalsegments.The balanced carrier in the family in which trisomy for 5q11.2-q13.3segregates with schizophrenia and renal anomalies carries a chromosome 5 deletedfor 5q11.2-q13.3 (Figure 1). This deleted chromosome was segregated into thesomatic cell hybrid HHW1O64 by Gilliam et a! (1989). Markers absent in theHHW1O64 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 the5q11.2-q13.3 region, while the markers D5S6, D5S39, D5S78, HEXB, DHFR,51. IntroductionD5S63 and D5S51 were assigned to within the deleted region.1.3 ISOLATION OF DNA MARKERSThe initial portion of my project involved the isolation of DNA fragmentsfrom within the 5q11.2-q13.3 region defined by the segmental trisomy. As indicatedpreviously, this region is of interest because it potentially contains genespredisposing to schizophrenia and hereditary renal adysplasia. The area is ofadditional 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 alsoserves to delineate the trisomic area.A variety of techniques had been devised for the isolation of human DNAfragments from chromosomal subregions prior to the development of polymerasechain reaction (PCR) techniques. Subtractive DNA cloning (Kunkel et a!., 1985;Nussbaum et a!., 1987) has been used for construction of recombinant librariescontaining regions of interest. This technique requires large amounts of startingDNA. Libraries have been constructed by the physical microdissection ofmetaphase chromosomes (Kaiser et a!., 1987), which involves complex DNAmanipulations. Libraries have also been made using DNA from somatic cell hybridscontaining 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.1.4PCRThe polymerase chain reaction (PCR) involves the exponential amplification61. Introductionof 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 diagrammaticrepresentation of this procedure is shown in Figure 2. The procedure involvesrepeated cycles consisting of: (1) denaturation of target DNA, (2) annealing of DNAprimers and (3) production of new DNA by extension from each primer. Athermostable DNA polymerase is used to enable the cycling to be carried outwithout the continual addition of polymerase (Saiki et a!., 1988). The first cycleconsists of a linear increase in the amount of target DNA, but each subsequent cycleresults in an exponential increase in the target sequence.1.5 AL U ELEMENTSA/u elements are human repetitive elements belonging to the SINE (shortinterspersed repeat) family of mammalian repetitive elements. A/u elements areapproximately 300 bp in length and consist of two directly repeating monomer units.A/u elements have a copy number between 7X105 and 9X105 in the human genome(Hwu et al., 1986). A/u elements have homology with the 7SL RNA, and it has beensuggested that A/u elements may represent defective 7SL RNA molecules that havebeen 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/usequence (Deininger et al., 1981; Kariya et a!., 1987). A/u-like elements have beendescribed in the genomes of other primates and in rodents. However, a high degreeof sequence divergence has occurred between the primate and rodent repeat units(Jelinek and Schmid, 1982).7Figure 2. Polymerase chain reaction (PCR)1. Introduction-Cycle 1J Cycle 2-1--1-Cycle 31-1--Exponential amplification of sequence between primers using the polymerase chainreaction. For simplicity, amplification is shown as starting from a single strand ofDNA. Primers are shown as arrows.81. Introduction1.6ALUPCRThe development of polymerase chain reactions using primers from humanspecific sequences such asAlu elements (Alu PCR) greatly facilitates the isolation ofhuman fragments from mixed DNA sources such as cell hybrids. Alu PCR involvesthe use of primers corresponding to the consensus sequence of the Alu family ofrepetitive elements in order to amplify the human DNA found between twoadjacent elements (Nelson et al., 1989, Brooks-Wilson et a!., 1990). Sufficientdivergence has occurred between human Alu elements and their rodent Alu-likehomologues to allow human-specific amplification between adjacent Alit elementsin 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 ofrecombinant libraries.Several different procedures have been reported involving the use ofAlitmediated PCR to isolate DNA fragments from specific chromosomal subregions.Regional localization of DNA fragments from the X chromosome was performed byAlu PCR amplification of YAC and phage isolates followed by hybridization toDNA from somatic cell hybrids (Nelson et a!., 1989). Fragments from chromosome10 were amplified byAlu PCR synthesis from a somatic cell hybrid and cloned(Brooks-Wilson et a!., 1990). Fragments from Xq28 have been isolated by detectionof differences visible on ethidium bromide stained gels between the PCR productsobtained from two related somatic cell hybrids using primers specific to Alit and tothe Li family of repetitive elements (Ledbetter et al., 1990). A fragment froml7pii.2 was isolated by hybridizing sub-fractions of the Alu PCR product from achromosome 17 hybrid to detect differences between the Alu PCR products from91. Introductionseveral chromosome 17 hybrids (Patel et a!., 1990).An additional procedure for the isolation of region-specific fragments, AluPCR differential hybridization, was developed as a part of this thesis (Bernard et a!.,1991a). Alu PCR differential hybridization is based upon the differentialhybridization of the Alu PCR products from two related somatic cell hybrids to achromosome specific phage library. A schematic representation of this technique isshown in Figure 3. Alu PCR differential hybridization was used in conjunction withtwo chromosome 5 hybrids, HHW1O64, the somatic cell hybrid which containschromosome 5 deleted for qll.2-q13.3, and GM1O114, which contains an intactchromosome 5 as the sole human component. A diagrammatic representation ofthe karyotype of the human chromosome present in each of these hybrids is shownin Figure 4. GM1O114 and HHW1O64 were used as substrates for anAlu PCRreaction. Differential hybridization of these Alu PCR products to a chromosome 5phage 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 PCRproduct from the chromosome 5 hybrid. Clones from within the deletion were thendetected by differential hybridization, since they hybridized to the Alu PCR productfrom the chromosome 5 hybrid but failed to hybridize to the Alu PCR product fromthe deletion chromosome 5 hybrid. This technique should be generally applicableto any somatic cell hybrid- deletion hybrid pair.101. Introductionh y b r i d zeAlu POR productfromintact chrom hybridh y b rid I zeMu POR productfromdeletion hybridFigure 3. Schematic representation ofAlu PCRdifferential hybridization procedurephage Hbraryhybridize Alu POR productfrom Intact chrom hybridpick positivespurify11Figure 4. Representation of human chromosomepresent in somatic cell hybrids HHW1O64 andGM1O1142223.123.223.331.131.231.33233.1. .,3435.135.235.313.3_13.2 I13.1111211.1_________11.213.3 I I2223.123.223.331.131.231.33233.133.233.33435.135.235.314GM1O11412HHW 1064Introduction15.315.215.115.315.215.11413.313.213.1121111.111.21213.113.213.314152114211. Introduction1.7 RADL4 TION HYBRID MAPPINGOrdering of human loci using radiation-induced breakage of humanchromosomes was introduced by Goss and Harris in 1975. This technique involvesthe production of human-rodent cell hybrids by the irradiation of humanlymphoblasts, 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 locusretention data. This technique was used to determine the order of four loci on thehuman X chromosome (Goss and Harris, 1975, 1977a). A modification of thismethod which removed the selective pressure for radiation hybrid growth was usedto 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 ofGoss and Harris (Cox et a!., 1990). Radiation hybrid mapping involves two majormodifications of the method of Goss and Harris. A somatic cell hybrid containing ahuman chromosome of interest is irradiated as a first step in radiation hybridmapping, rather than the human lymphoblasts used by Goss and Harris. Secondly,analysis of large quantities of marker retention data was made possible by thedevelopment of a series of algorithms (Cox et a!., 1990). The production of aradiation hybrid mapping panel involves the irradiation of a somatic cell hybrid linecontaining the human chromosome of interest followed by fusion of the irradiatedcell line with a rodent cell line. Fragments of the human chromosome present inthe 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 toanalyze 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 proportion131. Introductionof radiation hybrids. Markers further apart will be co-retained in a smaller fractionof radiation hybrids.Radiation hybrid mapping provides an alternate procedure for theproduction of long range maps of the human genome, and complements othermapping procedures such as meiotic linkage mapping, in situ hybridization and pulsefield gel electrophoresis (PFGE) (Stephens et a!., 1990). Radiation hybrid mappingalso affords several advantages when compared to alternate methods. In contrast tomeiotic linkage mapping, radiation mapping can be performed using non-polymorphic, moderately repetitive probes. Radiation mapping is not constrainedby restriction site placement, nor by meiotic cross-over frequencies. Radiationhybrid mapping was therefore felt to be the best method for rapidly determining theorder of the chromosome 5 clones obtained byAlu PCR differential hybridization.1.8 POLYMORPHISM SCREENINGThe majority of loci on the human linkage map are defined on the basis ofpolymorphisms detected within DNA, rather than the visible phenotypes most oftenfollowed in other organisms (Donis-Keller et a!., 1987). The more polymorphic asystem, 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 theiroffspring can be determined. The informativeness of a polymorphic system isreflected by the polymorphism information content, or PlC of the system (Botsteinet a!., 1980). The PlC value of a system is equivalent to the fraction of informativemeioses observed when a large number of unrelated individuals are scored. Theprobability that a given meiosis will be informative is therefore equal to the PlC for141. Introductionthe system typed. The algorithm for calculation of PlC is discussed in Appendix 1.The first polymorphisms detected within DNA were differences in restrictionenzyme digestion patterns commonly known as restriction fragment lengthpolymorphisms or RFLPs (Botstein et a!., 1980). This variation in restrictionfragment length can occur through the production or removal of a recognition sitefor a restriction enzyme, or through the insertion or deletion of DNA within arestriction fragment. Such DNA polymorphisms are common in human DNA, andcan be detected by hybridization with a DNA segment from within the variablerestriction fragment. While RFLPs are common in the human genome, they areunlikely to be highly polymorphic, since the production of more than two allelesrequires the presence of multiple events involving the same restriction fragment.Another type of DNA polymorphism has recently been detected whichinvolves variations in the number of simple sequence repeat units, such as (dCdA)n(dGdT)n ((GT)n; Weber and May, 1989). Simple sequence repeats areinterspersed throughout the genome, with a copy number of between 50,000 and100,000. Simple sequence repeats have been postulated to be enhancers of geneexpression (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 tohave no general function with respect to gene expression (Tautz and Renz, 1984;Levinson and Gutman, 1987). Slippage of DNA strands during replication, repair orrecombination is the most likely mechanism for expansion of simple sequenceelements throughout the genome (Tautz and Renz, 1984; Levinson and Gutman,1987). Variation of repeat number within an element is also postulated to occur151. Introductionthrough strand slippage (Weber, 1990).Amplification of a (GT) tract can be accomplished with PCR using primersimmediately flanking the tract (Weber and May, 1989). (GT) tracts with a repeatnumber (n) of between 11 and 15 are usually polymorphic, with the PlC increasingas the number of uninterrupted repeats increases (Weber, 1990). Tracts with 16 ormore repeats are invariably moderately to highly informative, with PlC values in the0.4 to 0.8 range (Weber, 1990). These sequences therefore serve as a valuablesource of highly informative DNA markers. These polymorphisms have the addedadvantage that they are PCR based, and can therefore be analyzed rapidly with aminimum amount of DNA. Multiple systems can be analyzed simultaneously if thePCR conditions are the same for all systems and the amplification products are non-overlapping in size. The majority of polymorphisms which were identified and typedduring the course of this thesis are of the (GT)n type.Alu elements are present in numerous copies in the human genome and alarge degree of sequence variation is present between elements (Kariya et al., 1987;Jurka and Smith, 1988). The dinucleotide CpG is a frequent site of polymorphicchange 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 todetect potential polymorphisms inAlu elements. The detection of polymorphismswithin Alu elements has been accomplished by amplification ofAlu elements usingunique flanking primers, followed by detection of differences by non-denaturingpolyacrylamide gel electrophoresis (Orita et a!., 1989, 1990) or conventionaldenaturing gels (Epstein et a!., 1990; Xu et a!., 1991). Polymorphisms involving the161. Introduction3’ polyA tail ofAlu elements have been detected by amplification of the polyA tailregion using a unique end-labeled primer and a primer specific to the 3’ end of anadjacent Alu element (Economou eta!., 1990; Zuliani and Hobbs, 1990). Thehypothesis that amplification of potentially polymorphic Alu polyA tails would bepossible using a unique primer flanking the polyA tail together with a primerspecific to the Alu consensus sequence was tested as a part of this thesis.1.9 LINKAGE MAPPINGGenetic crosses and backcrosses cannot, for obvious reasons, be performedby geneticists studying humans. Information on marker or disease locusrecombination rates and position must rely on available pedigrees. A variety ofmethods have therefore been developed to obtain the maximal amount ofinformation from observed marker segregations.Human linkage data are generally evaluated using a sequential probabilityratio, or likelihood test (Morton, 1955). The likelihood of obtaining an observedsegregation of two markers within a family is calculated for various values ofrecombination between the two markers. An odds ratio can then be calculated forthe various recombination fractions between the two markers. The numerator ineach odds ratio is the likelihood at each recombination fraction and thedenominator is the likelihood if the markers are unlinked. Since the number ofchildren in human families is generally quite small, it is usually difficult to obtain ameaningful odds ratio from a single family. However, since segregation withinunrelated families can be considered independent, the odds ratio at a givenrecombination fraction for several families is simply the product of the odds ratio at171. Introductionthat recombination fraction for each of the families. This method was first usedprior to the availability of calculators, and therefore calculations were simplified bytaking the logarithm of the odds ratio, or lod score (Morton, 1955). The lod scorefor several unrelated families typed for the same marker pair is therefore the sum ofthe lod scores obtained for each family. A lod score of 3, or odds of 1000:1 forlinkage, is generally considered sufficient evidence to state that two markers arelinked. A lod score of 2, or 100:1 odds for linkage, is considered to be suggestive oflinkage, and a lod score of -2, or 1:100 odds against linkage, is considered evidencethat 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 duPolymorphisme Humain (CEPH) was therefore organized to provide a set ofreference families (Dausset et al., 1990). CEPH families were selected on the basisof large sibship size and living parents and grandparents such that a maximalnumber of informative meioses could be obtained for each marker typed. CEPHprovides DNA from reference families to collaborating members and collectsgenotype data for markers typed.Lod scores for various values of recombination fractions between two locican readily be determined. However, lod score calculations become somewhattedious when large numbers of loci are compared in pairwise fashion andcalculations involving multiple loci are rather complex. A variety of computerprograms have therefore been developed to perform linkage calculations. Linkagecalculations in this thesis were performed using the computer program packageLINKAGE version 4.7 (Lathrop et a!., 1984, 1985; Lathrop and Lalouel, 1988).181. IntroductionTo accurately report recombination distances between loci, and to constructlinkage maps spanning several loci, a mapping function must be used to convert theobserved recombination frequency (e) to map distance, which is expressed in unitsof Morgans. One Morgan is the distance over which one cross-over event occurs per100 meioses (KIug and Cummings, 1983). The conversion from recombinationfraction to map distance is necessary for two major reasons. The first results fromthe fact that observable recombination events result from an odd number ofexchanges between loci, while an even number of events will be scored as absence ofrecombination. Secondly, cross-over events are not independent of one another, aphenomenon which is referred to as interference (Ott, 1985). The conversion tomap distance allows the production of linkage maps spanning multiple loci, sincemap distances are additive across contiguous segments.Various mapping functions have been proposed to convert recombinationfractions to map distances. The first mapping function was proposed by Haldane in1919, and was based on the assumption of no interference. This model thereforeassumes a random, independent distribution of chiasmata along a chromosome. Avariety of more complex mapping functions have since been derived to account forinterference. The Kosambi mapping function assumes that interference is directlyproportional to the recombination fraction between two loci (Kosambi, 1944).More complex estimates of interference have been incorporated into a variety ofmapping functions (Ott, 1985), however these functions are generally rathercumbersome and are not often used. Either the Haldane or the Kosambi mappingfunction is therefore commonly used for the analysis of human data. Derivations ofthe Haldane and Kosambi mapping functions are discussed in Appendix 1.191. IntroductionThe use of reference families such as the CEPH panel, together withcomputer program packages such as LINKAGE allow the placement of new geneticmarkers on linkage maps. If the markers are typed in disease families, informationcan 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 diseasegene and flanking markers. If physical localization information is available for theflanking markers, a general estimate can be made as to the physical distance adisease gene can be positioned within. Suitable steps can then be taken towards theisolation of the disease gene. The probability of positioning a disease gene on thelinkage map increases as the density of markers on the linkage map increases. Theuse of highly informative markers such as (GT) microsatellites also increases theprobability of detecting linkage. Linkage markers were developed during the courseof this thesis to increase the density of the linkage map of human chromosome 5 andto provide highly informative PCR based polymorphic systems.The research described in this thesis was designed to investigate a region ofhuman chromosome 5, qll.2-q13.3, defined by a segmental trisomy. This region wasof interest due to co-segregation of the 5q11.2-q13.3 trisomy with schizophrenia andrenal 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 oflinkage between markers within this region and chronic spinal muscular atrophy(SMA; Brzustowicz et a!., 1990; Melki et a!., 1990; Gilliam et a!., 1990). The initialobjective of this project was to isolate DNA fragments from within the 5q11.2-q13.3region. The next objective was to obtain order information on these DNAfragments. Polymorphism screening would then be carried out on clones of interest.201. IntroductionLinkage data obtained from the polymorphism screening would serve to position themarkers on the chromosome 5 linkage map. The linkage position of the newmarkers would then be used to delineate the breakpoints of the segmental trisomyand provide tools for the investigation of disease loci which lie near the newmarkers.212. MATERIALS AND METhODS2.1 MATERIALS2.1.1 Somatic cell hybridsThe somatic cell hybrid GM1O114 contains an intact human chromosome 5as the sole human component in a Chinese hamster background, and was obtainedfrom the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). Thehybrid HHW1O64 contains a human chromosome 5 with an interstitial deletion of5q11.2-q13.3 as the only human material in a Chinese hamster background (Gilliamet al., 1989). The derivative chromosome 5 in HHW1O64 was derived from abalanced carrier member of a family in which trisomy for the 5q11.2-q13.3 regioncosegregates with schizophrenia and renal anomalies (Bassett et a!., 1988;McGillivray et a!., 1990). The hybrid HHW213 contains a derivative chromosome 5deleted for approximately 95% of the q arm (Overhauser et a!., 1986a). The onlydetectable human DNA in HHW213 is an intact 5p, the centromere, and part ofband 5q11 (Overhauser et a!., 1986a). The human chromosome 5 is retained in thesomatic cell hybrids by complementation of the temperature sensitive rodent leucyltRNA synthetase gene (leuS) (Dana and Wasmuth, 1982).DNA samples from a chromosome 5 radiation hybrid mapping panelconsisting of 150 radiation hybrids were obtained from Dr. Ellen Solomon (ICRF,London, England). This panel was produced by irradiation of the somatic cellhybrid PN/TS-1, which contains an intact chromosome 5 as the only humancomponent in a Chinese hamster background, with approximately 50,000 rads of X222. Materials and Methodsrays. The radiation hybrids were produced by the fusion of the irradiated PN/TS-1cells with A23 cells, a Tk Chinese hamster ovary cell line.2.1.2 Phage librariesLAO5NSO1 is a flow sorted, chromosome 5, complete EcoRI-digest phagelibrary 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 SciencesDivision, Los Alamos National Laboratory (Los Alamos, NM) under the auspices ofthe National Laboratory Gene Library Project, which is sponsored by the U.S.Department of Energy.232. Materials and Methods2.2 METHODS2.2.1 Restriction enzyme digestionsRestriction enzyme digestions were carried out using 50 ng to 4 ug DNA, 1Xrestriction enzyme buffer, 0.1 mg/mi Bovine serum albumin (BSA), and ito 2units/ag restriction enzyme. Digests were performed by incubation of samples for 1hour to overnight at the temperature recommended for the restriction enzyme.Reactions were stopped by the addition of 1/4 volume stop buffer or by incubationat 65°C for 10 minutes.Restriction enzyme buffers (lOX)NaC1 Tris-HC1 MgC1 DTr KC1low- 100mM 100mM 100mMmedium 500 mM 100 mM 100 mM 100 mMhigh 1 M 500 mM 100 mM 100 mM -react4(BRL)- 200 mM 50 mM- 500 mMreactl0(BRL) 1.5 M 1 M 100 mMBuffers were filter sterilized by passing through 0.2 m filters.Stop buffer0.25% bromophenol blue0.25% xylene cyanol40% w/v sucrose in dH2O60 mM EDTA242. Materials and Methods2.2.2 Gel electrophoresisDNA samples were size fractionated by loading onto agarose gels andsubjecting the gels to a constant voltage. To produce a gel, agarose was dissolved inlx TBE buffer by boiling, then allowed to cool to approximately 40 C before beingpoured into a mold and allowed to solidify. Ethidium bromide was added to the gelat a concentration of 1 pg/mi to allow the visualization of DNA using UVillumination. Alu PCR products or restriction digested genomic DNA samples wererun on 0.8% agarose gels overnight at 30 V. Plasmid digests were run on 0.7% to1.0% 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 foragarose gels. ØX-174-RF DNA digested with HaeIII (Pharmacia) was used as a sizestandard for Nusieve gels. DNA samples were photographed after electrophoresisusing a 302 nm transilluminator.lox TBE buffer54 g Tris base27.5 g boric acid20 ml 0.5 M EDTA (pH 8.0)dH2O to 112.2.3 Southern blottingRestriction digested DNA or PCR products were transferred from agarosegels to nylon membranes using the .method of uthern (Southern, 1975). A252. Materials and MethodsPolaroid or negative was taken of the gel with a ruler present to determine thedistance of migration. Excess agarose was then trimmed away, and the gel wassoaked in 0.25 M HC1 for 15 minutes, 0.5 M NaOH/1.5 M NaC1 for 30 minutes and1 M Tris/l.5 M NaC1 for 30 minutes. The gel was washed with distilled water(dH2O) between each solution.The denatured DNA was then transferred from the agarose gel to a nylonmembrane. Either Genescreen (New England Nuclear) or Hybond (Amersham)was used for plasmid blots and Nytran (Schleicher & Schuell) or Hybond was usedfor blotting genomic DNA or PCR products. Unidirectional blots were made asfollows. Two pieces of identically sized Whatman 3 mm filter paper were placedinto a dish containing lox SSC. A glass plate was then put across the dish, and thefilter paper was folded over the glass plate such that both ends of the paperremained in the solution to create a wick. The treated agarose gel was then placedon top of the wet filter paper. A prewetted piece of membrane cut to the shape ofthe gel was placed on top of the gel. Two pieces of filter paper cut to the size of thegel were then placed on top of the membrane. Any excess area of wick uncoveredby the gel was covered with Saran wrap. A stack of paper towels approximately 20cm high was then placed on top of the gel. This transfer was done for 2 hours toovernight.Bidirectional blots were made by sandwiching the treated gel between twomembranes in an analogous fashion to the unidirectional blotting. Four pieces offilter paper and two pieces of membrane were cut to fit the gel, and pre-wetted. Apiece of Saran wrap was placed on the bench and 6 to 7 paper towels were placed ontop of the Saran wrap. Two pieces of filter paper, then a piece of membrane, then262. Materials and Methodsthe treated gel, then another piece of membrane, then two additional pieces of filterpaper were placed onto the paper towels. All of the paper towel unoccupied by gelwas covered with Saran wrap and a stack of paper towels was placed on top. A glassplate and a weight were placed on top of the paper towels. The transfer wasallowed to proceed for 2 hours to overnight.After transfer was completed, membranes were rinsed with dH2O to removeany agarose, placed between two pieces of filter paper, and baked for 1 1/2 to 2hours at 80°C.2.2.4 OligolabelingPCR products or phage DNA were labeled witha32P-dATP using therandom primer method (Feinberg and Vogelstein, 1984a,b). DNA samples werediluted to approximately 1 ng/il and boiled for 10 minutes, then placed on ice. Astandard labeling reaction comprised 30 j.Ll (30 ng) boiled DNA, 10 l OLB-A, 5jil 1 mg/mi BSA, 1 unit Kienow and 50 /.LCia32P-dATP. Reactions were alsoperformed using (1/2)X and 2X the standard reaction. Samples were held on iceuntil the addition of thea32P-dATP, and then incubated at room temperatureovernight.The labeling reaction was stopped by the addition of 1 volume NTSB, andthe unincorporated nucleotides removed by passing the reaction mixture through aSephadex G-25 spin column. Spin columns were made by placing a 1 ml pipette tipinto a collar formed by cutting the bottom and lid off a 1.5 ml eppendorf tube, thenplacing the collar and tip into a 12 X 75 mm culture tube. The bottom of the tip wasplugged with siliconized glass wool, and G-25 Sephadex (equilibrated in 1/5 TE)272. Materials and Methodswas added until the tip was full for a full reaction or until half full for a halfreaction. 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 columnspun again. The column was then washed with 2 volumes of dH2O and spun again.Reactions which had been doubled were divided in half and passed through 2 spincolumns. Final volumes of reactions were 200 l for full reactions, 100 l for halfreactions and 400 ul for doubled reactions.OLB-ASolutions A:B:C mixed in ratio of 100:250:150.Solution A Solution B1 ml 1.25 M Tris (pH 8); 0.125 M MgC1 2 M Hepes (pH 6.6)18 j.L1 2-mercaptoethanol5 l 100 mM dTTP (Pharmacia) Solution C5 jil 100 mM dGTP (“ “) Hexadeoxyribonucleotides5 ul 100 mM dCTP (“ “) (Pharmacia) suspended in TE at90 OD/ml.NTSB 1XTE20 mM EDTA 10 mM Tris (pH 8.0)2 mg/mi salmon sperm DNA 1 mM EDTA0.2% SDS282. Materials and Methods2.2.5 Preannealing with human DNARepetitive probes were prearmealed with a vast excess of shearednonradioactive human DNA (Litt and White, 1985). Typical prearinealingconditions consisted of 30 ng probe DNA and 200 j.g sheared nonradioactivehuman DNA in a total volume of 270 l and a final buffer concentration of 2XSSC. Samples were boiled for 5 minutes, and then preannealed at 65°C for 15minutes to 1 hour.20X SSC175.3 g NaC188.2 g sodium citratedH2O to 112.2.6 Prehybridization, hybridization and washingSouthern blots, plaque lifts or colony lifts were placed in heat-sealable bagsand prehybridized with hybridization solution at 65°C for 1 to 4 hours to block nonspecific probe binding. Prior to hybridization, probes were denatured by boiling andthen quick chilled on ice. Repetitive probes were preannealed with an excess ofnonradioactive human DNA (section 2.2.5). The probe was then added to thehybridization solution, and hybridization carried out overnight at 65°C.Hybridization and wash solutions were pre-warmed to 65°C before use. Filters werewashed twice for 15 minutes in lx SSC, 0.1% SDS and then twice for 30 minutes in0.2X 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. After292. Materials and Methodswashing, filters were dried briefly, then re-sealed in bags and exposed to Kodak XRP or X-AR film with DuPont Lightning Plus intensifying screens at -70°C or atroom temperature for varying time periods.Hybridization signal was stripped from blots by rinsing in 0.4 N NaOH at43°C for 30 minutes, followed by neutralization for 30 minutes at 43°C in 0.2 M TrispH 7.5, 0.2X SSC, 0.2% SDS. For the majority of blots, this treatment removed allsignals. Blots were then dried and re-used for hybridization. Blots were strippedand re-hybridized a maximum of 10 times.Hybridization solution6X SSC0.3% (w/v) SDS5X Denharts100 g/l salmon sperm DNA2.2.7 (GT) tract detection and isolationPoly(dA-dC)(dG-dT) (Pharmacia) was labeled using nick translation (Rigbyet a!., 1977) witha32P dATP and a BRL nick translation kit. Labeling conditionswere as specified by BRL. (GT)n tracts were detected by hybridization to thisprobe. Colony filters or blots were hybridized with nick translated probe overnightat 55°C, using hybridization solution lacking any competitor DNA. Blots werewashed for 1 hour at 65°C in 1X SSC, 0.1% SDS. Filters or blots were exposed toKodak RP film for 2 hours to overnight using DuPont Lightning Plus intensifyingscreens at room temperature.302. Materials and MethodsHybridization solution used for (GT)11 tract detection6X SSC0.3% SDS5X Denharts2.2.8 Phage manipulations2.2.8.1 Phage platingE.coli strain LE392 (Sambrook et aL, 1989) was used as host cells for phagegrowth. A 5 ml L-broth culture containing 0.2% maltose was inoculated using asingle colony from a freshly streaked plate of LE392 cells. The culture was grownovernight at 37°C, then the cells sedimented by spinning at 2500 rpm for 5 minutesin a clinical centrifuge. The bacterial pellet was resuspended in 1/2 volume 10 mMMgCl. For phage plating, 100 l of host cells were combined with up to 10 phageand incubated at room temperature for 15 minutes to allow the phage to infect thebacteria. 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°Covernight to allow plaques to develop. Confluently lysed plates (all bacteria onplate lysed) were produced using approximately io phage per plate.312. Materials and MethodsL-broth NZY broth5g yeast extract lOg NZaminelOg tryptone 5g yeast extract5g NaC1 5g NaC1lg D-glucose 2g MgCllldH2O lldH2O3001.Ll iON NaOH (pH to 7.2-7.4) 3001il iON NaOH (pH to 7.2-7.4)Autoclave 20 minutes at l5lbs pressure Autoclave 20 minutes at l5lbspressureTop agaroseAdd 7 g/l agarose to broth prior to autoclavingPlatesAdd 12 g/l agar to broth prior to autoclaving2.2.8.2 Plaque lifts and plaque purificationPlaque lifts (Benton and Davis, 1977) were made using the followingprocedure. 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 becomecompletely wet. The filter and plate was marked three times by stabbing with theneedle of a syringe containing India ink. The filter was removed from the plate andplaced DNA side up on top of Whatman filter paper soaked with the followingsolutions:322. Materials and Methods1) 1.5M NaC1, 0.5M NaOH; incubate for 4 minutes2) 1.5M NaC1, 1M Tris pH8; incubate for 4 minutes3) 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 byplacing 2 filters simultaneously on top of a plate.Phage were screened by hybridization of probes to plaque lifts. Plaqueswhich were positive were picked with a toothpick, diluted with ) diluent, replatedand re-screened. Three successive screens were carried out to obtain purifiedphage.), diluent10 mM Tris, pH 7.510 mM MgCl2.2.8.3 Small scale phage prepA confluently lysed NZY agarose plate was overlaid with 5 ml ice cold SMsolution and left at 4°C overnight. The overlay was collected, a few drops of CHC13added, and the tube spun at 2500 rpm for 5 minutes in a clinical centrifuge. Thesupernatant was transferred to a fresh tube and a few more drops of CHC13 added.The supernatant was then stored at 4°C until the phage prep was performed. Smallamounts 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, andincubated at room temperature for 10 minutes. 100 l of 2 M Tris; 0.2 M EDTA332. Materials and Methodswas then added, the tube mixed and incubated at 70°C for 10 minutes. 100 ul of5M CH3OOK, pH4.8 was added and the tube cooled in an ice-water bath for 30minutes. The tube was spun for 10 minutes in a microcentrifuge and thesupernatant decanted. Two phenol extractions and one Sevags extraction wereperformed. Two volumes of EtOH were added to the aqueous fraction and the tubespun for 5 minutes in a microcentrifuge. The supernatant was discarded, and thepellet rinsed in 70% EtOH and spun again. The pellet was resuspended in 50 j.LlTE containing 0.1 g/jil RNase and incubated at 37°C for 15 minutes to degradethe RNA.SM solution Sevags100 mM NaC1 24 volumes CHC138 mM MgSO4 1 volume isoamyl alcohol50 mM Tris (pH 7.5)0.01% (v/v) gelatin2.2.8.4 Large scale phage prepTwenty confluent plates were made for each phage stock from which largequantities of DNA were required. The plates were cooled at 4°C for at least onehour and then overlaid with Sml/plate ice cold Adiluent. Plates were left at 4°Covernight. The overlay was collected into large centrifuge tubes, a few drops ofCHC13 added and the tubes centrifuged at 10,000 rpm for 10 minutes in a Sorvallcentrifuge. One to two mis of the clear supernatant was saved for titering and theremainder transferred to clean centrifuge tubes. Large amounts of phage DNA342. Materials and Methodswere prepared using the following procedure (Davis et al., 1980). Solid NaC1 wasadded 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 thenadded to a final concentration of 10% w/v and dissolved by slow stirring. Thesolution 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 at4°C in a Sorval centrifuge. The supernatant was discarded and excess supernatantremoved by standing the centrifuge bottles upside down for 5 to 10 minutes. Anyresidual supernatant was then removed by gently wiping the sides of the bottles withKimwipes. The pellet was then gently resuspended in 9 ml )diluent using a widebore pipette. Residual bacterial debris was removed by centrifuging at 2500 rpm for10 minutes. The supernatant at this stage typically exhibited a bluish tinge due tothe suspended phage particles. 0.75 g/ml solid CsCl was added to the suspendedphage particles and mixed gently to dissolve. The phage suspension was transferredto ultracentrifuge tubes and heat sealed. The tubes were spun in the ultracentrifugeat 60,000 rpm for 16 to 20 hours at 20°C. After centrifugation, the phage particlescould be visualized as a blue-white band. This band was collected by removing thetop of the ultracentrifuge tube to allow air into the tube, and then puncturing theside 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 (Daviset 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 andthe mixture incubated for 30 minutes to several hours at room temperature. One352. Materials and Methodsvolume ofH20then 6 volumes of 95% EtOH were added. The DNA wasprecipitated by centrifugation for 1 minute in a microcentrifuge. The supernatantwas discarded and the DNA pellet resuspended in 1X TE.2.2.9 Plasmid manipulations2.2.9.1 LigationDNA fragments were subcloned into Bluescriptil by ligation. Vector andinsert DNA were restriction digested to produce complementary sticky or bluntends. Digests were stopped by heating at 65°C for 10 minutes. Ligations wereperformed by mixing vector and insert DNA in a 1:1 to 1:4 ratio in 1X ligationbuffer and 1 unit T4 DNA ligase in a reaction volume of 10 to 20 l. 50 to 100 ngof plasmid DNA were typically used for a ligation reaction. Samples were ligatedovernight at 14°C.lox ligation buffer500 mM Tris100 mM MgC110 mM ATP100 mM DTT1 mg/mi BSA362. Materials and Methods2.2.9.2 Production of competent cells and transformationDH5a cells (Sambrook et al., 1989) were made competent using thefollowing procedure (Hanahan, 1983). A culture tube containing 5 mIs of L-brothwas inoculated with a DH5a colony from a freshly streaked plate and grownovernight. 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 0D600 wasapproximately 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 10minutes at 4°C in a Sorval centrifuge. The supernatant was removed, and the pelletwashed with 150 ml pre-cooled 10 mM MgC1;10 mM MgSO4. The cells were thenspun at 2500 rev/mm for 10 minutes at 4°C in a Sorval centrifuge. The supernatantwas removed and the pellet resuspended in 100 ml pre-cooled FSB solution. Thecells were placed on ice for 15 minutes, then spun for 10 minutes at 2500 rev/mm at4°C in a Sorval centrifuge. The supernatant was removed, and the pelletresuspended in 24 ml of FSB solution. 0.84 ml of dimethylsulphoxide (DMSO) wasadded, the cells were mixed gently and incubated on ice for 10 minutes. Anadditional 0.84 ml DMSO was added, the cells mixed gently and incubated on ice for10 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 tocolonies/Lg supercoiled Bluescriptil.Ligated plasmids were transformed into competent DH5a cells using thefollowing procedure. Competent cells were thawed on ice, then 50 ul of cellsaliquoted into a chilled 17 X 100 mm polypropylene tube. 1 to 100 ng DNA wasadded to the cells and incubated for 30 minutes on ice. L-broth was added to a total372. Materials and Methodsvolume of 100 l and the cells heat-shocked at 42°C for 45 seconds. The cells wereplaced on ice for 2 minutes, then 0.1 to 1 ml L-broth was added and the cellsincubated at 37°C for one hour. Appropriate amounts of cells were plated onto XJAplates.SO media20 g bactotryptone5 g yeast extract10 mM NaCl2.5 mM KC110 mM MgCl10 mM MgSO4dH2O to 11 total volumeAutoclave for 20 minutes at 15 lbs pressureFSB Solution10 mM CH3OOK100 mM RbC145 mM MnCl210 mM CaCl23 mM hexamine cobalt chloride10% v/v glycerolAdjust pH to 6.4 usingconcentrated HC1. Filter sterilizeinto an autoclaved bottle.XJA platesPlates were made with L-broth media plus 12 g/l agar added before autoclaving.The following media supplements were added after autoclaving and cooling mediato approximately 50°C: 50 g/ml ampicillin, 40 ag/mi X-Gal (5-bromo-4-chloro-3-indolyl-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 ligationindependent cloning (LIC; Aslanidis and de Jong, 1990). This procedure generates382. Materials and Methodsrecombinants without using restriction enzymes or T4 DNA ligase. A PCR reactionis performed on both the vector and insert, followed by digestion with T4 DNApolymerase to produce long overhanging complementary tails. Vector and insertare then combined and transformed into bacteria. The PCR products generated byAlu-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 thePCR products using long wavelength UV illumination. The DNA was thenseparated from the agarose by spinning the agarose plug through glass wool for 1minute in a microcentrifuge. Single stranded tails were generated by treating thevector and insert DNA with 2 units T4 DNA polymerase in 1X T4 DNA polymerasebuffer with 0.5 mM dGTP present for the Alu-LIC PCR product and 0.5 mM dCTPpresent for the vector. Samples were incubated at 37°C for 20 minutes and thenheated to inactivate the enzyme for 10 minutes at 65°C. The T4 polymerase treatedproduct was purified by a phenol/Sevags extraction. DNA samples were thenprecipitated by the addition of 1/2 volume 7.5 M NH4OAc and 2 volumes 95%EtOH followed by centrifugation for 10 minutes at 4°C in a microcentrifuge. Thepellet was washed with 70% EtOH, reprecipitated by centrifugation in amicrocentrifuge for 5 minutes at 4°C, dried for 5 minutes in a vacuum desiccator andresuspended in TE. Vector and insert were mixed at a 1:3 ratio in 1X ligation bufferand incubated for 1 hour at room temperature. Transformations were carried out asusual.392. Materials and Methods5X T4 DNA polymerase buffer165 mM Tris pH 8330 mM CH3OOK50 mM MgCl2.5 mM DTT0.5 mg/mI BSA2.2.9.4 Colony screensColony screens for plasmids (Grunstein and Hogness, 1975) were made asfollows. A nitrocellulose filter was labeled, placed on top of a XIA plate andallowed to sit until the filter was thoroughly wet. A XIA plate for stock storage waslabeled in the same fashion as the nitrocellulose filter. Colonies were streakedusing a toothpick onto the nitrocellulose filter and the stock plate. Both plates wereincubated overnight at 37°C to allow growth of colonies. The stock plate was storedat 4°C. The nitrocellulose filter was removed from the MA plate and placed colonyside up on top of Whatman filter paper soaked with the following solutions:1)1.5 M NaC1, 0.5 M NaOH; incubate for 4 minutes2)1.5 M NaCl, 1 M Tris pH 8; incubate for 4 minutes3) 2X SSC; rinse (approximately 1 minute)Filters were then air dried and baked under vacuum for 1 1/2 to 2 hours at80°C. Colonies were screened by hybridization of probes to filters.402. Materials and Methods2.2.9.5 Plasmid minzrepSmall amounts of plasmid DNA were generated using an alkaline lysisminiprep procedure (Birnboin and Doly, 1979; Ish-Horowicz and Burke, 1981). 5mis of L-broth was innoculated with the colony of interest and then grown overnightat 37°C in the presence of 50 g/ml ampicillin. 0.5 ml of culture was added to 0.5ml of glycerol and saved as a stock. The remainder of the culture was centrifuged at2500 rpm for 10 minutes in a clinical centrifuge. The supernatant was removed, thepellet resuspended in 100 J.Ll solution I, transferred to an eppendorf tube andincubated at room temperature for 5 minutes. 200 jil of freshly prepared SolutionII was added, the tube inverted several times to mix the contents and then incubatedon ice for 5 minutes. 150 l Solution III was added, the tube inverted several timesto mix and then incubated on ice for 5 minutes. One volume of buffer-saturatedphenol was added and mixed and the tube was spun for 15 minutes in amicrocentrifuge. 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 amicrocentrifuge. The aqueous layer was removed and transferred to a fresh tube.1/10 volume of 7.5 M NH4OAc and 2 volumes of 95% EtOH were added and theDNA precipitated by centrifugation for 10 minutes at 4°C in a microcentrifuge. Thepellet was washed in 70% EtOH, then spun for 5 minutes at 4°C in amicrocentrifuge. The pellet was air dried for 10 to 15 minutes, then dessicated for 5minutes. The pellet was resuspended in 40 to 100 ul TE containing 0.1 g/lRNase. Samples were then incubated at 37°C for 15 to 30 minutes to remove RNA.Samples were stored at -20°C.412. Materials and MethodsAlkaline lysis solution I Alkaline lysis solution H50 mM glucose 0.2 N NaOH10 mM EDTA 1%SDS25 mM Tris pH 8.04 mg/ml lysozyme (Add powderedlysozyme to solution just prior to use)Alkaline lysis solution III60 ml 5M CH3OOK11.5 ml glacial acetic acid28.5 ml dH2O2.2.9.6 Plasmid large scale prepTo produce large quantities of plasmid DNA, a large scale alkaline lysisplasmid 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 scaleplasmid prep. 500 ml L-broth containing 50 jg/ml ampicillin was inoculated with acolony containing a plasmid of interest. The culture was grown at 37°C forapproximately 5 hours until the 0D600 was between 0.4 and 0.5, then 1 ml of 80mg/ml chloramphenicol was added and the culture incubated at 37°C for 12 to 16hours. The culture was centrifuged at 4000 rpm for 10 minutes at 4°C in a Sorvalcentrifuge, the supematant discarded and the pellet resuspended in 35 ml STE. Thesolution was transferred to smaller centrifuge tubes and re-centrifuged at 4000 rpmfor 10 minutes at 4°C in a Sorval centrifuge. The pellet was resuspended in 10 ml422. Materials and Methodsice cold Solution I, then incubated at room temperature for 5 minutes. 20 ml offreshly prepared Solution II was added, the contents mixed by inverting rapidlyseveral times and the tube stored on ice for 10 minutes. 15 nil of ice cold SolutionIII was then added, the tube inverted several times to mix and stored on ice for 10minutes. The bacterial debris was removed by centrifugation at 15,000 rpm for 25minutes at 4°C in a Sorval centrifuge. The supernatant was transferred into 2centifuge tubes, and 0.6 volumes of room temperature isopropanol was added toeach tube. The tubes were mixed gently and placed at room temperature for 15minutes. The DNA was precipitated by centrifugation for 10 minutes at 2500 rpm atroom temperature in a clinical centrifuge. The supernatant was discarded and thepellet air dried. The pellet was dissolved in 10 mi TE (pH 8.0) and 1 g/mi CsC1 and0.8 ml 10 mg/mi ethidium bromide added. The sample was placed in the dark for15 minutes, then transferred to a 16X76 mm polyallomer ultracentrifuge tube andheat sealed. The sample was then centrifuged at 60,000 rpm for 18 to 20 hours at20°C in an ultracentrifuge. The plasmid DNA band was visualized with a longwavelength UV transilluminator and extracted using a syringe. Ethidium bromidewas removed from the plasmid DNA by several extractions with butanol saturatedwith dH2O. The DNA was precipitated by adding 2 volumes of dH2O and 6volumes of EtOH, placing the sample at -20°C for 1 hour to several days andcentrifuging 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 2500rpm 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.432. Materials and MethodsSTE100 mM NaC110 mM Tris, pH 8.01 mM EDTA, pH 8.02.2.10 SequencingSequencing was performed on alkaline lysis miniprep DNA or M13mp18single stranded DNA by the dideoxy chain termination method (Sanger et al., 1977)usinga35S dATP and a Sequenase sequencing kit (U.S. Biochemical Corp.). Theprotocol used was a modification of standard techniques to allow sequencingthrough regions of high secondary structure.2.2.10.1 Denaturation of templatePlasmid miniprep DNA was denatured and precipitated prior to sequencing inthe following fashion. 18 j.Ll (5 to 10 J.Lg) of plasmid miniprep DNA was denaturedby the addition of 2 jil of 2 N NaOH; 2 mM EDTA and incubation at roomtemperature for 5 minutes. The sample was then neutralized by the addition of 2l of 2 M NH4OAc. 55 jil ice cold 95% EtOH was added and the sample placed at-70°C for 5 minutes. The denatured DNA was then precipitated by centrifugation ina microcentrifuge for 15 minutes. The supernatant was removed and the pelletwashed with 200 1 ice cold 70% EtOH. The sample was spun for 5 minutes in amicrocentrifuge and the pellet dried for 5 minutes in a desiccator. The pellet wasleft dry for up to 2 hours at -20°C before the sequencing reaction. The pellet wasresuspended in 6 to 6.5 jil dH2O immediately preceding the sequencing reaction.442. Materials and Methods2.2.10.2 Sequencing reactionsSequencing reactions were carried out on 5 to 10 ,.g denatured plasmidminiprep DNA or 2 g single stranded M13mp19 DNA. Plasmid or M13 DNA wasmixed with 5 to 10 pmoles sequencing primer in a total volume of 7 jLl. 1 ,ul ofdimethyl 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 andspun for 1 second in a microcentrifuge. 2 l of sequencing buffer was then addedand the sample incubated at room temperature for 5 minutes. 6.3 l of labelingmix 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 terminationmixes pre-warmed to 37°C. The termination tubes were then incubated at 37°C for3 to 5 minutes. The reactions were stopped by the addition of 4 l sequencing stopmix. Samples were stored at -20°C.Labeling mix1.025 jillOOmMDTT2.05 l 0.75 M dGTP; 0.75 jM dCTP; 0.75 M dTTP1 j1a35SdATP0.525 jil DMSO1.8 l 10 mM Tris pH 7.5; 5 mM DTT’; 0.5 mg/mi BSA0.25 j.Ll Sequenase- add just before ready to begin452. Materials and MethodsTermination mixtures Sequencing stop mix80 jM dGTP 95% formamide80 j.MdCTP 20 mM EDTA80 M dATP 0.05% Bromophenol Blue80 jtM dTl’P 0.05% Xylene Cyanol FF50 mM NaC18 M of one of ddGTP,ddCTP, ddTFP or ddATP2.2.10.3 SequencingprimersUniversal primers used for sequencing plasmids were T3 and T7 primers.M13mp19 was sequenced using the universal -40 primer. Universal sequencingprimers 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 theconsensus sequence of the Alu family of repetitive elements, and with uniqueprimers flanking (GT)n tracts orAlu 3’ tails. The sequences of these primers arelisted in section 2.2.11.6 (Specific PCR reactions and primer sequences).2.2.10.4 Denaturingpolyaciylamide gel electrophoresisSequencing reactions were subjected to denaturing gel electrophoresis usinga 6% Hydrolink (AT Biochemicals) sequencing gel. Sequencing gel systems were462. Materials and Methodsobtained from BioRad. One sequencing system consists of two 21 cm X 50 cmplates, one of which contains a buffer container, two 0.4 mm spacers, a 0.4 mmcomb, two clamps, a casting tray, a base, a stabilizer bar and electrical leads.6% Hvdrolink sequencing gel25.2 g urea3.6 ml lOX TBE6 ml 50% Hydrolirik Long Ranger solution30 ml dH2OThe gel ingredients were added to a flask in the order given and the urea wasdissolved by placing the flask in warm water. Sequencing plates were cleaned,spacers were sandwiched between the plates at either side and the plates clampedtogether on the sides. The bottom of the gel was sealed by placing 13 ml gelsolution, 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 intothe solution for approximately 1 minute such that the solution moved between theplates by capilliary action. The gel was then left for 2 minutes to ensure that thebottom seal was completely polymerized. To pour the gel, 26 j.Ll of TEMED and100 l of 25% APS was added to the remaining gel solution, and the solution waspipetted between the sequencing plates using a 25 ml pipette. The top of the gelwas then covered with the flat side of a sequencing comb, and the gel allowed topolymerize for 1 hour. The base was filled with 350 to 500 ml of 0.6X TBE and thegel 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 that472. Materials and Methodsthe teeth were placed slightly into the gel, producing wells for sample loading. Thegel was pre-run at 55 W until the gel temperature reached 50°C (approximately 1hour). 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. Sincesequencing proceeds in aS’ to 3’ direction, the sequence closer to the 3’ end can beobtained by increasing the run time. A second loading of certain samples wastherefore used to obtain sequence spanning a longer region. Up to 250 bp ofsequence could be obtained from a single load and up to 400 bp from a double load.22.1 0.5 Maniulation ofsequence dataSequence information was stored and manipulated using the computerprogram ESEE sequence editor (E. Cabot, Access Biosystems Inc., Burnaby, B.C.).482. Materials and Methods2.2.11 Polymerase chain reaction (PCR)PCR reactions (Saiki et at., 1985; Mullis et at., 1986; Mullis and Faloona, 1987) usingTaq 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 conditions10 ng plasmid DNA, 40 ng human lymphoblast DNA or 40 ng somatic cell hybridDNA50 mM Tris pH 8.00.05% Tween 200.05% NP4O2 mM MgCl0.2 mM each of dATP, dCTP, dGTP and dTT’Pprimer(s)- see below0.625 units of Taq DNA polymerasedH2O to a total reaction volume of 25 j.125 l mineral oil overlay2.2.11.2 Standard cycling conditionsThirty 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, thetubes were held at 72°C for 10 minutes and then stored at 4°C until removed fromthe thermal cycler. A Perkin-Elmer Cetus thermal cycler was used.492. Materials and Methods2.2.11.3 PCR primers- synthesis and punficationUnique primers for PCR were determined using the following criteria:1) Primer was approximately 20 bp in length and contained roughly equivalentfractions of A + T and G + C, such that Tm was approximately 58°C. Oligo T wasestimated 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 atendency to form primer dimers. Primers were screened for self-complementarityand dimer formation using the NAR program (Rychlik and Rhoads, 1989). Once apair of primers (primerl, primer2) had been determined for a given system, theirsequence was compared to ensure that a high frequency of primerl-primer2 dimerwould not occur.3) Primer did not have significant homology with sequences available through theEuropean Molecular Biology Laboratory (EMBL) sequence database version 27.The computer program FASTA version 1.2 (Pearson and Lipman, 1988) was used tosearch the primate portion of the EMBL database for sequence homologies witheach primer.Primers were purchased either from Bio-Synthesis Inc. (Lewisville, Texas) orthe UBC Oligonucleotide Synthesis Laboratory. Primers obtained from BioSynthesis Inc. had been gel filtration purified, desalted and lyophilized and weretherefore not purified further. Bio-Synthesis primers were resuspended in 1X TE,an 0D260 measurement made of a fraction of the sample, and the concentrationadjusted to 10OM assuming 1 0D260 33 j.Lg/ml. Primers were stored at -20°C.Primers obtained from the UBC Oligonucleotide Synthesis Laboratory were502. Materials and Methodsmade using an Applied Biosystems 380B oligonucleotide synthesizer. Primers fromthe UBC Oligonucleotide Synthesis Laboratory were purified by passing through aC-18 Sep-Pak cartridge in the following manner. The dried primer was resuspendedin 1.5 ml 0.5 M NH4OAc and then loaded onto a C-18 Sep-Pak cartridge which hadbeen pre-washed with 10 ml HPLC grade acetonitrile and 10 ml dH2O. Thecartridge was washed with 10 ml dH2O. The primer was then eluted with 1 ml 20%acetonitrile. A fraction of the sample was used to determine the quantity of primereluted by reading the 0D260 and the remainder of the sample dried using a SpeedVac. The purified primer was then resuspended in 1X TE at a concentration of100 M assuming 1 0D260 = 33 g/ml and stored at -20°C.2.2.11.4 Primer end-labelingPCR reactions used for the detection of polymorphisms were performed inthe presence of radioactive primers. Primers were radioactively end-labeled usingT4 polynucleotide kinase (PNK; Sambrook et a!., 1989). A typical end-labelingreaction consisted of 5 l 10 M primer, 1X PNK buffer, 25 j.Ci(32P) dATP,and 0.67 units of PNK in a total volume of 10 j.Ll. Reactions were also performedfor larger quantities of primer by increasing the amounts of all components by thesame factor. Primers were end-labeled for 45 minutes at 37°C and the reactionstopped by incubation at 65°C for 15 minutes. End-labeled primers were thenadded directly to PCR reactions.512. Materials and Methodslox PNK buffer500 mM Tris, pH 7.5100 mM MgCl50 mM DT1 mM spermidine2.2.11.5 Electrophoresis of radiolabeled PCR productsAfter 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 alleleswere determined relative to the sequenced allele using an M13 sequencing reactionas a secondary size standard.2.2.11.6 Specific PCR reactions and primer sequences1) Alu PCR.Alu PCR involves the use of primers corresponding to the consensussequence of the Alu family of repetitive elements to amplify the human DNA foundbetween 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 thesequence: 5’TCATGTCGACGCGAGACTCCATCTCAAA3’. The 3’ 18nucleotides of the A1S primer are homologous to the extreme 3’ end of the Aluconsensus sequence. Alu PCR reaction conditions were the standard conditionsusing 0.4 j.M A1S primer. Alu PCR cycling conditions consisted of 25 cycles of 1minute denaturation at 94°C, 1 minute annealing at 58°C, and 3 minutes extensionat 72°C, with an additional 10 second extension increase at 72°C each cycle. A final522. Materials and Methods72°C incubation for 10 minutes was performed at the end of the cycling.2) Alu-T3 andAlu-T7PCKAlu-T3 and Alu-T7 PCR involves reactions using anAlu-specific primer andeither of the sequencing primers T3 or T7. This type of PCR reaction was used toamplify 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 theTC-65 primer used by Nelson et al., 1989. The sequence of the TC65A primer is 5’TFGCAGTGAGCCGAGAT 3’. The TC65A primer is homologous to positions219 through 235 in the Alu consensus sequence (63bp to 46bp from the 3’ end of theAlu element). Standard PCR reaction conditions were used for Alu-T3 and Alu-T7PCR, with 0.4 JM of either A1S or TC65A primer and 0.4 JM of either T3 or 17primer added. Cycling conditions for Alu-T3 and Alu-T7 PCR were 25 cycles of 1minute denaturation at 94°C, 1 minute annealing at 45°C and 3 minutes extension at72°C, with an additional 10 seconds extension increase per cycle and a final 72°Cincubation for 10 minutes.3) Alu-LIC PCR and plasmid-LIC PCRPCR products suitable for LIC (section 2.2.9.3) were generated byamplification of 5PCR1 using the primer AlS-LIC and pUC19 using the primersPDJ8O and PDJ81 (Aslanidis and deJong, 1990). The sequence of the A1S-LICprimer is 5’ GATGGTAGTAGGCGAGACTCCATCTCAAA 3’. The sequence ofthe PDJ8O primer is 5’ CCTACTACCATCGGATCCCCGGGT 3’and the sequence532. Materials and Methodsof the PDJ81 primer is 5’ CCTACTACCATCGTCGACCTGCAG 3’. PCRconditions for the generation of LIC - compatible products were the same as thoseused forA!u PCR.4) D5S253 - amplification of (GT) tractPCR 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) end-labeled D5S253-GT and 10 pmoles each of D5S253-GT and D5S253-CA primers.5) D5S257 - amplification of (GT) tractPCR 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) end-labeled D5S257-GT primer and 10 pmoles each of D5S257-GT and D5S257-CAprimers.6) D5S260 - amplification of (GT) tractPCR 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) end542. Materials and Methodslabeled D5S260-GT primer and 10 pmoles each of D5S260-CA and D5S260-GTprimers.7) D5S262- amplification ofAlu 3’ tailPCR 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) end-labeled D5S262-GT primer and 10 pmoles each of D5S262-CA and D5S262-GTprimers. The primer D5S262-CA has homology with the 3’ end of the Alu consensussequence.8) D5S265- amplification ofAlu 3’ tailPCR primers flanking the Alu 3’ tail had the following sequences:D5S265-A = TC65A primerD5S265-T: 5’TrCAACCATITCCCCTGTrCG 3’The TC65A primer has a 2 bp mismatch with the sequence of the Alu element at theD5S265 locus. Standard reaction conditions were used with 0.5 Ci (1 pmole) endlabeled D5S265-T primer, 5 pmoles TC65A primer and 10 pmoles D5S265-Tprimer. Cycling conditions were 30 cycles of 1 minute denaturation at 94°C, 30second annealing at 54°C and 1 minute extension at 72°C, with a final 72°Cincubation for 10 minutes.552. Materials and Methods9) D5S266 - amplification ofAlu 3’ tailPCR 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 reactioncomponents 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 andD5S266-T primers.10) D5S268 - amplification of (GT) tractPCR 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) endlabeled D5S268-CA primer and 10 pmoles each of D5S268-CA and D5S268-GTprimers.562. Materials and Methods2.2.12 Alu PCR differential hybridizationPhage from the LAO5NSO1 or LAO5NLO3 libraries were plated at a densityof 1000 to 1500 plaques per 10 cm plate and plaque lifts were hybridized with thelabeled, preannealedAlu PCR product from the chromosome 5 somatic cell hybrid.Positive plaques were picked and stabbed multiple times onto a small area of a plateoverlaid with bacteria to produce extremely large plaques. Duplicate plaque liftswere made of the secondary plates, one lift hybridized with labeled, preannealedAlu PCR product from the chromosome 5 hybrid and the other lift hybridized withthe labeled, prearmealedAlu PCR product from the deletion 5 hybrid. Multipleexposures were made to compensate for overall differences in signal intensitybetween paired lifts. Phage which strongly hybridized with the chromosome Shybrid and failed to hybridize with the deletion 5 hybrid were picked and purified.2.2.13 Statistical analysis2.2.13.1 Radiation hybrid typing and analysisRadiation hybrid retention data were analyzed using the algorithms of Cox etal., 1990. Quattro-Pro compatible computer programs obtained from Dr. Cox wereused for entering and analysis of marker retention data.2.2.13.2 Polymorphism typing and analysisPolymorphisms 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,572 Materials and MethodsD5S37, 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 fordata analysis (Lathrop et a!., 1984, 1985; Lathrop and Lalouel, 1988). Additionaltyping of the (GT) repeat polymorphism within D5S39 (Mankoo et a!., 1991) wasperformed for CEPH families informative for D5S262 in order to maximize theinformation available for linkage analysis with D5S262.2.2.14 Somatic cell hybrid manipulations2.2.14.1 Cell cultureSomatic cell hybrids were grown in Dulbecco’s modified Eagle mediumsupplemented with 0.292 g/ml L-glutamine and 15% fetal bovine serum. Cells weresubcultured in the following fashion. The media was removed from the cells, andthe cells washed twice in 1X Hanks balanced salt solution. Cells were dislodgedfrom the sides of the flask by the addition of 0.25% trypsin in Hanks solution andincubation with periodic gentle shaking for approximately 5 minutes at 37°C. Anappropriate amount of cells was then transferred to a new flask, and fresh mediaadded. Cells were trypsinized the day before harvesting for chromosomes.582. Materials and Methods2.2.14.2 Somatic cell hybrid DNA preparationDNA was extracted from somatic cell hybrids in the following fashion. Cellswere precipitated by spinning for 5 minutes at 2000 rpm, washed by resuspending in0.85% (w/v) NaCJ, and reprecipitated by spinning for 5 minutes at 2000 rpm in aclinical centrifuge. One to 2 ml of 100 mM Tris; 40 mM EDTA pH 8.0 was added tothe cells. Cells were lysed by the injection of 1 volume of lysis solution using a 5 mlsyringe and a 16-18 gauge needle. An equal volume of TE saturated phenol wasadded 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 andthe upper aqueous layer removed with a large bore pipette and transferred to afresh 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 withphenol. The aqueous layers from two extractions were combined and extractedonce with phenol and once with Sevags. The volume of the final aqueous layer wasdetermined, 1/10 volume 4 M NH4OAc added and the sample mixed well. TheDNA was precipitated by the addition of an equal volume of isopropanol. DNA wascollected and recovered using a pasteur pipette with a curved end. The DNA on theend of the pipette tip was washed with 70% EtOH, then air dried. The DNA wasdissolved in 0.5 to 1 mliX TE by slow mixing overnight on a rotator at 4°C. Theyield of DNA was determined by taking an 0D260 of a fraction of the sample.DNA samples were stored at 4°C.592. Materials and MethodsLysis solution100 mM Tris pH 8.040 mM EDTA0.2% SDS2.2.14.3 Preparation and staining of metaphase chromosomesCells were harvested for metaphase chromosomes using the followingprocedure. Colcemid was added to 0.02 g/ml and the culture was incubated for 3hours to arrest cells in metaphase. The cells were detached from the flask usingtrypsin as described previously. Trypsinized cells were transferred to a centrifugetube and spun for 8 minutes at 1000 rpm in a clinical centrifuge. The majority ofsupernatant was removed and the cells resuspended in the residual supernatant bytapping gently. Cells were resuspended in approximately 5 ml 0.075 M KC1 bypipetting up and down with a pasteur pipette and incubated for 2 minutes at roomtemperature. Cells were precipitated by centriftigation at 1000 rpm for 8 minutes ina clinical centrifuge. The majority of supernatant was again removed and the cellsresuspended in the residual supernatant by tapping gently. Cells were graduallyresuspended in approximately 5 ml ice cold fix and left at room temperature for 10minutes. Cells were precipitated by centrifugation at 1000 rpm for 8 minutes in aclinical centrifuge. Cells were washed by resuspending in ice cold fix, thenprecipitated by centrifugation at 1000 rpm for 8 minutes in a clinical centrifuge. Ifthe suspension appeared to contain large amounts of debris, washing was repeated.Immediately prior to making slides, cells were resuspended in sufficient ice cold fixto create a milky colored solution. Slides were made by dropping the cell602. Materials and Methodssuspension onto a corner of a clean slide, then turning the slide so the solutionmoved down the slide. Amounts of cell suspension and slide angle were adjusteduntil chromosomes were well spread. Slides were made either immediately afterharvesting cells or the day after harvesting. Slides were air dried and stored incovered containers for approximately 1 week before staining.Metaphase spreads were Giemsa banded (G-banding) using conventionaltechniques (Verma and Babu, 1989). Slides were treated as follows:1) 0.1% trypsin; incubate for 10 to 15 seconds2) 1% CaC12; incubate for 1 minute3) dH2O; rinse twice4) Giemsa stain; incubate for approximately 1 minute5) dH2O; rinseTimes for trypsinizing and staining were adjusted to obtain optimal G-banding.Fix1 volume glacial acetic acid3 volumes methanolGiemsa stain2 ml Giemsa stain (Gurr’s improved R66)20 ml 0.025 M phosphate buffer pH 6.8 (Fisher)30 ml dH2O613. RESULTS3.1 CLONE ISOLA TIONAND LOCALIZATIONThe initial objective of my thesis project was to isolate DNA fragments fromwithin a region of human chromosome 5, qll.2-q13.3, defined by a segmentaltrisomy. This region was of interest due to the discovery of a Vancouver family inwhich the trisomic region co-segregated with schizophrenia and renal anomalies(Bassett et al., 1988; McGillivray et al., 1990). This region became of further interestdue to reports of linkage between markers within this region and chronic spinalmuscular atrophy (Brzustowicz et al., 1990; Melki et al., 1990; Gilliam et al., 1990).The technique ofAlu PCR differential hybridization (Bernard et a!., 1991a) wasdeveloped to preferentially isolate DNA fragments from within the 5q11.2-q13.3region. A schematic representation of the Alu PCR differential hybridizationprocedure is shown in Figure 3 (section 1.6). The localization of clones isolated byAlu PCR differential hybridization was confirmed by hybridization to the somaticcell hybrid GM1O114 (chromosome 5 hybrid) and absence of hybridization to thesomatic cell hybrid HHW1O64 (deletion 5 hybrid).3.1.1 Alu PCR differential hybridizationTable 1 shows a summary of results obtained using Alu PCR differentialhybridization in conjunction with the LAO5NSO1 and LAO5NLO3 phage libraries andthe somatic cell hybrids GM1O114 (chromosome 5 hybrid) and HHW1O64 (deletion5 hybrid). Two trials of the Alu PCR differential hybridization technique wereperformed with the LAO5NSO1 library and two trials were performed using the623. ResultsLAO5NLO3 library.Approximately 5 X io phage from the LAO5NSO1 chromosome 5 phagelibrary and approximately 1.3 X io phage from the LAO5NLO3 chromosome 5phage library were screened with the Alu PCR product from the chromosome 5hybrid. A total of 479 phage gave strong positive signals with the Alu PCR productfrom the chromosome 5 hybrid. The proportion of phage which hybridized with theAlu PCR product from the chromosome 5 hybrid was 0.4% for the LAO5NSO1library and 2.2% for the LAO5NLO3 library.Hybridization of duplicate filters (Figure 5) revealed a total of 73recombinant phage which hybridized to the chromosome 5 Alu PCR product anddid not hybridize to the deletion 5 Alu PCR product. When these phage werereplated and rescreened, 45 phage strongly hybridized to the chromosome 5 AluPCR product and did not hybridize to the deletionS Alu PCR product. Phage whichdid not give differential signals when replated fell into two categories, those whichno longer gave a strong signal with the Alu PCR product from either hybrid, andthose which gave equivalently strong signals with both hybrids. No furtherexperiments were performed with the phage which did not give differential signalsupon rescreening.The 45 recombinant phage which gave strong differential signals uponrescreening were plaque purified and phage minipreps were made. Phage from trial#1 were isolated and purified as an initial group, while phage from trials #2through #4 were isolated and purified as a second group. Localization tochromosome 5 and absence from HHW1O64 was later confirmed for 35/45 (78%)isolates (Table 1; section 3.1.3).633. ResultsTable 1. Clone isolation and localizationTrial Library #plaques positivea diffb rescreenC 10dscreened isolates signal confirmed1 LAO5NSO1 2.25X104 81 14 11 92 LAO5NSO1 2.8X104 116 6 3 33 LAO5NLO3 3.0X10 40 5 5 44 LAO5NLO3 1.0X104 242 49 26 19TOTAL 6.35X104 479 73 45 35a clones positive with Alu PCR product from chromosome 5 hybridb clones which exhibited differential signal on initial screenC clones which exhibited differential signal when re-screenedd clones for which localization to chromosome 5 and absence from HHW1O64 wasconfirmed by hybridization to Southern blots of the Alu PCR products from somaticcell hybrids HHW1O64 and GM1O114 (section 3.1.3)643. ResultsFigure 5. Detection of clones derived from 5q11.2-q13.3by differential hybridizationA B•:•.•• *eS.•...4\b••••.- .. ..••4S ODifferential hybridization signals obtained when Alu PCR products from thechromosome 5 hybrid and deletion 5 hybrid were hybridized to duplicate plaquelifts. (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 thedeletion 5 hybrid (HHW1O64). Arrows indicate three phage showing obviousdifferential hybridization which were picked for purification. Other apparentdifferences were less remarkable on the original autoradiographs.653. Results3.1.2 Alu PCR products from clones isolated by differential hybridizationThe 45 recombinant phage exhibiting strong differential signals were used assubstrates for Alu PCR reactions with the A1S primer. Since these clones wereisolated on the basis of hybridization to the Alu PCR product from the chromosome5 hybrid, each clone necessarily contains the 3’ end of anAlu element. The secondAlu element present in genomic DNA would also be present in each clone providedthe 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 30isolates therefore contain two Alu elements oriented such that the 3’ ends of theelements point towards each other, allowing amplification of the inter-Alu regionfound in genomic DNA. The inter-Alu region could be amplified from 46% ofisolates from the LAO5NSO1 library (Trials #1 and #2) and from 86% of isolatesfrom the LAO5NLO3 library (Trials #3 and #4).663. ResultsTable 2. Alu PCR products obtained from clonesexhibiting differential signalsTrial Lab Sizename inter-Aluelementa5PCR1 2.2kb5PCR25PCR3-5PCR4 2.2kb5PCR55PCR65PCR75PCR8-5PCR9 2.2kb5PCR1O 0.5kb5PCR112 A1u16 ndA1u19 1.9kb (1.7kb, 1.6kb, 0.7kb)A1u20 2.2kb3 A1u22 1.0kbA1u24 4.4kbA1u25 2.8kbA1u26 1.0kbA1u274 A1u28 4.4, 1.0A1u29 2.3kbA1u30 1.2kbA1u32 2.3kbA1u36 1.0kb (1.2kb, 1.4kb)A1u38 1.5kb673. ResultsTable 2. (con’t)Trial Lab Sizename inter-Aluelementa4 A1u41 4.4kbA1u43 2.8kbA1u47 3.0kbA1u48 1.0kbA1u52 2.5kbA1u54 2.8kbA1u55-A1u56 -A1u58 2.8kbA1u59 ndAlu6O 1.1kbA1u61 2.0kbA1u62 2.0kbA1u64 ndA1u66 2.8kbA1u67-A1u68 3.0kbA1u71 2.0kbA1u72 3.8kbA1u73 nda Size of product obtained byAlu PCRnd = no data-= no Alu PCR product was detectedSizes in brackets are of minor amplification products.683. Results3.1.2.1 Identification ofmultiple isolates and Alu-T3, Alu-T7 PCR for clones SPCR1 to5PCR11Clones 5PCR1 through 5PCR11 were the first group of phage isolated.Determination of clones isolated more than once (multiple isolates) for this groupoccurred at various steps during the purification and localization process. 5PCR4and 5PCR9 were found to be analogous to 5PCR1 by hybridization of the PCRproduct from 5PCR1 to the PCR products from 5PCR4 and 5PCR9. 5PCR5 and5PCR7 were initially postulated to be identical because they both contain a 3.8kbEcoRI fragment which hybridizes to a 2.9kbAlu PCR product from GM1O114. Theidentity of 5PCR5 and 5PCR7 was later confirmed by showing that the Alu-T7 PCRproduct from p5PCR5 (see below) hybridized to the 3.8kb insert from both phage.5PCR2 and 5PCR6 were initially assumed to be equivalent because they bothcontain two EcoRI fragments of 2.2 and 0.8kb, and hybridize to a 3.4kb Alu PCRfragment from GM1O114. 5PCR2 and 5PCR6 were later demonstrated to beidentical, since Alu-T7 PCR product from p5PCR2-1 hybridizes to the 2.2kb insertfrom each phage.The inserts from phage 5PCR1, 5PCR2, 5PCR3, 5PCR5, and 5PCR11 weresubcloned into Bluescript II as EcoRI fragments. All phage with the exception of5PCR2 contained a single EcoRI insert. Plasmid names from phage isolates withsingle EcoRI inserts are designated as phage names preceded by a “p” to denote theplasmid. As previously mentioned, phage 5PCR2 contained two EcoRI fragmentsof 2.2 and 0.8kb. Both of these fragments were subcloned into plasmids. The 2.2kbfragment was subcloned into plasmid p5PCR2-1 and the 0.8kb fragment subclonedinto plasmid p5PCR2-2. Plasmid preps from p5PCR2-1, p5PCR2-2, p5PCR3,693. Resultsp5PCR5, 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 clones5PCR1 through 5PCR11plasmid insert size Alu-T3 Alu-T7p5PCR2-1 2.2kb 0.4kb 1.2kbp5PCR2-2 0.8kb --p5PCR3 2.8kb 2.2kb-p5PCR5 3.8kb- 1.6kbp5PCR11 7kb - 1.4kb3.1.2.2 Identification of multiple isolates forphage A1u19 to Alu 73Isolates A1u16 through A1u73 were initially checked for identity with 5PCR1through 5PCR11 by hybridization ofAlu,Alu-T3 orAlu-T7 fragments from 5PCR1through 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. Cloneswere subdivided into two groups based on the size of their Alu PCR product. Thoseisolates which could not be Alu PCR amplified were included in both groups. Foreach 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 from703. Resultseach group. Each index clone was hybridized to the plaque lift from the appropriategroup. Multiple isolates were then determined on the basis of hybridizationpatterns.A summary of all multiple isolates obtained is shown in Table 4. For themajority of multiple isolates, all isolates in a group were from the same phagelibrary and produced the same size Alu PCR product, indicating that therecombinant phage were likely identical in nature. However, the Alu PCR productfrom A1u28 contained an extra 4.4kb product in addition to the 1kb productobserved for A1u22 and A1u48. A1u28 was therefore different from A1u22 andA1u48. Also, 5PCR11 was derived from the LAO5NSO1 library, while isolates A1u24and A1u59 were from LAO5NLO3, indicating that recombinant phage within thisgroup were not identical.To confirm that 5PCR11 and Alu24 contained overlapping sequences, theinter-Alu region from each of these isolates was hybridized to genomic DNA fromGM1O114. Both 5PCR11 and A1u24 hybridized to a 4.4kbAlu PCR product fromGM1O114, indicating that 5PCR11 and A1u24 contain inter-Alu element sequence incommon. The identity of these two phage was further confirmed by hybridization ofthe Alu-T7 PCR product from p5PCR11 to restriction enzyme digested DNA fromphage A1u24. The Alu-T7 PCR product from p5PCR11 hybridizes to a 7kb EcoRlfragment from phage A1u24, indicating that phage A1u24 likely contains the entiretyof the 7kb EcoRl insert from phage 5PCR11.All sets of multiple isolates are referred to in subsequent sections using thename of the lowest numerical isolate. A total of 30 distinct isolates were obtainedwhich gave differential hybridization signals.713. ResultsTable 4. Multiple IsolatesIsolate Library Equivalent phage Library5PCR1 LAO5NSO1 5PCR4, 5PCR9, A1u20 LAO5NSO15PCR2 LAO5NSO1 5PCR6 LAO5NSO15PCR5 LAO5NSO1 5PCR7, A1u16 LAO5NSO15PCR11 L4O5NSO1 A1u24, A1u59 LAO5NLO3A1u22 LAO5NLO3 A1u28, A1u48 LAO5NLO3A1u25 LAO5NLO3 A1u43, A1u54, A1u58 LAO5NLO3A1u62 LAO5NLO3 A1u71, A1u73 LAO5NLO3723. Results3.1.3 Confirmation of localization - Alu PCR localization blotsThe localization of each of the 30 distinct isolates to the deletion region wasconfirmed by hybridization of DNA from each recombinant phage to Southern blotsof the Alit PCR products from the chromosomeS and deletion 5 hybrids. Either theAlu PCR product, Alu-T7 PCR product, Alu-T3 PCR product or total phage DNAwas used as a hybridization probe (Table 5). Repetitive probes were preannealedwith a vast excess of unlabeled human DNA. Probes were classified as repetitive ifthey included the entire phage insert since each phage necessarily contained at leastone Alu element due to their method of isolation. Probes were also classified asrepetitive if initial hybridization of the Alu PCR product to an Alu PCR localizationblot produced multiple strong bands. The results of this localization are describedin Table 5. Examples of various probes hybridized to Alu PCR localization blots areshown in Figure 6. As is demonstrated in Figure 6, a single strongly hybridizingband was most prominent, with additional minor bands attributable to thehybridization of repetitive elements within the probe which were not completelyblocked by preannealing.Twenty non-identical isolates were present in the chromosome 5 hybrid andabsent from the deletionS hybrid. This corresponds to 35 of 45 phage isolated byAlu PCR differential hybridization if multiple isolates are included. Alu PCRdifferential hybridization therefore had success rate of 35/45 or 78%. Of the 10isolates which could not be localized to the deletion region, three (5PCR1O, A1u56and 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, andA1u68) were highly repetitive and therefore could not be classified.733. ResultsThe 20 non-identical isolates localized to the deletion region were assignedlocus names D5S205, and D5S251 through D5S269 (Table 5). Large scale phagepreps were made for each of these isolates.743. ResultsTable 5. Localization of clones by hybridization tosomatic cell hybrids GM1O114 and HHW1O64Locus Phage/Plasmid Probe Repet- Hybrid Hybridname itive GM1O114 HHW1O64D5S205 5PCR1 Alu PCR no + -D5S251 p5PCR2-1 Alu-T7 yes +D5S252 p5PCR3 Alu-T3 no + -D5S254 p5PCR5 Alu-T7 no + -D5S253 p5PCR11 Alu-T7 yes + -D5S255 A1u19 Alu PCR no +D5S256 A1u22 Alu PCR yes + -D5S257 A1u25 Alu PCR yes + -D5S258 Alu26 Alu PCR yes + -D5S259 A1u29 Alu PCR yes + -D5S260 A1u32 Alit PCR yes + -D5S261 A1u36 Alu PCR yes + -D5S262 A1u38 Alu PCR yes + -D5S263 A1u41 Alu PCR yes + -D5S264 A1u47 Alu PCR no + -D5S265 A1u52 Alu PCR yes + -D5S266 Alu6O Alu PCR yes + -D5S267 A1u61 Alu PCR no + -D5S268 A1u62 Alu PCR no + -D5S269 A1u66 Alit PCR yes + -5PCR8 phage yes - -A1u27 phage yes - -A1u55 phage yes - -A1u67 phage yes - -A1u72 Alu PCR no - -753. ResultsTable 5. (con’t)Locus Phage/Plasmid Probe Repet- Hybrid Hybridname itive GM1O114 HHW1O64A1u30 Alu PCR yes ? ?A1u68 Alu PCR yes ? ?5PCR1O Alu PCR yes + +A1u56 phage yes + +A1u64 phage yes + +763. ResultsFigure 6. Clone localization by hybridization toSouthern blots of hybrid Alu PCR products1 2 3 4 5 6 7 8 9 10 11 12 13 149—3.8——I—05—Localization of clones by hybridization to Southern blots of the Alu PCR products ofGM1O114 (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 thedeletion 5 hybrid. Lanes were hybridized as follows: (1, 2) Alu PCR product of thechromosome 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.773. Results3.1.4 Confirmation of localization - Genomic localization blotsThe localization of clones from loci D5S205, D5S251, D5S252, D5S254,D5S253, D5S255, D5S264, and D5S267 to the deletion region of chromosome 5 wasfurther 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 backgroundsignal and did not hybridize HHW1064 or hamster DNA (Figure 7). Similar resultswere obtained using probes from D5S255 and D5S264. Probes from D5S251,D5S253 and D5S267 produced discernable bands in the chromosome 5 hybrid DNAlane, no bands in the deletion 5 and hamster DNA lanes, and strong backgroundsignals in the human DNA lane. The localization to within the deletion region ofchromosome 5 was therefore confirmed for all isolates tested.3.1.5 Confirmation of localization - PCRThe localization of polymorphic systems associated with D55253, D5S257,D5S260, and D5S268 (section 3.3.1) was confirmed by amplification reactions usingthe HHW1O64 and GM1O114 hybrids as templates. Absence of amplification fromHHW1O64 and amplification from GM1O114 was demonstrated for all systems.783. ResultsFigure 7. Clone localization by hybridization toSouthern blots of hybrid genomic DNAA B1234 1234Confirmation of localization of phage isolates by hybridization to Southern blots ofEcoRI-digested genomic DNA. Lane 1, CHO (hamster); lane 2, GM1O114(chromosome 5 hybrid); lane 3, HHW1O64 (deletion 5 hybrid); lane 4, humanlymphoblast DNA. Panels were hybridized as follows: (A) Alu PCR product fromD5S205, (B) Alu-T3 product from D5S252, and (C) Alu-17 product from D55254.C1234Kb9.0—3.8—2.0—793. Results3.1.6 Majority of clones do not correspond to visible differences between hybrid AluPCR productsThe Alu PCR band to which each isolate corresponded was identified inorder to determine if the band was noticeable as a difference between the Alu PCRproducts of the chromosome 5 and deletion 5 hybrids. The Alu PCR products ofclones localized to the chromosome 5 deletion region were electrophoresed besidethe Alu PCR products of the chromosome 5 and deletionS hybrids (Figure 8). Themajority of isolates could not be detected as a difference between the Alu PCRproduct of the two hybrids. Several isolates, particularly those producing largeinter-Alit fragments, corresponded to bands which were not visible on the ethidiumbromide stained gel. Only the inter-Alu regions from loci D5S254 and D5S266appear to correspond to a visually detectable difference between the Alu PCRproducts of the two hybrids.803. ResultsFigure 8. Alu PCR productsEthidium bromide stained gel showing Alu PCR products from clones localized tothe deletion region compared with the Alu PCR products from the HHW1O64 andGM1O114 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 fromGM1O114. Lanes 4 to 19 contain the Alu PCR products from phage isolates. Lane4, 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).1 2 3 4 5 6 7 8 9 101112 13 14 15 16 17 18 19 2 91813. Results3.2 RADL4 TION HYBRID MAPPINGThe second goal of my thesis project was to physically map the 20 clonesisolated from the 5q11.2-q13.3 region defined by the segmental trisomy. Radiationhybrid mapping (Cox et a!., 1990) was chosen to accomplish this goal. A radiationhybrid mapping panel is produced by subjecting a somatic cell hybrid containing ahuman chromosome of interest to a high dose of X-rays, followed by fusion of theirradiated cells with a rodent cell line. Fragments of the human chromosome arenon-selectively retained in the radiation hybrids. Radiation hybrids can be analyzedfor the presence or absence of markers, with coretention of two markers anindication of the physical distance between the two markers. A series of algorithmsfor 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 hybridswas used to obtain order information on 18 clones isolated byAlu PCR differentialhybridization.3.2.1 Radiation hybrid typingTo determine the order of 18 of the 20 isolates which were localized to thedeletion region of chromosome 5 (Table 5), isolates were typed in a chromosome 5radiation hybrid panel consisting of 150 radiation hybrids. The Alu PCR productsfrom D5S252 and D5S263 gave very weak signals when hybridized to Alu PCRlocalization blots and therefore were not scored in the radiation hybrid mappingpanel. The inter-Alu region from the 18 remaining isolates was hybridized toSouthern 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.823. ResultsFigure 9 depicts the Alu PCR products from the amplification of hybrids 1 through20 (17 radiation hybrids). Comparable results were obtained for the remainder ofthe hybrids.Isolates which had previously been determined to be repetitive whenhybridized to Alu PCR localization blots were prearmealed with total human DNA.Isolates which gave multiple bands even after prearmealing due to their repetitivenature were hybridized to the Alu PCR hybrid blots singly. All other probes werehybridized 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 150hybrids are included in Appendix 2. D5S39 typing results obtained from Dr. EllenSolomon are also included in Appendix 2. Each of the 18 probes was nonselectivelyretained in 4.1% to 10.8% of the hybrids (Table 6).833. ResultsFigure 9. Alu PCR products for radiation hybrids 1through 201 2 3 4 5 6 7 8 9 10 1112 13 1415 16 1718 1920 2122Ethidium bromide stained gel showing Alu PCR products obtained from radiationhybrids. Lanes 1 and 21,Alu PCR product from GM1O114; Lane 2,Alu PCRproduct 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 containthe 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.84Figure 10. Probes from D5S260 and D5S262 hybridizedto radiation hybrids 1 to 201 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 171819 20213. ResultsAutoradiograph obtained by hybridization of probes from D5S260 and D5S262 toSouthern blot ofAlu PCR products from radiation hybrids. Lanes 1 and 21,AluPCR 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 theAlu PCR product obtained from radiation hybrids. Lane 3, hybrid 1; Lane 4, hybrid2; 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.I853. ResultsTable 6. Radiation hybrid retention frequenciesLocus Retention Radiation HybridsScoredD5S205 0.087 150D5S251 0.061 99D5S254 0.06 150D5S253 0.082 134D55255 0.075 133D5S256 0.097 134D5S257 0.097 134D5S258 0.073 150D5S259 0.043 117D5S260 0.067 150D5S261 0.053 150D5S262 0.108 148D5S264 0.054 149D5S265 0.090 133D5S266 0.04 1 98D5S267 0.048 84D5S268 0.073 150D5S269 0.05 100D5S39 0.127 150863. Results3.2.2 Twopoint radiation hybrid analysisTo determine the most likely order of the isolates, markers were analyzed ineach possible pairwise combination using the algorithm of Cox et a!., 1990. A lodscore of 3 or more was taken as significant evidence for linkage (Table 7). Thetwopoint results were used to subdivide the markers into four groups, as indicated inTable 7.873. ResultsTable 7. Twopoint radiation hybrid scores for markerslinked at lod >3Locus Distance LOD(cR5,üO) ScoreGroup #1D5S253- D5S257 80 3.56D5S257-D5S262 75 3.98D5S268-D5S262 95 3.13D5S260-D5S262 89 3.42D5S262-D5S39 97 3.36D5S39-D5S264 58 6.35Group #2D5S259-D5S258 63 3.58D5S258-D5S256 65 4.20D5S256- D5S265 81 3.50Group #3 Linked to 1 other markerD5S266-D5S261 42 3.37D5S255-D5S254 77 3.66Group #4 Linked to no other markerD5S205; D5S251; D5S267; D5S269Group #5 Untyped in radiation hybrid panelD5S252; D5S263883. Results3.2.3 Fourpoint radiation hybrid analysisCox et a!., 1990 devised algorithms for fourpoint radiation hybrid analysiswhich can be used to calculate the relative likelihoods of various marker orders andto determine radiation distances between markers. These fourpoint algorithmswere used for group #1 and #2 markers. The odds against inversion of adjacentmarker pairs was calculated by reversing the order of successive pairs of markersand calculating the likelihood of the order relative to the most likely order.Distances between markers in cR50,000 were calculated for the most likely markerorders.Group #1 consists of seven markers, five of which could be placed in a lineararray from the results of the twopoint analysis (D5S253-D5S257-D5S262-D5S39-D5S264). These 5 markers were analyzed in sets of 4 using the fourpoint algorithmsof Cox et al., 1990. The results of the overlapping fourpoint analyses performedinvolving D5S253, D5S257, D5S262, D5S39 and D5S264 are shown in Figure 11.Radiation distances and odds against inversion of adjacent markers for the mostlikely marker order are also presented in Figure 11. Markers at either end of thegroup (D5S253 and D5S264) were each present in only one of the sets of fourmarkers, so the odds of inversion of these markers was calculated using thepertinent fourpoint. Markers in the middle of the group (D5S257, D5S262 andD5S39) were present in both groups of four markers. Odds of inversion of thesemarkers 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 thefourpoint involving D5S253, D5S257, D5S262 and D5S39, while the odds ofinverting D5S262 and D5S39 was calculated from the fourpoint involving D5S257,893. ResultsD5S262, D5S39 and D5S264.The other two markers in group #1 (D5S268 and D5S260) were linked bythe twopoint data only to D5S262, one of the internal markers in the set. Fourpointanalyses involving both possible positions of D5S268 and D5S260 relative to D5S262would 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 forgroup #2 markers is D5S259-D5S258-D5S256-D5S265. The odds of inversion ofadjacent markers were calculated as described above for the group #1 markers.Radiation distances for this group and odds of inversion of adjacent markers for themost likely order of markers are shown in Figure 12.903. ResultsFigure 11. Fourpoint Radiation hybrid analysis -Group #1marker order odds Dl D2 D3D5S253-D5S257-D5S262-D5S39 1 80 75 97D5S257-D5S253-D5S262-D5S39 415D5S253-D5S262-D5S257-D5S39 3.5X10D5S253-D5S257-D5S39-D5S262 674D5S257-D5S262-D5S39-D5S264 1 75 97 58D5S262-D5S257-D5S39-D5S264 2.5X103D5S257-D5S39-D5S262-D5S264 7X105D5S257-D5S262-D5S264-D5S39 32D5S257-D5S262-D5S260-D5S39 1D5S257-D5S260-D5S262-D5S39 24D5S257-D5S262-D5S268-D5S39 1D5S257-D5S268-D5S262-D5S39 1.1Odds against a given order were calculated relative to all other permutations ofthe set of four markers. Distances between markers in cR50,000 are indicatedfor the most likely marker orders.most likely order:odds 415 3.5X105 7X105 32D5S253 D5S257 D5S262 D5S39 D5S264cR50,000 80 75 97 58Odds against inversion of marker pairs are indicated above the map, whiledistances between markers in cR50,0 0 are indicated below.913. ResultsFigure 12. Fourpoint Radiation hybrid analysis -Group #2marker order odds Dl D2 D3D5S259-D5S258-D5S256-D5S265 1 63 65 81D5S258-D5S259-D5S256-D5S265 35D5S259-D5S256-D5S258-D5S265 383D5S259-D5S258-D5S265-D5S256 294Odds against a given order were calculated relative to all other permutations ofthe set of four markers. Distances between markers in cR50,000 are indicatedfor the most likely order of markers.most likely order:odds 35 383 294D5S259 D5S258 D5S256 D5S265cR5O,000 63 65 81Odds against inversion of marker pairs are indicated above the map, whiledistances between markers in cR5O,000 are indicated below.923. Results3.3 POLYMORPHISMSA further aim of this thesis project was to place clones isolated from the5q11.2-q13.3 region onto the genetic linkage map of human chromosome 5. Thefirst step towards the accomplishment of this goal was to detect polymorphisms.Information obtained from radiation hybrid mapping was used to select clones ofparticular interest to use in the polymorphism screen. Screening was performed fora variety of types of DNA based polymorphisms. Polymorphism screening andtyping is described in general in section 3.3.1. Polymorphisms detected are thendescribed in further detail in sections 3.3.2. through 3.3.9.3.3.1 Polymorphism screening and typingPolymorphism screening was done for three types of polymorphisms;conventional RFLPs, simple sequence repeat polymorphisms and polymorphisms inthe polyA tails ofAlit elements. A summary of polymorphisms identified ispresented in Table 8. Polymorphisms detected were typed in the CEPH panel offamilies (Dausset et al., 1990). For each polymorphic system, hybridization toSouthern blots or PCR reactions were performed on CEPH parents. CEPH familiesfor which one or both parents were heterozygous were then typed. Theheterozygosity and polymorphism information content (PlC) of each marker systemwas calculated from allele frequencies of the CEPH parents (Table 8).933. ResultsTable 8. PolymorphismsLocus Polymorphism #Alleles Het PlCConventional RFLPD5S205 TaqI RFLP 4 0.42 0.39(GT)n TractsD5S253 (TG)23 8 0.78 0.75D5S257 (TG)12C(GT)8 5 0.35 0.29D5S260 (TG)CG(TG)11 5 0.75 0.69D5S268 (GT)16 7 0.72 0.68(GT) Tracts at 3’ end ofAlu elementsD5S262 (TA)3GA(TA)(TG)4A5(TG)5(TA)5- 2 0.06 0.06-TGGAA(TG)4( )A(T )D5S266 A21(TA)6G 10CATACA(TA)3 4 0.26 0.30Alu polyA tractA1u52A 3 0.47 0.41A1u52B 3 0.55 0.46Het = heterozygosityPlC = polymorphism information content943. ResultsScreening for conventional RFLPs was done for loci D5S205, D5S251,D5S252, D5S253, D5S254, D5S255 and D5S264. Probes were hybridized toSouthern blots of 5 unrelated CEPH individuals digested with BamHI, BgllI, EcoRI,Hindill, MspI, PstI, PvuII and TaqI. A TaqI polymorphism was detected forD5S205. Codominant inheritance was observed for D5S205 in all CEPH familiestyped.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 inthe human genome. Therefore, phage from all 20 loci within the deletion regionwere 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)ntract from each of these phage was subcloned into Bluescriptil and sequenced. Anautoradiograph of sequence obtained for the D5S257 (GT)n tract is shown in Figure13. Comparable sequencing autoradiographs were obtained for the remainder ofthe (GT)n tracts. Sequences of the six (GT)n tracts are presented in Table 8. TheSX (GT)n tracts isolated fell into two categories, those which were present asisolated tracts and those which were located at the 3’ ends ofAlu elements. Primersflanking the (GT)n tracts were used to amplify the tracts to check forpolymorphisms. All of the (GT) tracts amplified were polymorphic, with PlCvalues ranging from 0.06 to 0.75 (Table 8). Codominant Mendelian segregation wasobserved in CEPH families for all (GT)n tracts typed.953. ResultsFigure 13. Sequence of D5S257 (GT) tractTGCASequence surrounding D5S257 (GT)n tract. Autoradiograph was obtained bydenaturing polyacrylamide gel electrophoresis of a sequencing reaction on theplasmid p257RO.7 using the T7 sequencing primer.963. Results(GT)n tracts were not present within loci D5S259, D5S258, D5S256 andD5S265, which were of interest since they comprised radiation hybrid mappinggroup #2. Similarly, D5S255 and D5S254 were linked by radiation hybrid mappingbut did not contain (GT)n tracts. Alternate methods for generating polymorphismsfor these loci were therefore explored. The polyA tail ofAlu elements had beenreported to be polymorphic (Economou et al., 1990), and due to the method ofisolation each of these clones contained at least one Alit 3’ end. Therefore, Alitelements from within these isolates were subcloned into Bluescriptil and sequencewas obtained of the 3’ ends of the elements and flanking DNA. The insert fromphage 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 Hindlilfragments. Plasmids containing single Alit elements were identified byAlu PCR,since plasmids with a single Alu element produce a single band in either a TC65A-T3 PCR reaction or a TC65A-T7 PCR reaction. Plasmids containing a single Alitelement were then sequenced using TC65A, T3 or T7 as primers. A total of l2Alu3’ ends were sequenced, and 6 recognizable polyA tails were found. Sequences ofthe Alu elements with recognizable 3’ tails are shown in Table 9.973. ResultsTable 9. Sequence ofAlu polyA tailsLocus Plasmid Alu polyA tail sequenceD55254 p5PCR5 A15D55256 p22E1A8 7TA3G2D5S258 p26E2 AGACA8D5S258 p26H6 A8GAD5S259 p29H2 A7D5S265 p52H1 17CA54GC3tjTwo of the Alu 3’ tails were felt to be of sufficient length to warrantpolymorphism screening, namely the A15 tract found in p5PCR5 (D5S254) and theA17CA54GC36tract from p52H1 (D55265). A primer 3’ of the polyA tail wassynthesized for both tracts. The primer for D5S254 was named D5S254-T, and hadthe sequence 5’ TTAAAATGTATGCATTATIICTGGA 3’. The primer forD5S265 was called D5S265-T and had the sequence 5’TI’CAACCAIITCCCCTGTFCG 3’. The Alu element in p5PCR5 was thensequenced using the D5S264-T primer, and the Alu element in p52H1 sequencedusing the D5S265-T primer. Both Alu elements were found to have significanthomology to the TC65A primer. The Alu element in p5PCR5 had a 1 bp mismatchwith TC65A at the 5’ end of the primer, and the Alu element in p52H1 had two 1 bpmismatches with TC65A inside the primer. The sequence of the p5PCR5 Aluelement and surrounding region is shown in Figure 14, together with sequencealignment with the Alu consensus sequence. The sequence of the p52H1 Aluelement is shown in Figure 24 (section 3.3.7.).983. ResultsA PCR reaction using TC65A and each of the unique primers was carriedout to amplify the Alu polyA tracts for each locus. The Alu polyA tract presentwithin 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 genomicDNA using a variety of PCR reaction conditions and cycling conditions. The polyAtract corresponding to D5S254 was therefore not investigated further. The polyAtract present within D5S265 was amplifiable from both plasmid and genomic DNA(section 3.3.7).993. ResultsFigure 14. Sequence of D5S254 Alu polyA tract andflanking sequence and alignment with Alu consensussequencealucon 5’ GGCTGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGalucon 5’ GTGGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGalucon 5’ TGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCliii liii 11111 IllIllIllIllIll 11111111111D5S254 5’CCCCATCTC ACTAA TATACAAAAATTAGCCAGGCGTGGTGGCAT3’ GGGGTAGAG TGATT ATATGTTTTTAATCGGTCCGCACCACCGTAalucon 5’ GCGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCTGAGGCAGGAGAATCG111111111D5S254 5 ‘GTGCCTGTAGTCCCAGCTAGTCAGGTGGCTGAGGC AGGAGAATCA3’ CACGGACATCAGGGTCGATCAGTCCACCGACTCCG TCCTCTTAGTalucon 5’ CTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCACIIIIIItIIIIIIIIIIII,IIIIIIIIIIIIIIIIIIIIIIIIIID5S254 5 ‘ATTGAACCCGGGAGGTGGAGGCTGCAGTGAGCCGAGATTGTGCCACTGTAC3’ TAACTTGGGCCCTCCACCTCCGACGTCACTCGGCTCTAACACGGTGACATGalucon 5’ TGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAD5S254 5 ‘T CCTGCGTGGGCAACAGAGCGAGACTCCATCTCAAAAAAAAAAAAA3 ‘A GGACGCACCCGTTGTCTCGCTCTGAGGTAGAGTTTTTTTTTTTTTD5S254 5 ‘AATTCCAGAAATAATGCATACATTTTAAATAGCATGCCTTTCTGAGTTGTG3’ TTAAGGTCTTTATTACGTATGTAAAATTTATCGTACGGAAAGACTCAACACD5S254 5’ATGAAATCTTAT3 ‘TACTTTAGAATATC65A primer sequence is underlined in the Alu consensus sequence and D5S254-Tprimer sequence is underlined in the D5S254 sequence. The Alu consensussequence was obtained from the EMBL database version 27.1003. Results3.3.2 D5S205The 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 fromallele frequencies obtained by typing 80 CEPH parents (Table 10). The TaqIpolymorphism was detected by hybridization of the 2.2kb Alu PCR product fromphage 5PCR1 to Southern blots of CEPH genomic DNA digested with TaqI.Table 10. D5S205 Allele frequenciesAllele size (kb) frequencyAl 9kb 0.12A2 6kb 0.74A3 3.2kb 0.12A4 11kb 0.02D5S205 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 15.1013. ResultsFigure 15. D5S205 TaqI polymorphism9 kb -6 kb -bEEb ‘666±Segregation of the 9 kb and 6 kb alleles at the D5S205 locus in family 1349.1023. Results3.3.3 D5S253The polymorphism detected at locus D5S253 is a microsatellite repeat with aheterozygosity of 0.78 and a PlC of 0.75 calculated from allele frequencies obtainedby typing 77 CEPH parents (Table 11). The (GT) tract within D5S253 wassubcloned as a 3kb HindIll fragment into Hindill digested Bluescriptil to producethe plasmid p253H3.0. The region containing the D5S253-GT primer wassubcloned within a 500bp RsaI fragment into EcoRV digested Bluescriptil toproduce the plasmid p253R0.5. The sequence of the D5S253-GT primer wasdetermined from the plasmid p253R0.5 using T3 and T7 primers. The sequence ofthe D5S253-CA primer was then determined from the p253H3.0 plasmid using theD5S253-GT primer. The sequence surrounding the (GT)n tract (Figure 16) wasdetermined from the p253H3.0 plasmid using the D5S253-CA and D5S253-GTprimers. 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 toan amplification product of 126bp. D5S253 reference genotypes were as follows:133101 = A4,A4; 133102 = A1,A4. Segregation of the 124, 120 and 114 bp allelesin family 1340 is shown in Figure 17.1033. ResultsTable 11. D5S253 Allele frequenciesAllele size(bp) frequencyAl 126 0.045A2 124 0.097A3 122 0.026A4 120 0.38A5 118 0.17A6 116 0.11A7 114 0.16A8 102 0.0131043. ResultsFigure 16. D5S253 (GT)n tract and flanking sequence5’ AAAAATTAACTGGGTGTGGTAGTGTGCACCTGTGGTTCTAGCTACTCGGGAGGCTGAG3’ TTTTTAATTGACCCACACCATCACACGTGGACACCAAGATCGATGAGCCCTCCGACTCGTAGGAGGCTTGCTTGACCCCAGGAGGTCAAGGCTGTGGTGAGCTGAGATTGTACCATTGCATCCTCCGAACGAACTGGGGTCCTCCAGTTCCGACACCACTCGACTCTAACATGGTAACCACTCTAGCCTGGGCAACAGATCCAGACCTTGTCTCTAAATTAAAACAAAACAAAACCAAGTGAGATCGGACCCGTTGTCTAGGTCTGGAACAGAGATTTAATTTTGTTTTGTTTTGGTTACAAAAAAACAGCTGNATAAGGAAGGTTATAACAGAACACTGTTCTCTCTTTACACACACTGTTTTTTTGTCGACNTATTCCTTCCAATATTGTCTTGTGACAAGAGAGAAATGTGTGTGACACACACACACACACACACACACACACACACACACACATCACACGTACAGGAATTATTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAGTGTGCATGTCCTTAATAAATAACCTATCAGTTACATGGTGGTTTCACAGGTTTCAACTTCATCAACCCAGAACCACAATATTGGATAGTCAATGTACCACCAAAGTGTCCAAAGTTGAAGTAGTTGGGTCTTGGTGTTACACAGATTTTGGCTAGACTCTGACTCTCATCTACTAGTGATAACAACAAGTTCCCTGTGGGTGTCTAAAACCGATCTGAGACTGAGAGTAGATGATCACTATTGTTGTTCAAGGGACACCAGTTTATAGCCCACAGATTATCA3’TCAAATATCGGGTGTCTAATAGT5’Sequence surrounding (GT)n tract. Primer sequences are underlined.1053. ResultsFigure 17. D5S253 (GT)n polymorphism66EEEEr5124 bp -l2Obp--p114 bp -_____________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 thefigure. Minor bands present are shadow bands produced as an artifact of PCR.1063. Results3.3.4 D5S257The polymorphism detected at locus D5S257 (Bernard et a!., 1991b) is amicrosatellite repeat with a heterozygosity of 0.35 and a PlC of 0.29 calculated fromallele frequencies obtained by typing 79 CEPH parents (Table 12). The (GT) tractwithin D5S257 was subcloned as a 0.7kb RsaI fragment into EcoRV digestedBluescriptil to produce the plasmid p257R0.7. The sequence surrounding theD5S257 (GT)n tract (Figure 18) was determined from the plasmid p257R0.7 usingT3 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)12C(GT8whichcorresponds to an amplification product of 79bp. D5S257 reference genotypes wereas follows: 133101=A2,A2; 133102 = A2,A3. Segregation of the 79 and 77 bpalleles in family 1332 is shown in Figure 19.1073. ResultsTable 12. D5S257 Allele frequenciesAllele size(bp) frequencyAl 81 0.006A2 79 0.79A3 77 0.19A4 75 0.013AS 69 0.006Figure 18. D5S257 (GT) tract and flanking sequence5’ ATAGCACTTTTAAATTGTTCTCATTCCCTTTGCTTAAAGGTAAGGTTTTTGAGCATTT3’ TATCGTGAAAATTTAACAAGAGTAAGGGAAACGAATTTCCATTCCAAAAACTCGTAAATAATACATCAAGTTAAAGTGTCAATTGTAGGCTTTGGTAAGATAATATAGATCAGTCATAATTATGTAGTTCAATTTCACAGTTAACATCCGAAACCATTCTATTATATCTAGTCAGTATTTTGAAAAGGGAAATACACTGTGTGTGTGTGTGTGTGTGTGTGCGTGTGTGTGTGTGTGTAAACTTTTCCCTTTATGTGACACACACACACACACACACACACGCACACACACACACACAATCTGGAATTACTTTGTTGGAAT3’TAGACCTTAATGAAACAACCTTA5’Sequence surrounding (GT)n tract. Primer sequences are underlined.1083. ResultsFigure 19. D5S257 (GT) polymorphism79 bp -77 bp -6EbEb6EESegregation of the 79 and 77 bp alleles at the D5S257 locus in family 1332. Allelesizes 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.1093. Results3.3.5 D5S260The polymorphism detected at locus D5S260 (Bernard et a!., 1991c) is amicrosatellite repeat with a heterozygosity of 0.75 and a PlC of 0.69 calculated fromallele frequencies obtained by typing 69 CEPH parents (Table 13). The (GT) tractwithin 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 intoEcoRV digested Bluescriptil to produce the plasmid p260R0.5. The sequencesurrounding the D5S260 (GT)n tract (Figure 20) was determined from the plasmidp260R0.5 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 (TG)6CG(TG)11whichcorresponds to an amplification product of 146bp. D5S260 reference genotypeswere as follows: 133101 = A2,A4; 133102 = A3,A4. Segregation of the 152, 148 and146 bp alleles in family 1345 is shown in Figure 211103. ResultsTable 13. D5S260 Allele frequenciesAllele size(bp) frequencyAl 154 0.065A2 152 0.38A3 150 0.14A4 148 0.27A5 146 0.15Figure 20. D5S260 (GT)n tract and flanking sequence5’ CCAGTTTTCCTGAGAGTCATTAGCTCTGGCTTCTTGTGGATCCTTTCAGAAAGAATCT3’ GGTCAAAAGGACTCTCAGTAATCGAGACCGAAGAACACCTAGGAAAGTCTTTCTTAGAGTGTGTATATTTGCATGTGTGTGTGTGCGTGTGTGTGTGTGTGTGTGTGTGAATGGGTATCACACATATAAACGTACACACACACACGCACACACACACACACACACACACTTACCCATAATACACACTTGCATATGTCACAGTTGAATTTATTGTCTTGAATGGTAATAGTTAAGTCATTATGTGTGAACGTATACAGTGTCAACTTAAATAACAGAACTTACCATTATCAATTCAGTAAAATATATAG3’TTTATATATC5’Sequence surrounding (GT)n tract. Primer sequences are underlined.1113. ResultsFigure 21. D5S260 (GT)n polymorphism152 bp -148 bp -146 bp -6EE6EESegregation 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 thefigure. Minor bands present are shadow bands produced as an artifact of PCR.1123. Results3.3.6 D5S262The polymorphism detected at locus D5S262 is a microsatellite repeatlocated at the 3’ end of anAlu element with a heterozygosity of 0.06 and a PlC of0.06 calculated from allele frequencies obtained by typing 78 CEPH parents (Table14). The (GT) tract within D5S262 was subcloned as a 0.8kb SmaI-PstI fragmentinto SmaI-PstI digested Bluescriptil to produce the plasmid p262SP0.8. Thesequence surrounding the D5S262 (GT) tract was determined from the plasmidp262SP0.8 using the T7 primer. The GT tract was present at the 3’ end of anAluelement (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. Thesequence of the cloned product repeat unit was(TA)3GA(TA)3(TG)4(TA)5(TG)ATGGA (TG)3A(TG)6(TA)6TGwhich corresponds to an amplification product of 193bp. D5S262reference 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 frequenciesAllele size(bp) frequencyAl 193 0.97A2 185 0.031133. ResultsFigure 22. D5S262 (GT) tract and flanking sequencealigned with Alu consensus sequenceSequence surrounding (GT)n tract and alignment with Alu consensus sequence.Primer sequences are underlined. The Alu consensus sequence was obtained fromthe EMBL database version 27.1143. Resultsalucon 5’ GGCTGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGalucon 5’ GTGGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGalucon 5’ TGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCIII IIID5S262 5’TGGCAGCGT3 ‘ACCGTCGCAalucon 5’ GCGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCTGAGGCAGGAGAATCG1111111111 1111111111 1111111D5S2 62 5’ GTGCCTGTAATGGCAGCTACTCGNN GGNGAATTG3’ CACGGACATTACCGTCGATGAGCNN CCNCTTAACalucon 5’ CTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCAC11111111 111111 11111 IllIllIllIll II 11111111D5S262 5’ CTTGAACCTGGGAGGCAGAGGTGGCAGTGAGCCGATCTCATGCCACTGC3’ GAACTTGGACCCTCCGTCTCCACCGTCACTCGGCTAGAGTACGGTGACGalucon 5’ TGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAI IID5S262 5 ‘TTTCCATATATATATATA3’ AAAGGTATATATATATATD5S 262 5’ CACACACACACATACACANATATATATATACACACACATTCCATATATATA3’ GTGTGTGTGTGTATGTGTNTATATATATATGTGTGTGTAAGGTATATATATD5S 262 5’ TACACACACACATATATATATACACACACATATATATCTATATACTGTGTT3’ ATGTGTGTGTGTATATATATATGTGTGTGTATATATAGATATATGACACAAD5S 262 5’ AATTTAATGAGAGTTGGCAATTTTCTACAAGCAGTGTTATGAGGTATTG3’ TTAAATTACTCTCAACCGTTAAAAGATGTTCGTCACAATACTCCATAAC1153. ResultsFigure 23. D5S262 (GT)n polymorphismEE6666Eb0Segregation of the 193 and 185 bp alleles at the D55262 locus in family 35. Allelesizes 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.193 bp -1163. Results3.3. 7D5S265The subcloning and sequencing of the Alu polyA tract contained withinD5S265 was described in section 3.3.1. The sequence surrounding the D5S265A1upolyA 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 wasA17CA54GC,whichcorresponds to an amplification product of 172bp.The D5S265 primers were used for a PCR reaction involving the plasmidp52H1 and an amplification product of 172bp was obtained. An amplificationproduct of 172 bp was also obtained for GM 10114, and no amplification productwas observed when the HHW1O64 hybrid was used as a template. However, whenDNA samples from CEPH individuals were used as templates, two polymorphicsystems were amplified (Figure 25). The smaller system is referred to as AIu52Aand the larger system as AIu52B. When the PCR annealing temperature wasincreased, the intensity of the bands in each of the systems decreased by the sameproportion. The presence of the second system was therefore not due to detectionof a sequence with lower homology to the primers used. No amplification productwas observed using plasmid DNA or CEPH DNA as a template when TC65A wasnot added to the PCR reaction. Therefore, products observed were not due toamplification between two D5S265-T primers. The TC65A primer was unlabelled,indicating that amplification products observed were not due to priming between1173. Resultsadjacent TC65A sequences. The products observed for D5S265 were therefore dueto amplification between the D5S265-T and TC65A primers.1183. ResultsFigure 24. D5S265 Alu polyA tract and flankingsequence compared with Alu consensus sequencealucon 5’ GGCTGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGalucon 5’ GTGGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGalucon 5 ‘TGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGC1111111D5S265 5’CGGTGGCGC3’ GCCACCGCGalucon 5’ GCGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCTGAGGCAGGAGAATCGD5S265 5’ATGCCTGTAA CTCAGCT CTCAGGAGGCTGAGGC AGGAGAATCG3’TACGGACATT GAGTCGA GAGTCCTCCGACTCCG TCCTCTTAGCalucon 5’ CTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCACD5S265 5 ‘CTTGA CCTGGGAGGCAGAGGTTGC GTGAGCTGAGATAGCGCCATTGCAC3’ GAACT GGACCCTCCGTCTCCAACG CACTCGACTCTATCGCGGTAACGTGalucon 5’ TGCACTCCAGCCTGGGCGACAGAGCGAGACTC CGTCTCAI 11111111 11111111 1111111 111111D5S265 5’T CCAGCCTGA CGACAGAGTGAGACTCTTGTCTCAAAAAAAAAAAA3 ‘A GGTCGGACT GCTGTCTCACTCTGAGAACAGAGTTTTTTTTTTTTD5S2 65 5’ AAAAACAAAAACAAAAGCCCCAAAAAACAGTATTATCAACAAAATTGTAGG3’ TTTTTGTTTTTGTTTTCGGGGTTTTTTGTCATAATAGTTGTTTTAACATCCD5S265 5 ‘GGGCAAAAAGGAAAAAGGCAATCTGAACGAACAGGGGAAATGGTTGAATCC3’ CCCGTTTTTCCTTTTTCCGTTAGACTTGCTTGTCCCCTTTACCAACTTAGGD5S265 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-Tprimer sequence is underlined in the D5S265 sequence. The Alit consensussequence was obtained from the EMBL database version 27.1193. ResultsFigure 25. D5S265 Alu polyA tract polymorphisms12345678——186-177-172Amplification 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 areindicated on the left side of the figure. Minor bands present are shadow bandsproduced as an artifact of PCR.1203. Results3.3. Zi System A1uS2ASystem A1u52A consisted of 3 fragments of 172, 177 and 186 bp. CEPHparents observed had phenotypes of 172, 177/172, 186/172 and 186/177/172. Table15 lists offspring phenotypes observed for all informative matings.Various segregation models for the AIu52A system were tested to obtain thebest fit to the data. A single locus model with allele sizes of 186, 177 and 172bp isnot possible due to the presence of individuals with a 186, 177, 172 phenotype. Atotal of 5 families containing at least one 186, 177, 172bp individual were observed(2 parents and 15 children- Table 15).1213. ResultsTable 15. Observed matings - AIu52Aparental family offspring phenotypesphenotypes172 172,177 172,186 172,177,186172 X 172,1772 0 6 0 0104 0 10 0 01332 0 10 0 01344 6 2 0 01349 4 4 0 01362 1 9 0 01375 3 4 0 01418 3 5 0 01420 0 7 0 01333 0 9 0 01340 2 3 0 013291 3 2 0 013292 6 1 0 013293 1 5 0 0Total 29 77 0 0172 X 172,18617 1 0 3 01423 5 0 4 0Total 6 0 7 0172 X 172,177,1861346 0 5 3 01350 0 3 4 0Total 0 8 7 01223. ResultsTable 15 (con’t)parental family offspring phenotypesphenotypes172 172,177 172,186 172,177,186172,177 X 172,177102 0 13 0 01377 3 5 0 01408 2 3 0 01345 2 5 0 0Total 7 26 0 0172,177 X 172,186884 0 4 0 81421 0 0 3 51341 0 3 4 1Total 0 7 7 14172,177 X 172,177,18621 0 3 1 023 0 1 3 1Total 0 4 4 1? X 177,1721347 3 6 0 01424 0 8 0 01233. ResultsA slightly more complex model for segregation involving two loci, one ofwhich was polymorphic and the other of which was monomorphic for the 172 bpband was then tested for fit to the A1u52A data. Due to the presence of thepostulated constant 172bp band, a distinction could not be made as to whether thepolymorphic system involved three alleles of 186, 177 and 172 bp or three alleles of186, 177 and no amplification. The 172bp band is therefore indicated in bracketswhen referred to as part of the A1uS2A polymorphic system. The test for fit to thismodel was carried out as follows. Parental phenotypes were converted to the mostlikely genotypes using family segregation data. Allele frequencies for CEPH parentswere calculated based on genotypes inferred by the model (Table 16). Allelefrequencies were then used to predict expected CEPH parent phenotype valuesbased on Hardy-Weinberg equilibrium (Hedrick, 1983). AX2 test (Freedman et a!.,1980) was then used to compare observed and expected values, and a p valuecalculated. As seen in Table 17, the expected and observed values were closelycorrelated, with a 0.10< p <0.30 indicating that the data fit the single polymorphismmodel.1243. ResultsTable 16. Allele frequencies for polymorphic locuswithin A1u52A system assuming a single polymorphismAllele size(bp) frequencyAl (172) 0.675A2 177 0.260A3 186 0.065Table 17. Expected versus observed genotypes forpolymorphic loci within Alu52A system assuming asingle polymorphismgenotype expected observed(172),(172) 35 39(172),177 27 20(172),186 7 4177,177 5 8177,186 3 4186,186 0 1X2(4) = 5.69 0.1< p <0.31253. ResultsTo determine if CEPH offspring phenotypes followed Mendelian segregationassuming the single polymorphism model, a comparison was made betweenexpected and observed numbers of offspring for each parental mating (Table 18). Atest was used to compare expected and observed offspring numbers for eachmating class. A p value of greater than 0.10 was observed for all classes, indicatingthat the data fit the model well.Assuming the single polymorphism model, the polymorphic system within theD5S265 locus has a heterozygosity of 0.47 and a PlC of 0.41, using allele frequenciesobserved 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 theA1u52A locus in family 1349 is shown in Figure 26.1263. ResultsTable 18. Expected versus observed offspringphenotypes assuming a single polymorphism model forA1u52Apredicted parental offspring phenotypesgenotypes at thepolymorphic locus 172 172,177 172,186 172,177,186(i72)XL12Z) Obs 29 35 0 0(172) 177 Exp 32 32 0 0x2(1) = 0.56 0.30< p <0.50 families: 1344, 1349, 1362, 1375, 1418,1340, 13291, 13292, 13293UIZ)XJ17 Ohs 0 42 0 0(172) 177 Exp 0 all 0 0families: 2, 104, 1332, 1420, 1333U2Z)Xf.ii) Ohs 6 0 7 0(172) 186 Exp 6.5 0 6.5 0X2(1) = 0.077 0.70< p <0.90 families: 17, 1423Z)Xj77 Ohs 0 8 7 0(172) 186 Exp 0 7.5 7.5 0x2(1) = 0.067 0.70< p <0.90 families: 1346, 1350Ohs 7 13 0 0177 177 Exp 5 15 0 0x2(1) = 1.07 p 0.30 families: 1377, 1408, 13451273. ResultsTable 18. (con’t)predicted parental offspring phenotypesgenotypes at thepolymorphic locus 172 172,177 172,186 172,177,186U22)X177 Obs 0 13 0 0(172) 177 Exp 0 all 0 0family 102U7)X(172) Obs 0 3 4 1177 186 Exp 2 2 2 2x2(3) = 5 0.10< p < 0.30 family 1341Ui)X186 Obs 0 0 3 5177 186 Exp 0 0 4 4x2(1) = 0.5 p 0.50 family 1421mXU2z) Obs 0 4 0 8177 186 Exp 0 6 0 6x2(1) = 1.33 0.10< p <0.30 family 884U7)X177 Obs 0 4 4 1177 186 Exp 0 4.5 2.25 2.25x2(2) = 2.11 0.30< p <0.50 families: 21, 23Parental genotypes were predicted assuming a single polymorphism model.Expected values for offspring phenotypes were calculated assuming simpleMendelian segregation involving the predicted parental genotypes.1283. Results3.3. Z2 System A1u52BSystem A1u52B consisted of 3 fragments of 308, 310 and 312 bp. SystemA1u52B exhibited codominant Mendelian segregation for all CEPH families typed.A heterozygosity of 0.55 and a PlC of 0.46 was obtained from allele frequenciesobserved for 69 CEPH parents (Table 19). Segregation of the 308 and 310 bpalleles at the AIu52B locus in family 1349 is shown in Figure 26.Table 19. A1u52B Allele frequenciesAllele size(bp) frequencyAl 308 0.36A2 310 0.56A3 312 0.081293. ResultsFigure 26. A1u52A and AIu52B polymorphisms310 bp -_________________ _________________________________308 bp -Segregation of the 172 and 177 bp alleles at the A1u52A locus and 308 and 310 bpalleles at the Alu52B locus in family 1349. Allele sizes refer to predominant bandsand are indicated on the left side of the figure. Minor bands present are shadowbands produced as an artifact of PCR.1303. Results3.3.8 D5S266The polymorphism detected at locus D5S266 is a microsatellite repeatlocated at the 3’ end of anAlu element with a heterozygosity of 0.26 and a PlC of0.30 calculated from allele frequencies obtained by typing 53 CEPH parents (Table20). The (GT)n tract within D5S266 was subcloned as a 1.5kb Hindifi fragmentinto Hindifi digested Bluescriptil to form the plasmid p266H1.5. A portion of theDNA surrounding the (GT) tract was then subcloned as a 0.8kb XhoI-HindIIIfragment to produce the plasmid p266XHO.8 by digestion of p266H1.5 with XhoIand religating. A different portion of the DNA surrounding the (GT)n tract wassubcloned as a 0.4 kb HpaII fragment into Clal digested Bluescriptil to produce theplasniid 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 T7primers. As shown in Figure 27, the (GT) tract was at the 3’ end of anAluelement. The GT strand primer has homology with the Alit consensus sequence.The CA strand primer did not have significant homology with any sequences in theEMBL database. An additional Alu element was present in reverse orientationimmediately adjacent to the region to be amplified (Figure 27). The areasurrounding the D5S266 (GT)n polymorphism was therefore extremely rich inAluelements. Primer sequences were as follows:D5S266-GT: 5’CCTAGGTGATAGAGCAAGACC 3’D5S266-CA: 5’CAAATATAATACCACCI’CCAG 3’The sequence of the cloned product was21(TA)6G10CATACA(TA)3whichcorresponds to an amplification product of 123bp. D5S266 reference genotypes1313. Resultswere as follows: 133101 = A4,A4; 133102 = A4,A4. Segregation of the 123 and 129bp alleles in family 1375 is shown in Figure 28.The D5S266 polymorphism was somewhat difficult to amplify, and difficult toscore due to the presence of multiple shadow bands. Results obtained from theCEPH panel were therefore rather limited for this system.Table 20. D5S266 Allele frequenciesAllele size(bp) frequencyAl 129 0.085A2 127 0.057A3 125 0.009A4 123 0.821323. ResultsFigure 27. D5S266 (GT)n tract and flanking sequencecompared with Alu consensus sequenceSequence surrounding (GT)n tract and alignment with Alu consensus sequence.Primer sequences are underlined. The Alu consensus sequence was obtained fromthe EMBL database version 27.1333. Resultsalucon 5’ GGCTGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGalucon 5’ GTGGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGalucon 5’ TGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCalucon 5’ GCGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCTGAGGCAGGAGAATCGalucon 5’ CTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCACI I IIID5S266 5’TCGATGCCAC3 ‘AGCTACGGTGalucon 5’ TGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAD5S266 5 ‘TGCACTCCAGCCTAGGTGATAGAGCAAGACCCTGTCTCAAAAAAAAAAAAA3’ ACGTGAGGTCGGATCCACTATCTCGTTCTGGGACAGAGTTTTTTTTTTTTTD5S 266 5’ AAAAAAAATATATATATATATGTGTGTGTGTGTGTGTGTGCATACATATAT3’ TTTTTTTTATATATATATATACACACACACACACACACACGTATGTATATAD5S266 5 ‘ACACCGAAGCTGGAGGTGGTAGTTATATTTGTCAGCAAATTTTGCAACAAA3’ TGTGGCTTCGACCTCCACCATCAATATAAACAGTCGTTTAAAACGTTGTTTD5S266 5 ‘TTTAGGTTTTGCTTCAGAGTTGTCATTCCTTTTATGTTTCTTTTTTTTTTT3’ AAATCCAAAACGAAGTCTCAACAGTAAGGAAAATACAAAGAAAAAAAAAAAD5S266 5 ‘TGAGACAGAGTCTCAATCTGTCACCCAGGCTGGAGTGCAGTGGCACC3’ ACTCTGTCTCAGAGTTAGACAGTGGGTCCGACCTCACGTCACCGTGG111111 1111111 111111alucon 3 ‘ACTCTGCCTCAGAGCGAGACAGCGGGTCCGACCTCACGTCACGTCACCGCGD5S266 5’GTTGTCAGCTCACTGCAACCT3’3’ CAACAGTCGAGTGACGTTGGA5’alucon 3’ CTAGAGCCGAGTGACGTTGGAGGTGGAGGGCCCAAGTTCGCTAAGAGGACGalucon 3 ‘GAGTCGGAGTCGGAGGGCTCATCGACCCTAATGTCCGCGCGCGGTGGTGCGalucon 3 ‘GGCCGATTAAAAACATAAAAATCATCTCTGCCCCAAAGTGGTACAACCGGTalucon 3’ CCGACCAGAACTTGAGGACTGGAGTCCACTAGGTGGGTGGAGCCGGAGGGTalucon 3’ TTCACGACCCTAATGTCCGCACTCGGTGGTGCGGGTCGG’ 51343. ResultsFigure 28. D5S266 (GT)n polymorphismbEEbbbbESegregation of the 123 and 129 bp alleles at the D5S266 locus in family 1375. Allelesizes 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.1353. Results3.3.9 D5S268The polymorphism detected at locus D5S268 (Bernard et al., 1991b) is amicrosatellite repeat with a heterozygosity of 0.72 and a PlC of 0.68 calculated fromallele frequencies obtained by typing 80 CEPH parents (Table 21). The (GT)n tractwithin D5S268 was subcloned as a 0.4kb Alul fragment into EcoRV digestedBluescriptil to produce the plasmid p268A0.4. The sequence surrounding theD5S268 (GT)n tract (Figure 29) was determined from the plasmid p268A0.4 usingT3 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 isdeleted for one base relative to the correct sequence. The absence of this base didnot appear to affect the amplification of this polymorphic (GT) tract. Thesequence of the cloned product repeat unit was (GT)16 which corresponds to anamplification product of ll4bp. D5S268 reference genotypes were as follows:133101 = A4,A6; 133102 = A4,A6. Segregation of the 114, 118, 120 and 122 bpalleles in family 1332 is shown in Figure 30.1363. ResultsTable 21. D5S268 Allele frequenciesAllele size(bp) frequencyAl 124 0.019A2 122 0.038A3 120 0.038A4 118 0.33A5 116 0.31A6 114 0.25A7 112 0.012Figure 29. D5S268 (GT) tract and flanking sequence5’ TGCTCCTCAAAATGGCCCAATTATCAGGGGGACCAATCAGGCCATTTTTAACTTCATT3’ ACGAGGAGTTTTACCGGGTTAATAGTCCCCCTGGTTAGTCCGGTAAAAATTGAAGTAATTGATTAACATTTTAGTAATGTAACTAAATGCACACACACACACACACACACACACACACAACTAATTGTAAAATCATTACATTGATTTACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGACATACATACACTCATTTTCGCCTCACCTTTTGCAGGAAAAAAACAGAATT3’TGTATGTATGTGAGTAAAAGCGGAGTGGAAAACGTCCTTTTTTTGTCTTAA5’Sequence surrounding (GT)11 tract. Primer sequences are underlined.1373. ResultsFigure 30. D5S268 (GT) polymorphism6±6± 666±±Segregation of the 114, 118, 120 and 122 bp alleles at the D5S268 locus in family1332. Allele sizes refer to predominant bands and are indicated on the left side ofthe figure. Minor bands present are shadow bands produced as an artifact of PCR.1383. Results3.4 LINKAGE ANALYSISA goal 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. To achieve thisobjective, linkage analyses using likelihood tests (Morton, 1955) were performedwith each new polymorphic system in conjunction with previously positionedmarkers. New polymorphic markers were also positioned relative to each other.Linkage calculations were performed using the computer program packageLINKAGE version 4.7 (Lathrop et a!., 1984, 1985; Lathrop and Lalouel, 1988).3.4.1 Twopoint linkage analysisNew polymorphic markers were positioned relative to previously reportedchromosome 5 polymorphic markers (index markers). The index markers used foranalysis, D5S56, D5S59, D5S60, HPRTP2, D5S21, D5S76, D5S6, D5S39, D5S78,D5S71, D5S37 and D5S50, have been positioned on a chromosome 5 linkage mapby Weiffenbach et al., 1991.Twopoint linkage analyses were performed using the MLINK option of theLINKAGE program package version 4.7 (Lathrop et al., 1984, 1985; Lathrop andLalouel, 1988). New markers were initially subjected to a twopoint analysis with theindex markers D5S76, D5S6, D5S39, D5S78 and D5S71. These index markers werechosen 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 werealso tested in a twopoint analysis with DHFR, which is located within theapproximately 29cM region between D5S78 and D5S71, to ensure that linkage inthis interval was detected. Further twopoints were then carried out until each new1393. Resultsmarker was linked to at least two flanking index markers on each side of the newmarker or significant linkage (lod >2) could no longer be obtained. Linkage analysisplaced the new markers into three groups, those which were linked to markerswithin the 5q11.2-13.3 region (D5S201, D5S253, D5S260, D5S262 and D5S266),those which were unexpectedly linked to markers on 5p (D5S257 and D5S268), andthose which were unlinked to any chromosome 5 marker (AIu52A and AIu52B).For each group of chromosome 5 markers, new markers within each group weretested versus each other in a twopoint analysis to determine their relativepositioning.A1u52A and A1u52B were tested versus highly informative markers fromother chromosomes. Linkage was detected between A1u52A and markers onchromosome 2. A1u52B was found to be linked to markers on chromosome 17.Twopoint analyses for A1u52A and A1u52B were carried out using chromosome 2and 17 index markers until significant linkage (lod >2) could no longer be detected.Chromosome 2 index markers used (D2S51, D2S44, D2S54 and D2S43) have beenplaced on a linkage map by O’Connell et al. (1989). A linkage map of chromosome17, including index markers D17S34, D17S30, D17S31, D17S1 and MYH2, waspublished by Nakamura et al. (1988).For all loci pairs with a lod score of 2 or greater, the effective number ofrecombinants and effective number of informative meioses was calculated by themethod of Edwards (1976). The derivation of Edward’s formula is presented inAppendix 1. Table 22 contains lod score data for various recombination fractionsfor all twopoints with a lod of 2 or greater. Maximal likelihood values forrecombination fractions, maximal lod scores, effective numbers of recombinants and1403. Resultseffective numbers of informative meioses are also shown in Table 22.An estimate for the number of informative meioses scored for a polymorphicsystem can be obtained by a twopoint analysis involving the marker versus itself at arecombination fraction of 0. The method of Edwards (1976) can then be used toconvert the lod score obtained to the effective number of informative meioses. Thiscalculation was performed for all new polymorphic systems (Table 23). Theeffective number of informative meioses varied between a low of 24 for therelatively uninformative D5S262 locus to a high of 447 for the highly informativeD5S260 locus.141Table22. TwopointmeioticlinkageanalysiseMarkerloci0.050.100.150.200.250.30NRNEW5PMARKERSD5S268-D5S5613.7418.7619.8219.1317.3514.790.1519.8216925D5S268-D5S594.429.0510.4310.469.618.250.1710.5610217D5S268-D5S6021.8223.6722.9521.0518.3915.140.1023.6714815D5S268-HPRTP26.037.647.837.396.605.560.147.85639D5S268-D5S76-40.88-19.60-9.11-3.140.282.020.352.5512845D5S268-D5S6-24.01-6.541.565.687.487.690.287.7617850D5S257-D5S565.536.706.816.435.764.860.136.84517D5S257-D5S597.368.999.178.697.806.610.139.20699D5S257-D5S6011.4911.5910.859.728.326.740.0811.70655D5S257-HPRTP22.865.035.655.605.164.430.175.69559D5S257-D5S76-11.92-3.270.802.913.874.050.284.059326D5S257-D5S6-13.16-5.24-1.410.671.742.120.312.136620D5S268-D5S25725.1723.6521.4918.9816.1913.160.0325.351053Table22. (con’t)8Markerloci0.050.100.150.200.250.30NRNEW5QMARKERSD5S205-D5S76-8.10-0.153.314.875.315.000.255.329424D5S205-D5S613.9916.0215.8014.5912.7710.540.1216.1011414D5S205-D5S398.1210.9511.3410.679.367.620.1411.379113D5S205-D5S783.975.515.875.725.244.520.155.88508D5S205-DHFR6.345.745.094.423.712.9606.91230D5S205-D5S712.714.264.654.534.083.400.154.64406D5S205-D5S37-3.211.703.654.354.313.790.224.416113D5S205-D5S50-1.074.206.116.596.235.320.206.597916D5S253-D5S76-23.05-6.241.495.387.067.270.287.3416947D5S253.-D5S6-2.997.8912.0613.3412.9111.350.2113.3717236D5S253-D5S391.477.849.8710.099.327.970.1810.1510519D55253-D5S788.6411.4512.0211.6010.589.120.1512.0210215D5S253-DHFR11.5510.909.948.827.596.240.0411.61512D5S253-D5S71-1.691.903.393.963.983.600.214.005111D5S253-D5S377.2312.2913.5713.2211.919.960.1613.6012420D5S253-D5S5016.2222.0323.0121.9119.5916.430.1423.0318426Table22. (con’t)0Marker loci0.050.100.150.200.250.30NRNEW5QMARKERS-CONTINUEDD5S260-HPRTP25.088.289.138.948.176.960.169.168313D5S260-D5S2113.0115.5015.4414.2912.4910.210.1215.6611113D5S260-D5S767.6117.5220.6520.8219.2316.420.1821.0121839D5S260-D5S634.2036.5035.1532.0927.9422.960.1036.5122823D5S260-D5S394.9412.3414.5614.5513.2511.120.1714.7614324D5S260-D5S781.826.087.527.777.306.360.197.798717D5S262-D5S392.892.872.692.452.161.820.072.91151D5S266-D5S763.844.123.973.603.082.440.084.08232D5S205-D5525328.5028.0726.0923.3720.1616.540.0628.651429D55205-D5S260-9.86-0.443.715.606.145.780.256.1510827D5S205-D5S2623.693.543.252.872.441.960.053.69171D5S253-D5S260-31.91-10.59-0.634.546.997.640.307.6421464D5S260-D5S2622.142.342.272.071.791.450.112.34162D5S260-D55266-2.060.641.792.282.362.190.242.37389Table22. (con’t)eMarkerloci0.050.100.150.200.250.30ezNRCHROMOSOME2MARKERSAIu52A-D2S514.794.784.494.063.532.930.074.84252A1u52A-D2S4418.7517.6816.1314.3012.2710.040.0318.86782A1u52A-D2S549.018.287.386.415.394.330.029.22361A1u52A-D2S436.326.646.305.684.894.010.096.65394UICHROMOSOME17MARKERSAIu52B-D17S3411.3114.6915.1814.4212.8910.840.1415.2112217A1u52B-D17S3025.2228.0927.6025.5822.6018.880.1128.1718721AIu52B-D17S287.258.398.347.756.845.700.128.46607A1u52B-D17S3114.1614.6713.9512.6711.049.170.0914.71878AIu52B-D17S13.684.144.093.823.402.860.104.14263AIu52B-MYH24.345.025.034.734.253.610.135.07385=maximumlikelihoodvalueforrecombinationfraction2=maximumlodscoreR=effectivenumberof recombinants(Edwards,1976)N=effectivenumberof informativemeioses(Edwards,1976)3. ResultsTable 23. Effective number of informative meioses (N)for new polymorphic markersLocus Marker lod ND5S205 5PCR1 75.14 250D5S253 A1u24 131.85 438D5S257 A1u25 57.97 193D5S260 A1u32 134.63 447D5S262 A1u38 7.22 24A1u52A 44.07 146A1u52B 69.54 231D5S266 A1u60 17.11 57D5S268 A1u62 136.23 453Lod = lod score obtained at e = 0 for each marker versus itselfN = effective number of informative meioses for each system calculated using themethod of Edwards, 1976.1463. Results3.4.2 Multipoint linkage analysis3.4.2 1 ChromosomeS markersA general position for each of the new chromosome 5 markers was obtainedfrom twopoint linkage data. D5S266 and D5S262 could not be positioned on thechromosome 5 index map since they were each linked to only one index marker.Multipoint linkage analyses to position D5S253, D5S205, D5S260, D5S257 andD55S268 on the index map were performed using the CILINK option of theLINKAGE program package version 4.7 (Lathrop et al., 1984, 1985; Lathrop andLalouel, 1988). Index markers were included in the analysis if they gave a twopointlod score of 3 or greater with the new marker in question. Each new marker wasvaried across a region encompassing at least two index markers on either side of thenew marker and odds obtained for each order (Table 24). Recombination fractionswere obtained for the most likely marker order (Table 24).D5S268 could be unambiguously placed between index markers D5S60 andHPRTP2, since the odds against alternate locations were all greater than io6.D5S257 could be placed between D5S59 and HPRTP2, but could not be positionedrelative to D5S60 on the index map. However, when D5S268 was added to theindex map, D5S257 could be placed between D5S60 and D5S268 with odds ofgreater than io against alternate positions.D5S253 could be positioned between D5S78 and D5S71, with odds of greaterthan io against all other positions. D5S205 could not be positioned on the indexmap. However, when D5S253 was added to the index map, D5S205 could bepositioned between D5S78 and D5S253, with odds against alternate positions of1473. Resultsgreater than 102.D5S260 could be unambiguously placed on the chromosome 5 index mapbetween D5S21 and D5S76, with odds of greater than i06 against all other positions.D5S266 was significantly linked to D5S260 and index marker D5S76. D5S266 couldbe positioned distal of D5S260 by a multipoint analysis, but could not be positionedrelative to D5S76.D5S262 was linked to D5S205, D5S39 and D5S260. The order D5S260-D5S39-D5S205 could be determined from the multipoint data. The position ofD5S262 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 distancesshown in Figure 31 were calculated using Kosambi’s mapping function (Kosambi,1944).3.4.2.2 A!uS2A and A1u52BThe position of A1u52A with respect to chromosome 2 index markers andAlu52B with respect to chromosome 17 index markers was determined usingCILINK (Table 24). AIu52A could be positioned between markers D2S44 andD2S43, but could not be positioned relative to marker D5S254. A1u52B could bepositioned between index markers D17S28 and D17S1, but could not be positionedrelative to D17S31.148Table24.MultipointlinkageanalysisMarkerorder01e20405oddsD5S59-D5S60-D5S268-HPRTP2-D5S60.070.090.160.161D5S268-D5S59-D5S60-HPRTP2-D5S66.4X10D5S59-D5S268-D5S60-HPRTP2-D5S68.1X109D5S59-D5S60-HPRTP2-D5S268-D5S61.3X10’D5S59-D5S60-HPRTP2-D5S6-D5S2681.6X1026D5S59-D5S60-D5S257-HPRTP2-D5S760.080.070.210.161D5S257-D5S59-D5S60-HPRTP2-D5S768.9X103D5S59-D5S257-D5S60-HPRTP2-D5S7611D5S59-D5S60-HPRTP2-D5S257-D5S761.8X109D5S59-D5S60-HPRTP2-D5S76-D5S2577.6X1013D5S59-D5S60-D5S257-D5S268-HPRTP20.080.070.040.141D5S257-D5S59-D5S60-D5S268-HPRTP21.7X10 2D5S59-D5S257-D5S60-D5S268-HPRTP21.0X108D5S59-D5S60-D5S268-D5S257-HPRTP21.9XioD5S59-D5S60-D5S268-HPRTP2-D5S2572.1X101Table24. (con’t)Marker orderele2e3e4eoddsD5S39-D5S78-D5S253-D5S7 1-D5S370.110.120.180.021D5S253-D5S39-D5S78-D5S71-D5S371.4X10°D5S39-D5S253-D5S78-D5S71-D5S371.6X109D5S39-D5S78-D5S71-D5S253-D5S371.0X10 8D5S39-D5S78-D5S71-D5S37-D5S2532.5X1011D5S6-D5S39-D5S205-D5S78-D5S710.090.100.090.211D5S205-D5S6-D5S39-D5S78-D5S7 14.8D5S6-D5S205-D5S39-D5S78-D5S7 12.8X104D5S6-D5S39-D5S78-D5S205-D5S7136D5S6-D5S39-D5S78-D5S7 1-D5S2052.3X109D5S39-D5S78-D5S205-D5S253-D5S710.100.100.060.171D5S205-D5S39-D5S78-D5S253-D5S712.2X1011D5S39-D5S205-D5S78-D5S253-D5S7 13.0X102D5S39-D5S78-D5S253-D5S205-D5S711.2X103D5S39-D5S78-D5S253-D5S71-D5S2052.1X103Table24. (con’t)Marker order01e203e485oddsHPRTP2-D5S21-D5S260-D5S76-D5S6-D5S390.120.090.120.110.091D5S260-HPRTP2-D5S21-D5S76-D5S6-D5S394.8X1012HPRTP2-D5S260-D5S21-D5S76-D5S6-D5S391.6X109HPRTP2-D5S21-D5S76-D5S260-D5S6-D5S394.3X106HPRTP2-D5S2l-D5S76-D5S6-D5S260-D5S392.7X109HPRTP2-D5S21-D5S76-D5S6-D5S39-D5S2609.3X10I’ UiISD5S260-D5S76-D5S2660.180.091D5S266-D5S260-D5S761.3X104D5S260-D5S266-D5S762.6D5S260-D5S39-D5S205-D5S2620.170.140.051D5S260-D5S39-D5S262-D5S2051.03D5S260-D5S262-D5S39-D5S2051.18D5S262-D5S260-D5S39-D5S20510Table24. (con’t)Markerorder0102030405oddsD2S51-D2S44-AIu52A-D2S54-D2S430.100.030.020.131AIu52A-D2S51-D2S44-D2S54-D2S436X10D2S51-AIu52A-D2S44-D2S54-D2S43229D2S51-D2S44-D2S54-AIu52A-D2S4321D2S5 1-D2S44-D2S54-D2S43-A1u52A6X109D17S28-AIu52B-D17S31-D17S1-MYH20.110.060.080.101A1u52B-D17S28-D17S31-D17S1-MYH26X104D17S28-D17S31-A1u52B-D17S1-MYH21.1D17S28-D17S31-D17S1-AIu52B-MYH2537D17S28-D17S31-D17S1-MYH2-AIu52B2X106oddsoddsagainstanorderrelativetothemostlikelyorder3. ResultsFigure 31. Chromosome 5 multipoint linkage analysis -Summarypublished index map(Weiffenbach, 1991) New map5pter 5pterD5S59 D5S597 8D5S60 D5S607D5S25720 4D5S26814HPRTP2 HPRTP210 12D5S21 D5S21916 D5S26012D5S76 D5S7611 11D5S6 D5S68 9D5S39 D5S3912 10D5S78 D5S7810D5S20529 6D5S25318D5S71 D5S715qter 5qterMap distances in cM calculated using the Kosambi mapping function are indicatedto the right of each map. New markers placed on the linkage map are underlined.1533. Results3.5 HHW1O64 - CHARACTERIZATIONClones isolated byAlu PCR differential hybridization were assigned to the5q11.2-q13.3 region based on their absence of hybridization with the somatic cellhybrid HHW1O64. Genetic linkage results obtained with new polymorphic markersled to an examination of the DNA content of the HHW1O64 hybrid. Thepolymorphic systems isolated as a part of this thesis necessitated an inspection ofregions within both the p arm and the q arm of the human chromosome 5 present inthe HHW1O64 hybrid.3.5.1 HHJV1O64 Sp deletionTwo of the new polymorphic markers analyzed by meiotic linkage analysismapped to chromosome 5p (D5S257 and D5S268). Both of these markers had beenisolated on the basis of their absence of hybridization to the somatic cell hybridHHW1O64, which was believed to contain a chromosome 5 deleted only for 5q11.2-q13.3. The localization of D5S257 and D5S268 to Sp was therefore completelyunexpected. This localization was confirmed and the remainder of the isolateslocalized by hybridization ofAlu, Alu-T3 or Alu-T7 PCR products to Southern blotsofAlu PCR products from the somatic cell hybrids HHW213, GM1O114 andHHW1O64. GM1O114 contains a human chromosome 5 and HHW213 contains ahuman chromosome 5 consisting almost entirely of 5p material (Overhauser et a!.,1986a). Markers were classified as being derived from 5q11.2-q13.3 if theyhybridized GM1O114 and failed to hybridize to both HHW1O64 and HHW213.Markers were classified as located on 5p if they hybridized to GM1O114 andHHW213 and failed to hybridize to HHW1O64. Four of the twenty markers isolated1543. ResultsbyAlu PCR differential hybridization were located on 5p based on thishybridization scheme (Table 25). The remaining 16 were located within theexpected 5q11.2-q13.3 region. These results demonstrate that the somatic cellhybrid HHW1O64 contains a deletion on 5p in addition to the expected 5q11.2-q13.3deletion.D5S257 was physically localized using a somatic cell deletion mapping panelto 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 D5S257between HPRTP2 and D5S60, since HPRTP2 has been physically positioned to5pl4. (Overhauser et al., 1986b) and D5S60 has been cytogenetically mapped to5pl5.l (Weiffenbach et a!., 1991). The Sp deletion in the HHW1O64 hybridtherefore involves at least a portion of the 5pl5.l band.1553. ResultsTable 25. Marker localization to 5p or 5q11.2-q13.3Locus Probe LocationnameD5S205 5PCR1 5q11.2-13.3D5S251 5PCR2D5S252 5PCR3 5q11.2-13.3D5S254 5PCR5 5q11.2-13.3D5S253 5PCR11 5q11.2-13.3D5S255 A1u19 5q11.2-13.3D5S256 A1u22 5q11.2-13.3D5S257 A1u25 5p15.1D5S258 A1u26 5q11.2-13.3D5S259 A1u29 5q11.2-13.3D5S260 A1u32 5q11.2-13.3D5S261 A1u36 5q11.2-13.3D5S262 Alu38 5q11.2-13.3D5S263 A1u41D5S264 A1u47 5q11.2-13.3D5S265 A1u52 5q11.2-13.3D5S266 A1u60 5q11.2-13.3D5S267 A1u61 5q11.2-13.3D5S268 A1u62D5S269 A1u66 5q11.2-13.31563. Results3.5.2 HHW1O64 Sq deletionLinkage maps of human chromosome 5 report the following marker order:D5S21-D5S76-D5S6-D5S39 (Weiffenbach et a!., 1991; Westbrook et a!., 1991). The5q11.2-q13.3 deletion within HHW1O64 encompasses markers D5S6 and D5S39 butdoes not include markers D5S21 and D5S76 (Gilliam et al., 1989). The position ofthe proximal deletion breakpoint can therefore be assigned to between D5S76 andD5S6. The new marker D5S260 is not present in the HHW1O64 hybrid but maps tothe D5S21-D5S76 region based on linkage studies. The linkage results for D5S260are therefore inconsistent with the somatic cell hybrid data. Linkage and somaticcell hybrid data for markers D5S21, D5S76, D5S6, D5S39, and D5S260 weretherefore investigated.The somatic cell hybrid data obtained for hybrid HHW1O64 with markersD5S76 and D5S260 were re-examined. The first possibility addressed was thatD5S260 had been misappropriately assigned as absent from the HHW1O64. Thispossibility was excluded, since absence of D5S260 from the HHW1O64 hybrid wasconclusively 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 outsidethe deleted region was therefore investigated. D5S76 hybridized to a 10 kb TaqIfragment in HHW1O64, indicating that D5S76 maps outside the deleted region. Theinconsistency observed between the D5S260 linkage data and somatic cell hybridpositioning was therefore not due to errors in marker typing in HHW1O64.Another potential explanation for the mapping inconsistency observed forD5S260 is that D5S260 was incorrectly positioned by linkage mapping. This seemsunlikely, given the multipoint linkage analysis data placing D5S260 between D5S211573. Resultsand D5S76, with odds against all other orders of greater than i06 (Table 24).However, the position obtained for D5S260 is dependent on the order of the indexmarkers used for analysis. An incorrect position for D5S260 could be obtained ifthe order of index markers was incorrect. The odds for all possible orders of theindex markers D5S21, D5S76, D5S6 and D5S39 were therefore calculated using amultipoint analysis. As seen in Table 26, the most likely order for markers wasD5S21-D5S76-D5S6-D5S39, with odds against all other orders of greater than7X102. These linkage results are compatible with the published order of indexmarkers.An investigation of the linkage and somatic cell hybrid data involvingmarkers D5S21, D5S76, D5S6, D5S39, and D5S260 did not reveal any obviouserrors. The inconsistency between the positioning of D5S260 by linkage analysisand the absence of D5S260 from the somatic cell hybrid HHW1O64 may thereforeresult from unanticipated alterations in the HHW1O64 hybrid. The mappinginconsistency could be explained by the presence of a complex 5q deletion involvingtwo non-contiguous regions in the somatic cell hybrid HHW1O64. A deletion of theregion of DNA proximal to D5S76 in the HHW1O64 hybrid would explain theabsence of D5S260, while the deletion noticed on karyotyping the hybrid wouldinclude the markers distal to D5S76. Such a complex deletion would likely bemissed by cytogenetic analysis if the deleted region proximal to D5S76 was small.Such a complex rearrangement is the most likely explanation for the inconsistenciesin the D5S260 mapping data.1583. ResultsTable 26. Multipoint linkage analysis involving indexmarkers D5S21, D5S76, D5S6, and D5S39Marker order e1 e2 oddsaD5S21-D5S76-D5S6-D5S39 0.15 0.13 0.09 1D5S21-D5S76-D5S39-D5S6 7.0X102D5S21-D5S6-D5S39-D5S76 5. 1X103D5S21-D5S6-D5S76-D5S39 9. 1X10D5S21-D5S39-D5S6-D5S76 2.0X105all other orders > i09a odds against an order relative to the most likely order3.5.3 Cytogenetic analysis ofHHW1064An aliquot of the HHW1O64 hybrid cells used to prepare the hybrid DNAwas cytologically examined. The HHW1O64 hybrid cells contained a single humanchromosome 5 and the only cytologically detectable deletion was the expectedconstitutional deletion of 5q11.2-q13.3 (Figure 32). Approximately 1/3 of allmetaphases examined exhibited a partial or complete breakage of the humanchromosome 5 near the centromere on the long arm of the chromosome, in a regionconsistent with the Sq deletion breakpoints.1593. ResultsFigure 32. Metaphase chromosomes from somatic cellhybrid HHW1O64LIcc(IArrow indicates human chromosome 5 with constitutional deletion for 5q11.2-q13.3.1603. Results3.6 CHARACTERIZATION OF CHROMOSOMAL REARRANGEMENTSEGREGATING IN TRISOMYFAMILYThe somatic cell hybrid HHW1O64 carries a deleted chromosome 5 derivedfrom a carrier female of the family in which schizophrenia and renal anomalies cosegregate with trisomy for the 5q11.2-q13.3 region (Bassett et al., 1988; McGillivrayet al., 1990). The chromosome 5 present in HHW1O64 was demonstrated to containa 5p deletion and likely contains a complex rearrangement on 5q. The derivativechromosome 5 present within the carrier female could contain rearrangementsidentical to that found in HHW1O64, or could contain the expected 5q11.2-q13.3deletion as the sole rearrangement. An investigation of the segregation of the newpolymorphic markers in the trisomic family was therefore carried out to determinethe DNA content of the rearrangement segregating in this family..3.6.lSpThe somatic cell hybrid HHW1O64 contains a deletion on the p arm ofchromosome 5. The 5p deletion could have arisen during the generation of theHHW1O64 hybrid, or could be part of a complex constitutional rearrangement in thecarrier individual from whom HHW1O64 was derived. To determine if there was aconstitutional deletion of 5p in addition to the 5q11.2-q13.3 deletion in the carrierindividual who was the source of the chromosome 5 in HHW1O64, D5S257 andD5S268 were analyzed in the carrier’s family. D5S257 was partially informative andD5S268 was fully informative in this family (Figure 33). Segregation data indicatethat the carrier individual does not have a constitutional deletion on Sp. TheHHW1O64 hybrid therefore contains a secondary, non-cytologically visible deletion1613. Resultsof the p arm of chromosome 5 in addition to the expected constitutional 5q11.2-q13.3 deletion.3.6.1 SqThe HHW1O64 hybrid may contain a complex rearrangement involving the5q11.2-q13.3 region, with deletions of the regions on both sides of the markerD5S76. The new marker D5S260 maps proximal of D5S76 and the new markerD5S253 maps distal to D5S76. The family segregating for the segmental trisomywas therefore typed for the polymorphic loci associated with D5S260, D5S76 andD5S253 (Figure 33). D5S76 and D5S260 were partially informative in this familyand D5S253 was uninformative as all individuals were homozygous for the l2Obpallele. Segregation data indicate that the rearrangement in the trisomic familyinvolves D5S260. D5S76 segregates in a normal Mendelian fashion, indicating thatthis locus is not involved in the rearrangement.1623. ResultsFigure 33. Family segregating for segmental trisomytyped for D5S268, D5S257, D5S76 and D5S260D5S268 120,114 124,120 126,114 126,124 124,124D5S260 154,150 154,152,150 150,150 152,150 152,150D5S76 io,io 14,10 14,10 14,10 no data• 46, XY, der(1) nv Ins46, XX, nv ins(1;5)(qe23q13.3q11.2)El 46, XVSizes of alleles at D5S268, D55257, D5S76 and D5S260 loci are indicated undersymbols for individualsD53257 77,77 77,77 79,77 79,77 79,771634. DISCUSSION4.1 CLONE ISOLATIONA variety of techniques have been developed for the isolation of humanDNA fragments from chromosomal subregions. Prior to the invention of PCR,recombinant libraries containing regions of interest were produced bymanipulations 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 largequantities of genomic DNA, while libraries produced by physical microdissection ofmetaphase chromosomes (Kaiser et a!., 1987) require complex manipulations.Libraries produced from somatic cell hybrids containing all or part of a singlehuman chromosome (for example; Scambler et a!., 1987) must be screened in orderto isolate human DNA sequences from the rodent background. Region-specifichuman fragments were therefore difficult to obtain prior to the use of PCR.Alu mediated PCR has certain advantages over conventional DNA fragmentisolation. Human-specific fragments can be directly isolated from somatic cellhybrids using Alu PCR. Conventional selection procedures for distinguishinghuman and rodent isolates are therefore eliminated. Alu PCR can be performedwith small quantities of starting DNA. Regional localization ofAlu PCR derivedclones can be accomplished by hybridization to the Alu PCR products from somaticcell hybrids. Since the complexity of DNA in anA!u PCR reaction is reducedrelative to the somatic cell source, localization ofAlu PCR derived clones can beaccomplished in a shorter length of time than for conventional Southern blot1644. Discussionanalysis.Four techniques for the isolation ofAlu PCR fragments from a specificchromosomal region were reported prior to my report onAlu PCR differentialhybridization. The first technique reported, Alu PCR on YACs or phage followedby 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 bycloning of the Alu PCR material (Brooks-Wilson et a!., 1990), requires a somatic cellhybrid containing only the region of interest. The two other techniques reported(Ledbetter et a!., 1990; Patel et a!., 1990) require the detection of visible differencesbetween PCR products from various hybrids. The Ledbetter et a! (1990) techniqueinvolves the isolation of differences between the Alu-L1 PCR products of twosomatic cell hybrids which are visible on an ethidium bromide stained gel. Theprocedure reported by Patel et a! (1990) is rather complex and involves thefollowing steps: (1) A!u PCR on a monochromosomal hybrid, (2) separation of thefragments generated by agarose gel electrophoresis, (3) division into subfractions bycutting out agarose plugs from various sizes ofAlu PCR product, and (4)hybridization of the subfractions to the A!u PCR product of various hybrids in orderto detect differences by hybridization. This procedure therefore also relies on thepresence of visually detectable differences between the PCR products of varioushybrids.Alu PCR differential hybridization requires the segregation of a chromosomecontaining a deletion of the region of interest into a somatic cell hybrid. Suchdeletion chromosomes are often readily available from patients with geneticdiseases. The other components used for this procedure, a chromosome specific1654. Discussionphage library and a somatic cell hybrid containing an intact copy of the chromosomeof interest, are readily available for all human chromosomes. Alu PCR differentialhybridization therefore complements and extends the previously existing methodsfor isolation of region-specific Alu PCR fragments.The derivation of the Alu PCR differential hybridization technique dependedin a large part on the materials which were available at the time the technique wasdeveloped. 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 deletionfor 5q11.2-q13.3. Two chromosome 5 phage libraries, LAO5NSO1 and LAO5NLO3were also available. The technique ofAlu mediated PCR (Nelson et a!., 1989,Brooks-Wilson et al., 1990) was reported soon after my thesis research started. Astrategy for the isolation of clones within 5q11.2-q13.3 was therefore developedwhich made use of all of these components. This technique was called Alu PCRdifferential hybridization to highlight the use ofAlu PCR products derived fromdifferent sources as hybridization probes.Alu PCR differential hybridization is applicable to two somatic cell hybridswhich differ only in that one contains a deletion of the region of interest. When thistechnique was used in conjunction with chromosome 5 materials, isolate localizationcould be confirmed by hybridization to Alu PCR localization blots for 35/45 (78%)of clones isolated byAlu PCR differential hybridization (Table 1). Alu PCRdifferential hybridization is therefore highly effective for the isolation of regionspecific clones.Alu PCR differential hybridization should be generally applicable to any1664. Discussionhybrid - deletion hybrid pair. However, the proportion of isolates localized to thedeleted region will depend on the size of the region which is deleted. Alu elementsappear to be deficient in Giemsa (G) positive bands and enriched in G negativebands (Korenberg and Rykowski, 1988). Therefore, the number ofAlu PCR isolateslocalized to the deleted region will also depend on the chromosomal composition ofthe hybrids involved. The cytologically visible deletion within HHW1O64encompasses approximately 10% of chromosome 5, and contains both G positiveand G negative bands (Gilliam et a!., 1989). A total of 479 chromosome 5recombinant clones were screened for differential hybridization, and 35 were absentfrom the HHW1O64 hybrid (Table 1). Twenty-five of these 35 isolates were from 5qand 10 were from 5p. The cytologically visible deletion therefore accounted for25/479 (5%) of isolates and 10/479 (2%) were from a non-cytologically detectable parm deletion. The fraction of isolates from Sq was close to that expected based onthe cytologically determined size of the deletion. The large fraction of p armisolates is likely due to an abundance ofAlu elements within this region.Flow sorted chromosome 5 libraries were used rather than genomic phagelibraries to minimize the number of phage screened, and to reduce the number offalsely positive phage in the initial screen. False positives could arise due toincomplete preannealing of repetitive elements present in the Alu PCR product ofthe chromosome 5 hybrid. These false positives should be eliminated by thesecondary screen, since the Alu PCR product from the deletion 5 somatic cell hybridwould be expected to contain the same unblocked repetitive elements. Conversely,some isolates located within the deletion region could be missed if they containedrepetitive elements which were not preannealed.1674. DiscussionThe large insert phage library LAO5NLO3 was of greater value for Alu PCRdifferential hybridization than the LAO5NSO1 library for several reasons. A largerproportion of phage from the LAO5NLO3 library were positive when hybridized withthe Alu PCR product of the chromosome 5 hybrid (2.2% compared with 0.4% forLAO5NSO1), thereby decreasing the number of phage in the initial screen. Theproportion of clones containing the 3’ ends of both flanking Alu elements was alsohigher in the LAO5NLO3 library than the LAO5NSO1 library. The inter-Alu regioncould be amplified from 15/15 clones obtained from the LAO5NLO3 library butfrom only 1/5 clones from the LAO5NSO1 library. The use of a large insert phagelibrary therefore reduces the effort required for clone isolation and localization.Isolate location was confirmed by hybridization to cell hybrid genomic DNAfor 8/8 clones tested (section 3.1.4). Absence of amplification from the somatic cellhybrid 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 wereinappropriately assigned to the deletion region using Alu PCR localization blots.Such isolates could arise due to polymorphic differences between chromosomes inthe placement or structure ofAlu elements. Differences in the degree ofAlu PCRamplification between the two hybrids could also lead to falsely assigned clones. Noisolates were found which had been falsely assigned by the Alu PCR localizationblots. Therefore, differences between GM1O114 and HHW1O64 inAlu placementorAlu PCR amplification do not appear to be frequent.The AlS primer used for Alu PCR was from the extreme 3’ end of the Aluconsensus sequence, and therefore generates a minimum ofAlu-homologousmaterial (Brooks-Wilson et al., 1990). The use of the A1S primer therefore allows1684. Discussionthe production of unique Alu PCR products. However, only 5/l6Alu PCR productsisolated byAlu PCR differential hybridization were unique (Table 5). Thisrepetitive nature of the Alu PCR products suggests a potential clustering ofrepetitive elements in the inter-Alu region. The nature of the repetitive elementspresent within the Alu PCR products was not pursued further, since their presencedid not hinder the isolation of clones byAlu PCR differential hybridization.Only 2/20 isolates located within the deletion region were discernible as adifference between the Alu PCR product of the chromosome 5 hybrid and thedeletion 5 hybrid on an ethidium bromide stained gel (section 3.1.6). The methodof differential hybridization therefore can be used for isolation of non-visuallydiscernible differences between two related somatic cell hybrids.In summary, Alu PCR differential hybridization is a highly specific methodfor the isolation of region-specific DNA fragments which complements and extendspreviously existing methods. Localization of clones was rapidly confirmed byhybridization to the Alu PCR products of the somatic cell hybrids. Alu PCRdifferential hybridization should be generally applicable to any somatic cell hybrid-deletion hybrid pair.4.2 RADIATION HYBRID MAPPINGThe recently developed procedure of radiation hybrid mapping allows theordering of widely spaced markers in the human genome (Cox et a!., 1990).Radiation hybrid mapping complements and extends conventional mappingtechniques, such as meiotic linkage mapping, PFGE with rare-cutting enzymes, andin situ hybridization. Radiation hybrid mapping also has certain advantages since it1694. Discussioncan be performed with small, moderately repetitive non-polymorphic markers, anddoes not depend on recombination frequencies or restriction enzyme sitepositioning. Radiation hybrid mapping was therefore selected for physicallyordering clones isolated byAlu PCR differential hybridization.To type the chromosome 5 radiation hybrid mapping panel each of thehybrids was amplified using Alu PCR, and then Southern blotted. The Alu PCRproducts from the clones isolated byAlu PCR differential hybridization were thenhybridized to the Southern blots of the hybrid Alu PCR products. The use ofAluPCR allows rapid typing of markers, since the decreased complexity ofAlu PCRproducts relative to hybrid genomic DNA allows a reduction in exposure time.Since radiation hybrids can be somewhat unstable, it is recommended that a singlebatch of hybrid DNA be used to screen all probes (Cox et a!., 1990). If multipleinvestigators wish to screen the same batch of hybrid DNA, it is useful if each screencan be accomplished with a minimal amount of DNA. The use ofAlu PCR enablesscreening of a large number of probes with a very small amount of radiation hybridDNA. Alu PCR also offers the advantage that probes which are highly repetitive onhybridization to genomic DNA may appear non-repetitive or only slightly repetitivewhen used to screenAlu PCR products, due to the reduced complexity of the AluPCR product relative to the genomic DNA source. Time spent isolating uniqueprobes can therefore be reduced. Obviously, care must be taken that the sameprimer(s) used to isolate the Alu PCR probe are used to amplify the radiationhybrids.D5S39 was typed by Dr. Solomon by hybridization to Southern blots ofgenomic DNA from the radiation hybrids. AnAlu PCR isolate was not available for1704. DiscussionD5S39, so the genomic typing results were used for this probe. The sensitivity oftyping using hybrid genomic DNA may be somewhat different from that obtained byusing hybrid Alu PCR products. However, these differences were not large, sincecomparable retention frequencies were obtained for D5S39 and the Alu PCRisolates. As will be discussed later, the positioning of D5S39 by radiation mappingwas comparable to that obtained by meiotic linkage analysis. Therefore, inclusionof this probe in the radiation hybrid analysis did not appear to cause anydiscrepancies.Radiation hybrid mapping in conjunction with Alu PCR was used to rapidlyobtain radiation hybrid retention data for clones isolated byAlu PCR differentialhybridization. The algorithms of Cox et a!., 1990 (Appendix 1) were used foranalysis of the radiation hybrid retention data. Markers were initially analyzed inpairs using twopoint algorithms. A lod score of 3 was considered evidence ofsignificant linkage between markers. Using this criteria, markers could be placedinto four groups. Group #1 consists of 7 linked markers, group #2 of 4 linkedmarkers, group #3 of 2 pairs of linked markers, and group #4 of 4 markers unlinkedto any other marker (Table 7). Each marker was significantly linked to a maximumof two other markers. The order of markers suggested by the twopoint analysis wastherefore assumed to be the most likely. The relative likelihoods of inversion ofpairs of markers was then calculated for groups #1 and #2 using fourpointalgorithms. 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 ingroup #1 could not be positioned. Radiation hybrid mapping was therefore usefulfor grouping markers and obtaining a rough order of certain groups of markers.1714. DiscussionThe fact that not all markers could be ordered by radiation hybrid mappingreflects in part the density of markers used. The cytologically visible deletion in thesomatic cell hybrid HHW1O64 spans approximately 10% of chromosome 5, orroughly 20Mb. The 18 markers typed in the chromosome 5 radiation panel wereexpected to be located within this 20Mb region. As will be discussed later, the 18markers actually span a much larger region, since 3 of the markers were located on5p. The density of markers used to screen the chromosome 5 radiation panel wastherefore relatively low.The amount of radiation used to create the chromosome 5 radiation hybridmapping panel was another reason why linkage was not obtained between allmarkers typed in the radiation panel. Marker retention frequencies vary accordingto the amount of radiation used to make a panel. Cox et a!. (1990) used 8,000 radsto create their radiation hybrid panel and obtained marker retention frequencies of32% to 59%. The chromosome 5 radiation panel was made using 50,000 rads andmarker 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 toobtain overlapping retentions and therefore significant linkage.Radiation hybrid distances indicate the frequency of breakage betweenmarkers after exposure to a given amount of x-rays. Radiation distances betweensignificantly 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 100cR50000 limit (100% frequency of breakage between markers).Radiation hybrid mapping was used to position 18 new chromosomeS1724. Discussionmarkers and D5S39 into 4 groups. This information was then used to determinewhich markers to investigate further. Radiation hybrid mapping therefore serves asa valuable tool for the rapid ordering of non-polymorphic loci.4.3 POLYMORPHISM SCREENING AND LINKAGE ANALYSISThe loci used for polymorphism screening were determined largely by theresults of the radiation hybrid mapping. Radiation hybrid group #1 consisted of 7markers, 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 interestto 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 mapclose to the SMA disease gene. Polymorphisms from radiation hybrid groups #2and #3 were of interest to allow comparison of radiation and linkage maps, and toposition these groups within the 5q11.2-q13.3 region.During the course of this thesis, screening was done for three types ofpolymorphisms; conventional RFLPs, polymorphic (GT)n microsatellite repeats andpolymorphisms inAlu polyA tails. Screening for conventional RFLPs was doneprior to radiation hybrid mapping and before much information was available on(GT)n polymorphisms. RFLP screening was therefore done with the isolates fromthe firstAlu 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 forD5S205 (section 3.3.2). D5S205 was mapped by linkage analysis between D5S78and D5S71.1734. DiscussionSubsequent to the screen for conventional RFLPs, the radiation hybridmapping was finished and extensive information was available on theinformativeness 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 ormore repeats would be detected (Weber and May, 1989), since tracts with morethan 10 repeats had been reported to often be polymorphic (Weber, 1990). A totalof 6 (GT) tracts were detected, 5 of which were from radiation hybrid group #1and one which was from radiation hybrid group #3. All of these tracts weretherefore of interest, and were tested to determine if they were polymorphic. All ofthe tracts were polymorphic, with PlC values ranging between 0.06 and 0.75 (Table8).(GT)n tracts isolated fell into two categories, four tracts which were presentas isolated tracts and two which were at the 3’ ends ofAlu elements. The fourisolated (GT)n tracts amplified well and alleles were easy to score. D5S253 andD5S268 had uninterrupted GT runs of 16 and 23 and were highly informative, withPlC values of 0.75 and 0.68, respectively. D5S260 and D5S257 had 11 and 12uninterrupted GT residues, and had PlC values of 0.69 and 0.29. These four tractstherefore followed the general rules for informativeness determined by Weber(1990), namely that tracts of 16 or more uninterrupted GT repeats would be highlyinformative, while tracts with repeat numbers of between 11 and 15 would be highlyvariable in terms of their PlC values.Linkage mapping positioned D5S253 between index markers D5S78 andD5S71 (Figure 31). D5S253 is currently the closest marker to the distal 5q11.2-1744. Discussionq13.3 deletion breakpoint, and provides a highly informative marker within the 29cM gap between D5S78 and D5S71 (Weiffenbach et a!., 1991). D5S260 linkagemaps between index markers D5S21 and D5S76 (Figure 31). This positioning wasunexpected, since both D5S21 and D5S76 map outside the 5q11.2-q13.3 deletionregion (Gilliam et a!., 1989). The positioning of the (GT) polymorphismsassociated with D5S257 and D5S268 was also unexpected, since both D5S257 andD5S268 were found to linkage map to 5p (Figure 31). As will be discussed later, thediscovery of these three markers led to an analysis of the HHW1O64 hybrid and thedeletion chromosome 5 from which the hybrid was derived.The two (GT)11 tracts at the ends ofAlu elements were of two types. TheD5S262 tract was a cluster of short repeats of TG and TA, and the longestuninterrupted repeat was 6 units long (Figure 22). Although this system waspredicted to be uninformative using Weber’s (1990) rules, the tract was investigatedbecause of its position in radiation hybrid group #1. The D5S262 polymorphismwas, as expected, relatively uninformative, with the least common allele just abovethe 0.01 frequency level normally used to define a system as polymorphic (Hedrick,1983). The probability that D5S266 would be polymorphic was much harder toassess, 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 expectedfor a (GT) tract of 10 repeats (Weber, 1990). The presence of the Alu polyA tailmay therefore have contributed to the level of informativeness. Neither D5S262 norD5S266 could be accurately positioned on the chromosomeS linkage map. D5S262was insufficiently polymorphic to allow placement. Amplification of the D5S266(GT) tract and 3’ Alu region resulted in the production of a large amount of1754. Discussionshadow bands, positioned at 1 bp intervals from approximately 2 bp above to 10 bpbelow the major band amplified (Figure 28). Similar shadow bands are generallyobserved at 2 bp intervals for most isolated (GT)n tracts. The shadow bands are anin vitro artifact, since they are also seen for amplifications involving plasmidtemplates. For the majority of (GT)n systems, these shadow bands do not interferewith typing. However, the presence of excessive shadow bands made typingextremely 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 hybridgroup #2 and a portion of group #3. Alternate methods for detection ofpolymorphism were therefore investigated for these loci. Since clonescorresponding 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 forthe 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 andHobbs, 1990). The technique described by Economou et a! (1990) involveddetection of polymorphisms inAlu polyA tails by amplification using a primerspecific to the Alu element in question along with a primer from the non-repetitiveregion flanking the Alu element. The hypothesis was tested that this procedurecould be modified slightly, such that the Alu primer was derived from the Aluconsensus sequence, rather than the sequence of the specific Alit element.To isolate Alu elements, portions of the recombinant phage from loci ofinterest were subcloned into plasmids, and the plasmids screened for the presenceof a single Alu element. Sequence for the Alu polyA tail was then obtained using1764. Discussionthe TC65A primer, which is homologous to the Alu consensus sequence. Since onlya few polymorphisms involving Alu polyA tails have been detected thus far,guidelines have not been established for estimating the probability that a tract willbe polymorphic. Taking (GT) tracts as an example, the polymorphism level ofAlupolyA tails is likely proportional to the number of repeat units. Tracts from D5S254and D55265, with 15 and 17 residues respectively, were therefore investigated forpotential polymorphisms. A specific amplification product for the D5S254 AlupolyA tail was not obtained under a variety of amplification conditions. This wassomewhat unexpected, since the TC65A primer had only one mismatch at the 5’ endwith the sequence of the D55254 Alu element. An appropriately sized amplificationproduct could be obtained using plasmid or phage template, which implies that theproblem did not involve the primers or PCR system. One possible explanation isthat the phage could have been rearranged during cloning, potentially due torecombination betweenAlu elements. The sequence obtained could therefore bedifferent from that present in genomic DNA. Although each unique primer used forPCR was carefully screened for secondary structure and for sequence homologies,the primer flanking the Alu element could have been the problem if it either wouldnot bind to the target sequence due to secondary structure constraints, or wassomewhat repetitive, and bound to numerous places elsewhere in the genome. TheD5S254 polyA tail tract was not investigated further.The Alu polyA tract from D5S265 was amplifiable with the expected 172 bpsize from both plasmid and genomic DNA. Additional amplification products wereobserved in genomic samples at 177 and 186 bp, which were initially assumed torepresent alleles at the D5S265 locus. However, CEPH individuals were observed1774. Discussionwith a phenotype of 186,177,172, (Table 15) which is not possible under a singlelocus model. A variety of segregation models were therefore examined for fit to thedata. The model which best fit the data was found to be two loci, one of which waspolymorphic and the other which was monomorphic for the 172 bp band (singlepolymorphism model). The polymorphic locus was found to be in Hardy-Weinbergequilibrium assuming a single polymorphism model, which would be highly unlikelyif the model was incorrect. Also, phenotype frequencies for all CEPH offspring fitthose expected for a single polymorphism model for all parental mating classes.Linkage analysis demonstrated that the polymorphic locus was located onchromosome 2.An additional polymorphic system with fragment sizes of 308, 310 and 312bpwas observed using the TC65A and D5S265-T primers (Figure 25). The largersystem was not amplifiable from the GM1O114 hybrid, indicating that theamplification products observed were not from chromosome 5. Linkage analysisdemonstrated that this system was located on chromosome 17.Three systems were amplified using the TC65A and D5S265-T primers, onefrom each of chromosomes 2, 5 and 17. No amplification product was observedusing D5S265-T primer alone and the TC65A primer was unlabelled. The systemsobserved for D5S265 were therefore due to amplification between the D5S265-Tprimer and the TC65A primer. As the annealing temperature for PCR wasincreased, the amount of product obtained decreased in an identical fashion foreach of the three systems. Therefore, the primer binding sites for these threesystems were very similar or identical.The three systems amplifiable using the D5S265-T and TC65A primers likely1784. DLccussionarose due to duplication by an unknown mechanism. One possible duplicationmechanism would involve Alu element transposition. This mechanism is unlikely,however, since Alu elements are thought to transpose utilizing an RNAintermediate (Ulla and Tschudi, 1984; Chen et a!., 1985) and the transpositionalevent postulated here would necessarily include flanking sequences. The formalpossibility exists that the D5S265-T primer sequence serves a functional role onchromosomes 2, 5 and 17. This seems somewhat unlikely, however, due to the factthat the primer was derived from anonymous sequence. A more likely explanationis that the D5S265-T primer is located within a low copy repetitive element whichhad not yet been sequenced when the database search was made. The juxtapositionof the D5S265-T primer sequence with the 3’ end of anAlu element could havearisen 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 ofrepetitive elements (Weiner et al., 1986) provides support for this type ofmechanism. The presence of sequences homologous to D5S256-T on chromosome2 together with an identical size amplification product to that obtained forchromosome 5 could then be explained by a duplication event of a low copyrepetitive element containing the Alu element. The chromosome 17 locus could bethe result of an independent Alit insertional event, or could be due an additionaltransposition event of the low copy repeat containing the Alu element. Furtherevidence for the common origin of the chromosome 2 and 5 systems, with a moredistant origin for the chromosome 17 system is the appearance of the amplificationproducts for the various systems. The chromosomes 2 and 5 systems exhibited alarge number of shadow bands, similar to that observed for the D5S266 system,1794. Discussionwhile the chromosome 17 system was relatively devoid of shadow banding. Whilethe difference in the proportion of shadow bands could be a technical artifact, itcould also be due to sequence differences within the amplified products from thevarious loci, with fewer repeats present in the polyA tail from chromosome 17.For the D5S265 Alu polyA tail amplification, difficulties experienced weredirectly related to the primer flanking the Alu element. The presence of twomismatches between the TC65A primer and the Alu sequence did not appear tohave any detrimental effect. The causes of the amplification problems for theD5S254 system are more difficult to ascertain, but are unlikely to solely involve themismatch present between the TC65A primer and the D5S254A1u sequence. AlupolyA tail polymorphism detection using a primer specific to the Alu consensussequence 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 duringthe 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 mostinformative, followed by (GT)n tracts with between 10 and 15 repeats. (GT) tractsassociated with Alu polyA tails were more difficult to type due to the presence ofexcessive shadow banding. Alu polyA tracts can serve as sources of polymorphismswhen (GT)n tracts are unavailable, but do not seem generally attractive due topotential problems with shadow bands and difficulties with amplification of the tractof interest.1804. Discussion4.4 COMPARISON OF MAPPING METHODSPositional information on markers isolated byAlu PCR differentialhybridization was initially obtained using radiation hybrid mapping. Linkagemapping was then performed for markers of interest to place them relative to otherpolymorphic markers. Figure 34 depicts results obtained for markers which weremapped using both methods, which included index marker D5S39 and new markersD5S257, D5S268, D5S260, and D5S253. All markers mapped using both methodswere present within radiation hybrid group #1. The orientation of radiation hybridgroup #1 as indicated in Figure 34 was based on the somatic cell hybrid localizationof markers D5S39 to 5q11.2-q13.3 (Gilliam et a!., 1989) and D5S257 to 5pl5.1 (JohnMcPherson, pers. comm.). Although D5S262 and D5S264 were not positioned onthe linkage map, they are shown in the radiation map in Figure 34 to preventdiscontinuities. D5S260 and D5S268 could not be positioned relative to D5S262 byradiation hybrid mapping, and are therefore indicated in brackets immediatelyadjacent to D5S262 on the radiation map.The order of markers obtained by radiation hybrid mapping was analogous tothat obtained by linkage mapping for D5S257, D5S260, D5S268 and D5S39. Thesole discrepancy between the two maps was the position of D5S253, which wasplaced between D5S78 and D5S71 by linkage mapping, but was positioned next toD5S257 by radiation hybrid mapping. Somatic cell hybrid localization haspositioned D5S78, D5S71 and D55253 onto 5q (Tables 25 and 27), while D5S257has been placed on 5p (Table 25). The linkage placement of D5S253 betweenD5S78 and D5S71 is therefore confirmed by the somatic cell hybrid localizationdata, indicating that D5S253 was incorrectly positioned by radiation hybrid mapping.1814. DiscussionWhile the map order obtained by radiation hybrid mapping is very similar tothat obtained using linkage analysis, inconsistencies exist when distances betweenmarkers are considered. D5S257 maps to 5pl5.l and D5S262 maps to 5q11.2-q13.3by somatic cell hybrid mapping (Table 25). The distance between D5S257 andD5S262 therefore encompasses approximately 1/4 of chromosome 5, and yet thesemarkers were linked by radiation hybrid mapping. It seems highly unlikely thatD5S257 and D5S262 would be linked by radiation hybrid analysis given that noradiation hybrid linkage was detected for the marker pairs D5S257-D5S268 andD5S205-D5S253, which are located close to each other on the linkage map. Thisdifference in detection of radiation hybrid linkage can be partially attributed todifferences 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 couldcompletely explain the radiation hybrid linkages obtained.1824. DiscussionFigure 34. Comparison of radiation hybrid map andmeiotic linkage mapPublished Linkage map Radiation hybrid mapOrder5pterD5S59D5S253D5S60D5S257 D5S257D5S268HPRTP2D5S21D5S260 D5S262 (D5S260; D5S268)D5S76D5S6D5S39 D5S39D5S264D5S78D5S205D5S253D5S71qter1834. Discussion4.5 CHARACTERIZATION OF HHWJO64 AND SEGMENTAL TRISOMYThe somatic cell hybrid HHW1O64 was demonstrated to contain a deletionfor 5q11.2-q13.3 by the absence of hybridization of markers known to be presentwithin this interval (Gilliam et a!., 1989). Lack of hybridization with HHW1O64 wastherefore used as a basis for the isolation of probes from 5q11.2-q13.3 byAlu PCRdifferential hybridization. As a result of genetic linkage analysis with newpolymorphic markers, the DNA content of the HHW1O64 hybrid was examined.The polymorphic systems isolated as a part of this thesis necessitated an inspectionof regions within both the p arm and the q arm of the human chromosome 5 presentin the HHW1064 hybrid. An examination was also made of the derivativechromosome 5 present in the carrier female from whom HHW1O64 was derived.Linkage data on two of the clones isolated byAlu PCR differentialhybridization (D5S257 and D5S268) unequivocally positioned them on the p arm ofchromosome 5. HHW1O64 therefore carries a non-cytologically visible deletion onthe p arm of chromosome 5 in addition to the expected constitutional 5q11.2-q13.3deletion. The position of the 5p secondary microdeletion was partially delineatedby physical mapping of D5S257 using a chromosome 5 somatic cell hybrid mappingpanel (John McPherson, pers comm). D5S257 maps to a tightly defined region of5p15.l within region I as defined by Overhauser et a!., 1987. The HHW1O64 5pmicrodeletion therefore encompasses at least part of the 5pl5.l band.To confirm the 5p localization of D5S257 and D5S268 markers and tolocalize the remainder of the new markers, each isolate was hybridized to HHW213,a hybrid which contains predominantly 5p material. Four out of twenty distinctisolates were found to be located on the p arm of chromosome 5 (Table 25). When1844. Discussionmultiple isolates are included, 10/35 (29%) of isolates were from the non-cytologically detectable deletion within 5p. The large number of p arm isolatescompared to the number of isolates obtained from the cytologically detectable 5qdeletion is likely due to the relative Alu-richness of the two regions, with the area ofthe p arm deletion being particularly rich inAla elements.The 5q deletion within the HHW1O64 hybrid involves markers D5S6 andD5S39 but does not include markers D5S76 and D5S21 (Gilliam et al., 1989). Themarker D5S260 was positioned between D5S21 and D5S76 by multipoint linkagemapping, but is not present within the somatic cell hybrid HHW1064. Anexamination of the somatic cell hybrid and linkage data was performed in anattempt to resolve this inconsistency. D5S260 was clearly demonstrated to be absentfrom 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 weredetected.Linkage results for the D5S21-D5S39 region were closely examined todetermine any potential errors. The consensus order obtained from linkage analysiswas D5S21-D5S76-D5S6-D5S39 (Weiffenbach, et a!., 1991; Westbrook et a!., 1991;this thesis). However, a careful examination of these data revealed certainproblems with the linkage map in this region. Weiffenbach et a!., 1991 used theBUILD option of CR1-MAP to produce a multipoint linkage map of markerspositioned with greater than 1000:1 odds against all other positions. However, thispaper also reports data using the FLIPS option of CR1-MAP, which gives the oddsof inversion of 1:1.6 for D5S76 and D5S6 and odds of “better than 1:1000” forinversion of the entire D5S76-D5S6-D5S39 segment (Weiffenbach et a!., 1991). The1854. Discussionlinkage map reported by Westbrook et al, 1991 was made by the compilation of datafrom both CEPH families and other families. While the order of markers wasreported to be the same for all sources of data, the relative odds of inversion ofmarkers in each study was not indicated (Westbrook et a!., 1991). Finally, twopointlinkage data obtained for D5S260 placed D5S260 closer to both D5S21 and D5S6than to D5S76 (Table 22). These results suggest a problem with the linkage data forD5S76. A careful examination of the linkage data therefore suggests that the orderof markers in the vicinity of D5S76 is somewhat questionable.As the number of polymorphic markers identified in humans increases, it hasbecome evident that data errors are a limiting factor in the production of multipointlinkage maps (Keats et al., 1991; Sheilds et a!., 1991). Data errors can include errorsin reading or entering data, interchange or mislabelling of samples ormisinterpretation of bands or reactions (Keats et al., 1991). Data errors can alterboth distances between markers and marker order. Duplicate typing of all loci willcontrol for data error, but is not feasible for the majority of systems due to time andcost constraints. Retesting of all apparent recombinants over small distances and alldouble recombinants over moderate distances is helpful but is not done routinelydue to logistic constraints (Keats et a!., 1991; Shields et a!., 1991).Alternate mapping methods can often provide information regardingpotential errors in linkage maps. Physical localization data for chromosome 5 indexmarkers from published reports were therefore examined (Table 27). D5S21 waspositioned within 5pl3-pll using a somatic cell hybrid localization panel(Overhauser et a!., 1987). D5S6 and D5S39 were mapped by in situ hybridization to5q12-q13.1 and 5q13, respectively (Mattei et al., 1991). The order of these markers1864. DiscussionObtained 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 acombination of linkage data reported by Leppert et a!., 1987 and the presence ofD5S76 in the HHW1O64 somatic cell hybrid observed by Gilliam et a!., 1989.Physical localization data for D5S76 which are clearly distinguishable from linkagedata are therefore not available. While the physical localization results are clearlyincomplete without data on D5S76, no evidence was observed against the linkageorder D5S21-D5S76-D5S6-D5S39. Also worthy of mention is the HHW1O64 dataplacing D5S21 and D5S76 outside the deletion while D5S6 and D5S39 are locatedwithin the deleted region (Gilliam et a!., 1989). Any inversion of marker ordersincluding D5S76 such as D5S21-D5S6-D5S76-D5S39 or D5S21-D5S39-D5S6-D5S76implies the presence of two noncontiguous deletions in the HHW1O64 hybrid. TheHHW1O64 hybrid data therefore lend support to the D5S21-D5S76-D5S6-D5S39order.In the absence of evidence to the contrary, the somatic cell hybrid HHW1O64contains a complex deletion in the 5q11.2-q13.3 region, encompassing at least twonon-contiguous segments on either side of D5S76. A diagrammatic representationof the derivative 5 chromosome postulated to be present in the HHW1O64 hybridshown in Figure 35. Cytological locations for index markers were used to determineapproximate breakpoint positions (Table 27).1874. DiscussionTable 27. Physical location of index markerslocus cytological location referenceD5S59 5pl5.2-5p15.l Weiffenbach et a!., 1991D5S60 5pl5.l Weiffenbach et a!., 1991HPRTP2 5pl4 Overhauser et a!., 1986bD5S21 5pl3-pll Overhauser et a!., 1987D5S76 5cen-qll.2 Bishop and Westbrook., 1990D5S6 5q12-q13.1 Mattei et al., 1991D5S39 5q13 Mattei et a!., 1991D5S78 5q11.2-q13.3 Gilliam et al., 1989D5S71 5q14-q21 Bishop and Westbrook., 1990D5S37 5q21 Stewart et a!., 1987A breakage of the human derivative chromosome 5 present withinHHW1O64 was detected at or near the 5q11.2-q13.3 deletion breakpoint inapproximately 1/3 of metaphases examined. Since a common fragile site is notpresent at or near this region of chromosome 5 (McAlpine et al., 1990), this fragilityappears to be a specific to the derivative chromosome 5. Fragility at translocationbreakpoints has been documented in the lymphocytes of various translocationcarriers (for example, Juberg et al., 1983; Drets and Therman, 1983), and isgenerally attributed to the juxtaposition of regions of DNA not normally found nextto each other. The fragility at the 5q11.2-q13.3 deletion breakpoints in theHHW1064 hybrid may have been due to such DNA interactions or may merely havebeen an artifact of culture. Fragility at this site was not examined in the carrierfemale from whom HHW1O64 was derived. However, the carrier female was1884. Discussionphenotypically normal, indicating that the postulated fragile site had no clinicalsignificance.The cytogenetically balanced carrier from whom HHW1O64 was derived hasa deleted chromosome 5 and a chromosome 1 with an insertion of chromosome 5material. This individual is a member of a family in which segmental trisomy for5q11.2-q13.3 segregates with schizophrenia and other anomalies (Bassett et a!.,1988; McGillivray et a!., 1990). Segregation for the (CA) polymorphismsassociated with D5S268 and D5S257 was studied in this family to determine if the5q11.2-q13.3 deletion chromosome in the carrier individual was also deleted for 5p.Segregation at the D5S76, D5S253 and D5S260 loci was also investigated todetermine if the 5q deletion was the result of a complex rearrangement. TheD5S268 locus was fully informative in this family (Figure 33). The polymorphismsassociated with D5S257, D5S76 and D5S260 were partially informative and D5S253was 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 5from his mother and a normal chromosome 5 from his father (Figure 33). Theaffected son is disomic at the D5S268 locus, inheriting the maternal 124 allele. Ifthe D5S268 locus were translocated to the der(1) chromosome then the affected sonwould have been trisomic for D5S268. It is unlikely that the D55268 locus is deletedfrom the der(5) chromosome and inserted elsewhere in the genome since the intactchromosome 5 transmitted to each of the sons carries a different maternal D5S268allele necessitating at least one recombination event. Therefore, the chromosomalrearrangement segregating in this family does not involve 5p.1894. DiscussionThe deletion 5 chromosome present within the carrier female contains the 10kb D5S76 allele, since this allele is present in the HHW1O64 hybrid. The normalchromosome 5 present within the carrier female therefore carries the 14 kb D5S76allele, which is passed to both sons. Normal Mendelian segregation is thereforeobserved at the D5S76 locus (Figure 33). The carrier female has 150 and 152 bpalleles at the D5S260 locus, one of which is located on her normal chromosome 5and the other which is on her derivative 1 chromosome. She passes both alleles atthe D55260 locus to her affected son, since the affected son is trisomic at theD5S260 locus (Figure 33). The trisomic region therefore includes the D55260 locusbut does not contain the D5S76 locus. The deletions on either side of D5S76 in thederivative chromosome 5 present in the somatic cell hybrid HHW1O64 weretherefore derived from the carrier female.The chromosomeS rearranged as a part of the segmental trisomy may havehad a marker order of D5S21-D5S260-D5S76-D5S6, which is identical to thatobtained in the CEPH panel. Given this marker order, the deletion of D55260 andD5S6 without the deletion of D5S76 requires a complex rearrangement.Alternatively, if the chromosome 5 carried an inverted marker order of D5S21-D5S76-D5S260-D5S6, a single deletion event would explain the removal of D5S260and D5S6 without deletion of D5S76. This inversion event could have arisen whenthe complex rearrangement occurred in the family segregating for the segmentaltrisomy, or could be present in an ancestral chromosome. A distinction between thevarious origins of the rearrangement could be made by determining the orientationof D5S76 on the deleted chromosome.The results presented as a part of this thesis indicate that a de novo1904. Discussiondeletion is present in the HHW1O64 somatic cell hybrid. Results were also obtainedsupporting the hypothesis that a complex rearrangement involving 5q11.2-q13.3 ispresent in HHW1O64 and is segregating in the trisomic family from whichHHW1O64 was derived. The presence of a complex rearrangement segregating inthe trisomic family has important implications regarding the position of the putativegenes for schizophrenia susceptibility and renal development. The region involvedin the segmental trisomy, and thus the region to which these genes would beassigned, can now be expanded to include regions both proximal and distal toD5S76. Linkage studies involving schizophrenia and hereditary renal adysplasiamust therefore include markers both proximal and distal to D5S76. Diagrammaticrepresentations of the derivative chromosome 5 postulated to be present withinHHW1O64 and the carrier female member of the trisomic family are shown inFigure 35.1914. DiscussionFigure 35. Derivative chromosome 5 present inHHW1O64 and carrier female from whom HHW1O64was derived‘I.,23.123.223.331.131.231.33233.1932.33435.135.235.35 marker HHW1O64 carrier female15.315.215.11413.313.213.1121111.111.21213.113.213.3III141521D5S59H- D5S60D5S257D5S268HPRTP2D5S21D5S260D5S76D5S6D5S39D5S78D5S205D5S253D5S71D5S37I present in derivative chromosome 5• absent from derivative chromosome 51924. DLccussion4.6 CONCLUSIONSThe research described in this thesis generated a variety of results relevant tothe field of human molecular genetics. A new technique, Alu PCR differentialhybridization, was developed for the isolation of region-specific human DNAfragments from mixed DNA sources. This technique is applicable to any hybrid-deletion hybrid pair and complements and extends previously reported techniques.Alu PCR differential hybridization was used to isolate clones from a region of thehuman genome, 5q11.2-q13.3, defined by a segmental trisomy. This region was ofinterest due to co-segregation of the trisomic region with schizophrenia and renalanomalies 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 muscularatrophy 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 wasdetermined using radiation hybrid mapping. While a linear order could not bedetermined for all clones using radiation hybrid mapping, insights were maderegarding parameters required to observe significant linkage in the radiation hybridmapping panel. Order information obtained from radiation hybrid mapping wasused to select markers to screen for polymorphisms. Polymorphic systems detectedallowed the characterization of the HHW1064 somatic cell hybrid, which is of valueto other investigators utilizing this hybrid. The new polymorphic systems providedinformation regarding the segmental trisomy segregating in the Vancouver family.These results have important implications regarding the positioning of genes forschizophrenia and hereditary renal adysplasia postulated to be present within the1934. Discussiontrisomic region. Polymorphic markers isolated during the course of this thesis alsoprovide useful markers for linkage studies involving disease genes in the vicinity ofthe new markers.4.7 SUMMARY1. Alu PCR differential hybridization, a novel technique for the isolation of region-specific human DNA fragments was devised. This technique should be generallyapplicable to any somatic cell hybrid-deletion hybrid pair.2. Alu PCR differential hybridization was used in conjunction with two chromosome5 somatic cell hybrids (GM1O114 and HHW1O64) to isolate 20 clones absentfrom the HHW1O64 hybrid.3. Radiation hybrid mapping together with Alu PCR was used to rapidly obtainpositional information on 18 of the new isolates.4. Clones of interest were screened for the presence of three types ofpolymorphisms; 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 detectedat the D5S253 locus. D5S253 is currently the closest marker to the distalbreakpoint within the 5q deletion region. D5S253 is located in an approximately29 cM gap between D5S78 and D5S71.1944. Discussion6. A highly informative (GT)n tract was detected in locus D5S260. D5S260 is notpresent in the somatic cell hybrid HHW1O64, but linkage maps between the indexmarkers D5S21 and D5S76, which are located outside the region deleted in theHHW1O64 hybrid. Examination of the HHW1O64 typing data and linkage datafor markers in this region of chromosome 5 suggests that the HHW1O64 hybridcontains a complex deletion encompassing two non-contiguous fractions on eitherside of D5S76.7. A moderately informative RFLP marker (D5S205) and two moderatelyinformative (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 TC65Aprimers was found to detect three systems, one on each of chromosomes 2, 5 and17. The chromosome 5 system was monomorphic, while the chromosome 2 and17 systems were polymorphic and moderately informative. One possibleexplanation for the observed amplifications is that D5S265-T primer was within alow copy repetitive element into which anAlu element had transposed.9. The highly informative (GT)n tract polymorphism associated with D5S268 andthe moderately informative (GT)n tract polymorphism associated with D5S257were unexpectedly found to linkage map to chromosome 5p.10. The regional localization of the remainder of isolates was performed using the1954. Discussionsomatic cell hybrid HHW213, which contains mainly 5p material. Four out oftwenty of clones isolated byAlu PCR differential hybridization were located onchromosome 5p. The HHW1O64 hybrid therefore contains a non-cytologicallydetectable deletion on chromosome 5p.11. The carrier individual from whom HHW1O64 was derived does not have a 5pdeletion, based on segregation analysis of D5S268 and D5S257 in her family.Segregation analysis for D5S76 and D5S260 in this family was used todemonstrate that the complex rearrangement on 5q postulated for HHW1O64was derived from the balanced carrier.4.8 PROPOSALS FOR FURTHER RESEARCHListed below are several experiments which could be performed to extendthe results obtained in this thesis.1. Isolate more clones from the 5q11.2-q13.3 region using Alu PCR differentialhybridization, prescreen these isolates for the presence of (GT) tracts anddevelop more polymorphisms for this region.2. Perform Alu PCR differential hybridization using different somatic cell hybrids todelineate the limits of resolution of this technique, and/or to isolate clones usefulfor the mapping of different disease genes.1964. Discussion3. Perform Alu PCR differential hybridization using cosmids as the source of clonedDNA in an attempt to increase the amount of DNA present for each isolate. AluPCR differential hybridization would presumably work with gridded cosmidarrays, which will soon be generally available for all human chromosomes.4. Obtain more information on radiation hybrid typing of index markers such asD5S6 in the mapping panel which was used for this thesis. The presence ofadditional markers would increase the density and presumably increase thechance of detecting significant linkages.5. Map isolates in a different radiation hybrid mapping panel. A panel which wasmade using a lower dose of radiation would presumably allow the linkage of allmarkers 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 familiessegregating for schizophrenia. This study would investigate the formal possibilitythat the postulated locus for schizophrenia within 5q11.2-q13.3 was race-specific.7. Perform linkage analysis in hereditary renal adysplasia pedigrees to look forlinkage to the 5q11.2-q13.3 region.8. Isolate more Alu polyA tracts, and develop PCR systems for amplification ofpolyA tails. Primers from the Alu consensus sequence with varying amounts of1974. Discussionhomology to the Alu element in question could then be tested to determine theamount of homology necessary for tract amplification. General rules for theinformativeness ofAlu polyA tails could also be formulated.9. Sequence more of the region around Alu element in p52H1 (D55265) todetermine if any evidence exists for the postulated low copy repetitive element.Evidence for such an element could also be demonstrated by the hybridization ofvarious regions to Southern blots of genomic DNA.10. 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Primer-directedenzymatic amplification of DNA with a thermostable DNA polymerase.Science 239:487-491ST. CLAIR, D., BLACKWOOD, D., MUIR, W., BAILLIE, D., HUBBARD, A.,WRIGHT, A., AND EVANS, H.J. (1989). No linkage of chromosome 5q11-q13 markers to schizophrenia in Scottish families. Nature 339:305-309.SAMBROOK, J., FRITSCH, E.F., AND MANIATIS, T. (1989). “Molecular cloning:A laboratory manual,” 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York.SANGER. F., NICKLEN, S., AND COULSON, A.R. (1977). DNA sequencing withchain-terminating inhibitors. Proc. NatL Acad. Sci. USA 74:5463-5467.SCAMBLER, P1, ESTIVILL, X., BELL, G., FARRALL, M., McLEAN, C.,NEWMAN, R., LIEFLE, P.F.R., FREDERICK, P., HAWLEY, K.,WAINWRIGHT, B.J., WILLIAMSON, R., AND LENCH, N.J. (1987).Physical and genetic analysis of cosmids from the vicinity of the cystic fibrosislocus. Nucleic Acids Res. 15:3639-3652.SHIELDS, D.C., COLLINS, A., BUETOW, K.H., AND MORTON, N.E. (1991).Error filtration, interference, and the human linkage map. Proc. NatL Acad.Sd. USA 88:6501-6505.SHERRINGTON, R., BRYNJOLFSSON, J., PETURSSON, H., P0 TI ER, M.,DUDLESTON, K., BARRACLOUGH, B., WASMUTH, J., DOBBS, M.,AND GURLING, H. (1988). Localization of a susceptibility locus forschizophrenia on chromosome 5. Nature 336:164-167.SHETH, P., ABDELHAK, S., BACHELOT, M.F., BURLET, P., MASSET, M.,HILLAIRE, D., CLERGET-DARPOUX, F., FREZAL, J., LATHROP,G.M., MUNNICH, A., AND MELKI, J. (1991). Linkage analysis in spinalmuscular atrophy, by six closely flanking markers on chromosome 5. Am. .1Hum. Genet. 48:764-768.SLIGHTOry, J.L., BLECHL, A.E., AND SMITHIES, 0. (1980). Human fetal Gand .globin genes: Complete nucleotide sequences suggest that DNA canbe exchanged between these duplicated genes. Cell 21:627-638.SOUTHERN, E.M. (1975). Detection of specific sequences among DNA fragmentsseparated by gel electrophoresis. .1 Mol. 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Genomics 7:524-530.WEIFFENBACH, B., FALLS, K., BRICKER, A, HALL, L., McMAHON, J.,WASMUTH, J., FUNANAGE, V., AND DONIS-KELLER, H. (1991). Agenetic linkage map of human chromosome 5 with 60 RFLP loci. Genomics10:173-185.WEINER, A.M., DEININGER, P.L., AND EFSTRATIADIS, A. (1986). Nonviralretroposons: Genes, pseudogenes, and transposable elements generated bythe reverse flow of genetic information. Ann. Rev. Biochem. 55:631-661.WESSEL, H.B. (1989). Spinal muscular atrophy. Pediatric Annals 18:421-427.WESTBROOK, C.A., NEUMAN, W.L., HEWITr, J., KIDD, KK., LE BEAU,M.M., AND WILLIAMSON, R. (1991). Report of the chromosome 5workshop. Genomics 10:1105-1109.XU, G., NELSON, L., O’CONNELL, P., AND WHITE, R. (1991). AnAlupolymorphism intragenic to the neurofibromatosis type 1 gene (NFl). Nucl.Acids Res. 19:3764.ZULIANI, G., AND HOBBS, H.H. (1990). A high frequency of lengthpolymorphisms in repeated sequences adjacent to Alit sequences. Am. JHum. Genet. 46:963-969.208APPENDIX 1. ALGORIThMS1. Radiation Hybrid MappingRadiation hybrid mapping is a statistical method for the production ofphysical maps of mammalian chromosomes (Cox et a!., 1990). This method is basedon the assumption that the further apart two markers are on a chromosome, thehigher the probability of a radiation-induced breakage between them. Thefrequency of breakage between two markers can be estimated in the followingfashion (Cox et a!., 1990).For two markers, A and B, the observed segregation can be defined in terms of fourunknowns: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 AP1J3 = probability of retention of a fragment containing both A and BThe fraction of hybrids retaining marker A but not B is therefore equivalent tobreakage between A and B plus retention of the fragment containing A and non-retention of the fragment containing B, ie.(A+B-) = 8PA(l-PB)TSimilarly,(A-B +) = ePB(l-PA)T(A+B+) = ePAPB +(1-e)PT(A-B-)= e(l-Pj(l-PB) + (1-e)(1-P)T209Appendix 1. AlgorithmsThese 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-2AB)A and B can be estimated from the observed marker segregation data as RA andRB where RA is equal to the fraction of all hybrids analyzed for marker A thatretain marker A and RB is equal to the fraction of all hybrids analyzed for marker Bthat retain marker B. e can then be calculated from the observed data, and variesbetween 1 (markers are always broken apart) and 0 (markers are never brokenapart). A mapping function D -ln(1-e), which is analogous to the Haldanemapping function for meiotic linkage mapping, is then used to convert the frequencyof breakage into an estimate of the distance between markers (D). D is expressedin centiRays (cR) with the amount of radiation used to make the radiation hybridpanel indicated as a subscript ie cR50,00 means 50,000 rads were used to make thepanel. A distance of 1 cR50000 is therefore equivalent to a breakage frequencybetween two markers of 1% when 50,000 rads are used.The odds of obtaining a given distance between two markers relative to theodds that the markers are unlinked (e =1) can be calculated in a similar fashion tomeiotic linkage mapping (Cox et al., 1990). A lod score can then be obtained fromthis 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 mostpossible order of four markers, and the odds for inversion of marker pairs.210AppendL 1. Algorithms2. Mapping Functions - Haldane and KosambiMapping functions are used to convert observed recombination fractions (e)into recombination distances (d), such that for any contiguous map regiondli = d1 + d2 + ... + d. It has been observed that recombination fractions followthe general formulae:(1)The corresponding equation for map distance can be written:(2) d12=d+2If 0 is then thought of as a function of recombination distance ie e =f(x), where x isequated with map distance, equation (1) becomes,(3) f(x1+x2) = f(x1) + f(x2) - pf(x)f(x)When (3) is rearranged, divided by x2, and the limit taken as x2 -> 0, the left handof side of the equation can be written as the standard definition for a derivative.(4) lim f(1±2)-f x= f(,{1-pf(x)}X2->0 X2 X2solving the differential using the fact that f(x)/x -> 1 as x -> 0 leads to:(5) f’(xl) = 1-pf(x)or,(6) .=l-pedxHaldane (1919) then integrates with respect to e, to obtain:(7) x =- 1. ln(1 - p0) or 0 = .1 (1 - eP9p p211AppendL 1. AlgorithmsHaldane then makes the observation that when x is very large, e approaches 0.5asymptotically. He therefore replaces p with 2 to obtain:(8) x =- 1 ln(1 - 28) or e = 1 (1 -e292 2which is the Haldane mapping functionKosambi (1944) makes the hypothesis that p can be replaced by 4e, which whensubstituted into (6), leads to the differential equation(9) de=1-492 or dx = dedx 1-4e2This differential can be easily solved using standard tables to give(10) x= j. ln 1 + 2e or e =1 tanh (2x)4 [ 1-28 2which is the Kosambi mapping functionMore complex mapping functions have since been derived in an attempt toobtain 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 tothe data.212Appendix 1. Algorithms3. Polymorphism Information Content (PlC)The PlC of a marker indicates the probability that any meiosis will beinformative, based on observed allele frequencies (Botstein et al., 1980). If thesystem is in Hardy-Weinberg equilibrium, the expected proportions of each parentalgenotype can be calculated from observed allele frequencies. For each mating class,the frequency of mating can expressed as the product of the two parental genotypicfrequencies. The probability that an offspring will be informative can bedetermined for each mating class by inspection of the parental genotypes. Thepercentage of informative meioses for each mating class is the product of the matingfrequency and the probability that an offspring will be informative. The PlC is thencalculated by summing this product for all mating classes. Mathematically, thesimplest expression of PlC is { 1 - (the proportion of uninformative matings)} or,n n-i nPlC = 1 - E Pi2 - E E 2pp1i=1 i=1 j=i+1Where j represents the allele frequency of the th allele.213Appendix 1. Algorithms4. Effective number of Informative Recombinants and MeiosesFor organisms in which genetic analysis is carried out by planned crosses, thenumbers of recombinants and non-recombinants can be directly counted. Forhuman genetic analysis, however, linkage analysis is carried out using likelihoodanalysis with both phase known and phase unknown families (Morton, 1955). Amethod was therefore developed for determining the numbers of recombinants andnon-recombinants in phase known families which would give the same lod score asthat observed (Edwards, 1976). These values were termed effective numbers ofrecombinants and non-recombinants, the sum of which is necessarily the number ofeffective number of informative meioses (Edwards, 1976). Based upon thesequential likelihood analysis of Morton (1955), Edwards (1976) derived thefollowing equations for calculation of effective numbers:(1) 1 s { e [log e - log 0.5] + (1- e) [log (1-e) - log (0.5)] }(2) r=se(3) n=s-rWhere,e = most likely recombination fraction1 = lod score at most likely value of es = effective number of meiosesr = effective number of recombinantsn effective number of non-recombinants214APPENDIX 2. RADIATION HYBRID DATA1 = probe present in hybrid0 = probe not present in hybridND = no dataHYBRID D5S205 D5S251 D5S254 D5S253 D5S255 D5S256 D5S2571 0 0 0 0 0 0 02 0 0 0 1 0 0 03 0 0 0 0 0 0 04 0 0 1 0 1 0 05 0 0 0 0 0 0 06 0 0 0 0 0 0 08 0 0 0 0 0 0 010 0 0 0 1 0 1 112 1 0 0 1 1 1 113 0 1 0 0 0 0 114 0 0 0 0 0 0 015 0 0 0 0 0 0 016 0 0 0 0 0 0 017 0 0 0 0 0 0 018 0 0 0 0 0 0 119 0 0 1 0 0 0 020 0 0 0 0 0 0 021 0 0 0 0 0 0 022 0 0 1 0 1 0 023 0 0 0 0 0 0 025 0 0 0 1 0 1 126 0 0 0 0 0 0 027 0 0 0 0 0 0 028 0 0 0 0 0 0 029 0 0 0 0 0 0 030 0 0 0 0 0 0 031 0 0 0 0 0 0 032 0 0 0 0 0 0 033 0 0 1 0 1 0 034 0 0 0 0 0 0 035 0 0 0 0 0 0 037 0 0 0 0 0 0 038 0 0 0 0 0 0 039 0 0 0 0 0 0 040 0 0 0 0 0 0 0215Appendix 2. Radiation hybrid dataHYBRID D5S205 D5S251 D5S254 D5S253 D5S255 D5S256 D5S25741 0 0 0 0 0 0 042 0 0 0 0 0 0 043 0 0 0 0 0 0 044 0 0 0 0 0 0 045 0 0 0 0 0 0 046 0 0 0 0 0 0 047 1 0 1 0 1 1 048 0 0 0 0 0 0 049 0 0 0 0 0 1 050 0 0 0 0 0 0 051 0 0 0 0 0 0 052 0 0 1 0 0 1 153 0 0 0 0 0 0 154 0 0 0 0 0 0 055 1 0 0 1 0 1 056 0 0 0 0 0 0 057 0 ND 0 0 0 0 058 0 ND 0 0 0 0 059 0 ND 0 0 0 1 060 0 ND 0 0 1 0 061 0 ND 0 0 0 0 063 0 ND 0 0 0 0 064 0 ND 0 0 0 0 065 0 ND 0 0 0 0 066 0 ND 0 0 0 0 067 0 ND 0 0 0 0 068 0 ND 0 0 0 0 069 1 ND 0 0 0 0 071 1 ND 1 0 0 0 072 1 ND 0 0 0 0 073 0 ND 0 0 0 0 074 0 ND 0 0 0 0 075 0 ND 0 0 0 0 076 0 0 0 0 0 0 077 0 0 0 0 0 0 078 0 0 0 0 0 0 081 0 0 0 1 0 0 082 0 0 0 0 0 0 083 0 0 0 0 0 0 084 0 0 0 0 0 0 085 0 0 0 0 0 0 086 0 0 0 1 0 0 187 0 0 0 0 0 0 088 0 0 0 0 0 0 089 0 0 0 0 0 0 090 0 1 0 1 0 0 0216Appendix 2. Radiation hybrid dataHYBRID D5S205 D5S251 D5S254 D5S253 D5S255 D5S256 D5S25792 0 0 0 0 0 1 093 1 0 0 0 0 0 094 1 0 1 0 0 0 095 0 0 0 0 0 0 096 0 ND 0 0 ND 0 097 0 ND 0 0 ND 0 098 0 ND 0 0 ND 0 099 0 ND 0 0 ND 0 0100 0 ND 0 0 ND 0 1101 0 ND 0 0 ND 0 0104 0 ND 0 0 ND 1 0105 0 ND 0 0 ND 0 0106 0 ND 0 0 ND 0 0107 0 ND 0 0 ND 0 0109 0 ND 0 0 ND 0 0110 0 ND 0 0 ND 0 0111 0 ND 0 0 ND 0 0112 0 ND 0 1 ND 0 1113 0 ND 0 1 ND 0 0114 0 ND 0 0 ND 0 0115 0 ND 0 0 ND 0 0117 0 ND 0 ND 0 ND ND120 0 ND 0 ND 0 ND ND121 0 ND 0 ND 0 ND ND122 0 ND 1 ND 1 ND ND123 0 ND 0 ND 0 ND ND124 0 ND 0 ND 0 ND ND125 1 ND 0 ND 0 ND ND126 0 ND 0 ND 0 ND ND127 0 ND 0 ND 0 ND ND128 0 ND 0 ND 0 ND ND129 0 ND 0 ND 0 ND ND130 0 ND 0 ND 0 ND ND131 0 ND 0 ND 0 ND ND132 1 ND 0 ND 1 ND ND133 0 ND 0 ND 0 ND ND134 0 ND 0 ND 0 ND ND135 0 ND 0 0 0 0 1136 0 0 0 0 0 0 0137 0 0 0 0 0 0 0139 0 0 0 0 0 0 0140 0 1 0 0 0 1 0141 0 0 0 0 0 0 0143 1 0 0 0 0 0 0144 0 0 0 0 0 0 0145 0 0 0 0 0 0 0217AppendL 2. Radiation hybrid dataHYBRID D5S205 D5S251 D5S254 D5S253 D5S255 D5S256 D5S257146 0 0 0 0 0 0 1149 0 0 0 0 0 0 0150 0 0 0 0 0 0 0151 0 1 0 0 0 0 0152 0 0 0 0 0 0 0153 0 0 0 0 0 0 0154 0 0 0 0 0 0 0155 0 0 0 0 0 0 0156 0 0 0 0 0 0 0157 0 1 0 0 0 0 0158 0 0 0 0 0 0 0159 1 1 0 1 1 1 1160 0 0 0 0 0 0 0162 0 0 0 0 0 0 0163 1 0 0 0 0 0 0164 0 0 0 0 0 1 0165 0 0 0 0 0 0 0166 0 0 0 0 1 0 0167 0 0 0 0 0 0 0168 0 0 0 0 0 0 0172 0 0 0 0 0 0 0y 0 0 0 0 0 0 0z 0 0 0 0 0 0 0218Appendb 2. Radiation hybrid dataHYBRID D5S258 D5S259 D5S260 D5S261 D5S262 D5S264 D5S2651 0 0 0 0 0 0 02 0 0 0 0 0 0 13 0 0 0 1 0 0 14 0 0 0 0 0 0 05 0 0 0 0 0 0 06 0 0 0 0 0 0 08 0 0 0 0 0 0 010 1 0 0 0 1 0 112 1 1 0 0 1 1 113 0 0 0 0 1 0 014 0 0 0 0 0 0 015 0 0 0 0 0 0 016 0 0 0 0 0 0 017 0 0 0 0 0 0 018 0 0 0 0 1 1 119 0 0 0 0 0 0 020 0 0 0 0 0 0 021 0 0 0 0 0 1 022 0 0 0 0 0 0 123 0 0 0 0 0 0 025 0 0 0 0 0 0 026 0 0 0 0 0 0 027 0 0 0 0 0 0 028 0 0 0 0 0 0 029 0 0 0 0 0 0 030 0 0 0 0 0 0 031 0 0 0 0 0 0 032 0 0 0 0 0 0 033 0 0 0 0 0 0 034 0 0 0 0 0 0 035 0 0 0 0 0 0 037 0 0 0 0 0 0 038 0 0 0 0 0 0 039 1 0 0 0 0 0 040 0 0 0 0 0 0 041 0 0 0 0 0 0 042 0 0 0 0 0 0 043 0 0 0 0 0 0 044 0 0 0 0 0 0 045 0 0 0 0 0 0 046 0 0 0 0 0 0 047 0 0 0 0 0 0 048 0 0 0 0 0 0 049 0 0 0 1 0 0 050 0 0 0 0 0 0 051 0 0 0 0 0 0 0219Appendix 2. Radiation hybrid dataHYBRID D5S258 D5S259 D5S260 D5S261 D5S262 D5S264 D5S26552 1 1 0 1 0 0 053 0 0 0 1 1 1 054 0 0 0 0 0 0 055 1 0 0 0 0 0 056 0 0 0 0 1 0 057 0 0 0 0 0 0 058 0 0 0 0 0 0 059 1 1 1 0 0 0 160 0 0 0 0 0 0 061 0 0 0 0 0 0 063 0 0 0 0 0 0 064 0 0 0 0 0 0 065 0 0 0 0 0 0 066 0 0 0 0 0 0 067 0 0 0 0 0 0 068 0 0 0 0 0 0 069 0 0 0 0 1 0 071 0 0 0 1 0 0 072 0 0 0 0 0 0 073 0 0 0 0 0 0 074 0 0 0 0 0 0 075 1 0 0 0 0 0 076 0 0 0 0 0 0 077 0 0 0 0 0 0 078 0 0 0 0 0 0 081 0 0 1 0 0 0 082 0 0 0 0 0 0 083 0 0 0 0 0 0 084 0 0 0 0 0 0 085 0 0 0 0 0 0 086 0 1 0 0 0 0 087 0 0 1 0 1 0 088 0 0 0 0 0 0 089 0 0 0 0 0 0 090 0 0 1 1 1 1 092 0 0 0 0 0 0 193 0 0 0 0 0 0 094 0 0 0 0 0 0 095 0 0 0 0 0 0 096 0 ND 0 0 0 0 ND97 0 ND 0 0 0 0 ND98 0 ND 0 0 0 0 ND99 0 ND 1 0 1 1 ND100 0 ND 0 0 0 0 ND101 0 ND 0 0 0 0 ND104 0 ND 1 1 1 0 ND220Appendix 2. Radiation hybrid dataHYBRID D5S258 D5S259 D5S260 D5S261 D5S262 D5S264 D5S265105 0 ND 0 0 0 0 ND106 0 ND 0 0 0 0 ND107 0 ND 0 0 0 0 ND109 0 ND 0 0 0 0 ND110 0 ND 0 0 0 0 ND111 0 ND 0 0 0 1 ND112 0 ND 0 0 0 0 ND113 0 ND 0 0 0 0 ND114 0 ND 0 0 0 0 ND115 0 ND 1 0 0 0 ND117 0 ND 0 0 0 0 0120 0 ND 0 0 0 0 0121 0 ND 0 0 0 1 0122 0 ND 0 0 0 0 0123 0 ND 0 0 ND 0 0124 0 ND 0 0 ND 0 0125 1 ND 0 0 1 0 0126 0 ND 0 0 0 0 0127 1 ND 0 0 1 0 0128 0 ND 0 0 0 0 0129 0 ND 0 0 0 0 0130 0 ND 0 0 0 0 0131 0 ND 0 0 0 0 0132 0 ND 0 0 0 ND 1133 0 ND 0 0 0 0 0134 0 ND 0 0 0 0 0135 0 0 1 0 1 0 0136 0 0 0 0 0 0 0137 0 0 1 0 0 0 0139 0 0 0 0 0 0 0140 1 1 1 0 1 0 1141 0 0 0 0 0 0 0143 0 0 0 1 0 0 0144 0 0 0 0 0 0 0145 0 0 0 0 0 0 0146 1 0 0 0 0 0 0149 0 0 0 0 0 0 0150 0 0 0 0 0 0 0151 0 0 0 0 0 0 0152 0 0 0 0 0 0 1153 0 0 0 0 0 0 0154 0 0 0 0 0 0 0155 0 0 0 0 0 0 0156 0 0 0 0 0 0 0157 0 0 0 0 0 0 0158 0 0 0 0 0 0 0221Appendk 2. Radiation hybrid dataHYBRID D5S258 D5S259 D5S260 D5S261 D5S262 D5S264 D5S265159 0 0 0 0 1 0 1160 0 0 0 0 0 0 0162 0 0 0 0 0 0 0163 0 0 0 0 0 0 0164 0 0 0 0 0 0 0165 0 0 0 0 0 0 0166 0 0 0 0 0 0 0167 0 0 0 0 0 0 0168 0 0 0 0 0 0 0172 0 0 0 0 0 0 0y 0 0 0 0 0 0 0z 0 0 0 0 0 0 0222Appendix 2. Radiation hybrid dataHYBRID D5S266 D5S267 D5S268 D5S269 D5S391 0 0 0 0 02 0 1 0 0 03 0 0 0 0 04 0 0 0 0 05 0 0 0 0 06 1 0 0 0 08 0 0 0 0 010 0 0 0 0 012 0 0 0 0 113 0 0 1 0 014 0 0 0 0 015 0 0 0 0 016 0 0 0 0 017 0 0 0 0 018 0 0 0 0 119 0 0 1 0 020 0 0 0 0 021 0 0 0 0 122 0 0 0 0 023 0 0 0 0 025 0 0 0 1 026 0 0 0 0 127 0 0 0 0 028 0 0 0 0 029 0 0 0 0 030 0 0 0 0 031 0 0 0 0 032 0 0 0 0 033 0 0 0 1 034 0 0 0 0 035 0 0 0 0 037 0 0 0 0 038 0 0 0 0 039 0 0 1 1 040 0 0 0 0 041 0 0 0 0 042 0 0 0 0 043 0 0 0 0 044 0 0 0 0 045 0 0 0 0 046 0 0 0 0 047 0 0 1 0 048 0 0 0 0 049 1 0 0 0 050 0 0 0 0 051 0 0 0 0 0223Appendix 2. Radiation hybrid dataHYBRID D5S266 D5S267 D5S268 D5S269 D5S3952 1 0 0 0 053 0 1 1 1 154 0 0 0 0 055 0 0 0 0 056 0 0 1 0 157 ND ND 0 ND 058 ND ND 1 ND 059 ND ND 0 ND 060 ND ND 0 ND 061 ND ND 0 ND 063 ND ND 0 ND 064 ND ND 0 ND 065 ND ND 0 ND 066 ND ND 0 ND 167 ND ND 0 ND 068 ND ND 0 ND 069 ND ND 0 ND 071 ND ND 0 ND 072 ND ND 0 ND 073 ND ND 0 ND 074 ND ND 0 ND 075 ND ND 0 ND 076 ND 0 0 0 077 ND 0 0 0 078 ND 0 0 0 081 ND 0 0 0 082 ND 0 0 0 083 ND 0 0 0 084 ND 0 0 0 085 ND 0 0 0 186 ND 0 0 1 087 ND 0 0 0 088 ND 0 0 0 089 ND 0 0 0 090 ND 1 1 0 192 ND 0 0 0 093 ND 1 0 0 094 ND 0 0 0 095 ND 0 0 0 096 ND ND 0 ND 097 ND ND 0 ND 098 ND ND 0 ND 099 ND ND 1 ND 1100 ND ND 0 ND 0101 ND ND 0 ND 0104 ND ND 0 ND 1224Appendix 2. Radiation hybrid dataHYBRID D5S266 D5S267 D5S268 D5S269 D5S39105 ND ND 0 ND 0106 ND ND 0 ND 0107 ND ND 0 ND 0109 ND ND 0 ND 0110 ND ND 0 ND 0111 ND ND 0 ND 1112 ND ND 0 ND 0113 ND ND 0 ND 1114 ND ND 0 ND 0115 ND ND 0 ND 0117 0 ND 0 ND 0120 0 ND 0 ND 0121 0 ND 0 ND 1122 0 ND 0 ND 0123 0 ND 0 ND 0124 0 ND 0 ND 0125 ND ND 0 ND 0126 0 ND 0 ND 1127 0 ND 0 ND 0128 0 ND 0 ND 0129 0 ND 0 ND 1130 0 ND 0 ND 0131 0 ND 0 ND 1132 0 ND 1 ND 0133 0 ND 0 ND 0134 0 ND 0 ND 0135 0 ND 0 0 1136 0 ND 0 0 0137 0 ND 0 0 0139 0 ND 0 0 0140 0 ND 0 0 0141 0 ND 0 0 0143 1 ND 0 0 0144 0 ND 0 0 0145 0 ND 0 0 0146 0 ND 0 0 0149 0 ND 0 0 0150 0 ND 0 0 0151 0 ND 0 0 0152 0 ND 0 0 0153 0 ND 0 0 0154 0 ND 0 0 0155 0 0 0 0 0156 0 0 0 0 0157 0 0 0 0 0158 0 0 0 0 0225Appendix 2. Radiation hybrid dataHYBRID D5S266 D5S267 D5S268 D5S269 D5S39159 0 0 1 0 0160 0 0 0 0 0162 0 0 0 0 1163 0 0 0 0 0164 0 0 0 0 0165 0 0 0 0 0166 0 0 0 0 0167 0 0 0 0 0168 0 0 0 0 0172 0 0 0 0 0y 0 0 0 0 0z 0 0 0 0 0226

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