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Physical mapping and clone isolation from the telomeric region of human chromosome 8p Ma, Lingli 1996

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P H Y S I C A L MAPPING A N D C L O N E ISOLATION F R O M THE T E L O M E R I C REGION OF H U M A N C H R O M O S O M E 8p by LINGLI M A B.Sc.(Honours), Queen's University, 1993 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES M E D I C A L GENETICS P R O G R A M M E We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A D E C E M B E R 1996 ©Lingli Ma 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of MI oj i \ . 6 ) t l/t *f)Y S The University of British Columbia Vancouver, Canada Date be>£"> , DE-6. (2/88) ABSTRACT Physical maps provide rapid access to any chromosomal region of interest, and serve as essential tools for the localization of genes. Of particular interest is the observation that gene clustering occurs in telomeric areas, although few genes have been mapped to the telomeric end of human chromosome 8p. In an effort to refine the physical map of 8pter-8p23, radiation hybrid mapping was used to determine the chromosomal order of ten sequence tagged sites (STSs) mapping to this interval. Four out of ten loci were positioned at 1,000:1 odds against order inversion, corroborating previously established linkage data. Secondly, STS content mapping in yeast artificial chromosomes (YACs) aided in the identification of potentially overlapping YACs , and a preliminary Y A C contig of the region was generated. Six Y A C s constituting a "minimum tiling path" were selected. As cosmids provide the necessary reagents for detailed positional cloning strategies and genomic sequencing, the primary objective of this project was to isolate bins of cosmids mapping to 8pter-8p23. To achieve this goal, Alu PCR products derived from the minimum tiling path Y A C s were used as hybridization probes against a chromosome 8 specific cosmid library. The initial screening identified an average of over 300 cosmids per Y A C . However, when a subset of positive cosmids was digested with EcoRl , Southern blotted and re-probed with the Alu PCR products, a false positive rate of 55% was observed. Probing the blots with total human D N A detected numerous fragments containing repetitive DNA. As a final ii analysis to confirm the identity of specific clones, cosmid EcoRl fragments were hybridized onto Southern blots containing EcoRl digested Y A C DNA. Fragments which clearly demonstrated sequence homology to the corresponding Y A C were strongly positive with the Y A C Alu PCR probe but negative with the total human D N A probe in the preceding experiment, suggesting that unique sequences were being isolated. In contrast, sequence homology was not demonstrated by fragments that were strongly positive with both the Alu PCR and total human D N A probes. A single cosmid was also identified which confirmed the overlap relationship between two Y A C s that was originally suggested by STS content mapping. iii TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vi List of Figures vii Acknowledgements ix Chapter 1: Introduction 1 1.1 Introduction 1 1.2 Human Chromosome 8 5 1.3 Linkage Mapping 6 1.3.1 D N A Polymorphisms 8 1.3.2 Current Status of the Human Genetic Linkage Map 10 1.4 Physical Mapping 11 1.4.1 Radiation Hybrid Mapping 13 1.4.2 The Yeast Artificial Chromosome System 17 1.4.3 Current Status of the Human Physical Map 21 1.4.4 Sequencing 22 1.5 Strategies for the Isolation of Sequence-Ready Cosmids 26 Chapter 2 : Materials and Methods 30 2.1 Radiation Hybrid Mapping 30 2.1.1 Chromosome 8 Radiation Hybrid Panel 30 2.1.2 STS Content Mapping in Radiation Hybrids 30 2.1.3 R H Mapping Analysis 33 2.2 Yeast Artificial Chromosome Analysis 35 2.2.1 CEPH Mega Y A C s 35 2.2.2 Preparation of Y A C D N A 35 2.2.3 PCR Amplification 37 STS Content Mapping 37 Alu?CR 38 Y A C Vector Arm PCR 38 iv 2.3 Cosmid Analysis...., 39 2.3.1 Human Chromosome 8 Cosmid Library 39 2.3.2 Isolation of Cosmid D N A 39 2.3.3 Preparation of High Density Cosmid Library Filters 42 2.4 Restriction Enzyme Digestion 42 2.5 Agarose Gel Electrophoresis 43 2.6 Southern Blotting 44 2.7 Extraction of EcoRl Fragments 45 2.8 Oligolabeling 46 Chapter 3 : Results 49 3.1 Radiation Hybrid Mapping 49 3.1.1 Two-Point Radiation Hybrid Analysis 51 3.1.2 Multipoint Radiation Hybrid Analysis 53 3.2 STS Content Mapping in Y A C s 56 3.3 Identification of Cosmids Contained Within Minimum Tiling Path YACs 60 3.3.1 Y A C A lu PCR Products Onto High Density Cosmid Colony Filters.. 60 3.3.2 Y A C Alu PCR Products Onto EcoRl Digested Cosmid D N A 63 3.3.3 Individual Cosmid EcoRl Fragments as Probes Onto Y A C EcoRl Southern Blots 74 Chapter 4: Discussion 79 4.1 Radiation Hybrid Mapping 79 4.1.1 Two-Point Radiation Hybrid Analysis 79 4.1.2 Multipoint Radiation Hybrid Analysis 80 4.2 Y A C Contig Construction 83 4.3 Effectiveness of Using Y A C Alu PCR Products as Probes For the Isolation of Cosmid Subsets 85 4.3.1 Initial Screening 85 4.3.2 Secondary Screening 87 4.3.3 Confirmation of the Identity of Selected Cosmids 89 4.4 Conclusions 92 4.5 Proposals for Future Research 94 References 95 Appendix 1 R H Mapping Data 106 Appendix 2 Positive Cosmids Identified with Minimum Tiling Path Y A C s 108 Appendix 3 Published Work Done Prior to the Completion of this Thesis 116 LIST OF TABLES Table 1 STS Marker Information 32 Table 2 Radiation Hybrid Retention Frequencies 49 Table 3 Two-Point LOD Scores, Breakage Frequencies and Distance Estimates 52 Table 4 Maximum Likelihood Placements of Six Loci With Respect to Four Core Loci. . . 54 Table 5 Top Five Locus Orders Obtained from Maximum Likelihood Analysis of Ten Loci Simultaneously 54 Table 6 Y A C Chimaerism, Sizes, and Numbers of Positive Cosmids Identified Per Y A C 62 Table 7 Subset of Eighteen Y A C Specific Cosmids Chosen for Further Analysis 64 Table 8 Summary of Results of Individual Cosmid EcoRl Fragments Probed Onto Y A C EcoRl Southern Blots 78 VI LIST OF FIGURES Figure 1 Cytogenetic Map of Chromosome 8p, with Corresponding Linkage Map 3 Figure 2 The pYAC4 Yeast Artificial Chromosome Cloning Vector 20 Figure 3 The sCos-1 Cosmid Cloning Vector 25 Figure 4 STS Content Mapping on the Radiation Hybrid Panel Using Marker D8S596.. 50 Figure 5 STS Content Mapping in Y A C s Using Marker D8S201 57 Figure 6 A Preliminary Y A C Contig Map of the Telomeric Region of Chromosome 8p.. 58 Figure 7 Comparison of Linkage, RH, and STS Content Maps 59 Figure 8 Ethidium Bromide Stained Agarose Gel of Alu PCR Products Derived from Y A C 787cl 1 60 Figure 9 Example of a High Density Cosmid Filter ("L") Probed with Alu PCR Products from Y A C 967c 11, and Corresponding Positives 61 Figure 10 Ethidium Bromide Stained Agarose Gel of Eighteen Y A C Specific Cosmids Digested with EcoRl 65 Figure 11a EcoRl Digested Cosmids Probed with Total Human D N A 67 l i b EcoRl Digested Cosmids Probed with Alu PCR Products from Y A C 967c 11 68 11c YACyRM2205 69 l i d Y A C 7 8 7 c l l 70 l i e YAC810f8 71 1 If Y A C 693dll 72 l l g YAC741h4 73 vii Figure 12 Ethidium Bromide Stained Agarose Gel of Y A C D N A Digested with EcoRl ...74 Figure 13a Southern Blot of Y A C EcoRl Digests Probed with 151H12E4.0 76 13b Probe23D5E1.7 76 13c Probe 136G1E2.9, Showing Overlap Between Y A C s 741h4 and 787cl 1 77 13d Probe 168 A2E6.0 77 Figure 14 Possible Explanation For the Overlap Relationship Between Y A C s 741h4and787cll 91 viii ACKNOWLEDGEMENTS I would like to express my sincere thanks and appreciation to my supervisor, Dr. Stephen Wood, for his continuous guidance and support. I am grateful to the members of my supervisory committee, Drs. Carolyn J. Brown and Muriel J. Harris, for their constructive criticism and advice. Special thanks to Mike Schertzer, for his patience, humour, and technical and academic expertise. Tanya Nelson, Leah Debella, Richard Bruskiewich, Gurjodh Singh and Karim Damani all provided helpful suggestions and assistance throughout the course of my research. Finally, I would like to thank my family and friends in Toronto and Vancouver for their optimism and encouragement. I am especially grateful to Yuri Ma, Shannon Young, Dave Dyment, and Ted Everson. ix 1 Chapter 1 Introduction 1.1 Introduction Two important and related goals of the Human Genome Project include the development of both genetic linkage maps and physical maps of each human chromosome. These maps will have a profound impact on all aspects of our understanding of molecular genetics and inherited disease, primarily by facilitating the identification of disease causing genes. From the genetic linkage map, the relative positions of inherited disease loci with respect to polymorphic DNA markers will become rapidly accessible, whereas the physical map will greatly aid in the determination of the precise, physical locations of these loci. Relatively little mapping information is available for the telomeric end of human chromosome 8p, despite evidence that it represents a potentially gene-rich region (Craig and Bickmore, 1994). In order to facilitate the future localization of genes, cDNAs and polymorphic markers in this region, the overall objective of this project was to refme the physical map of 8pter-8p23 (Figure 1), using a combination of complementary techniques and resources. Initially, radiation hybrid mapping was employed in an effort to establish the chromosomal order of ten previously localized sequence tagged sites (STSs). This was followed by STS content mapping in yeast artificial chromosomes (YACs), to identify a minimum set, or "minimum tiling path" of overlapping YACs that collectively span the entire region of interest. Since smaller clones are needed for detailed analyses and high Chapter 1. Introduction 2 resolution mapping, the principal goal of this thesis was to isolate bins of cosmids contained within this interval, by hybridization of Alu PCR products derived from the minimum tiling path Y A C s onto a chromosome 8 specific cosmid library. Furthermore, the effectiveness of using this particular technique for identifying cosmid subsets was evaluated. The rationale behind this study is presented in the remainder of this chapter. Chapter 1. Introduction 3 23.3 23.2 23.1 22 21.3 21.2 21.1 12 11.2 11.1 y 8p D8S356 D8S264 D8S201 D8S518 WT67 D8S277 D8S349 D8S252 D8S520 D8S550 D8S265 D8S21 D8S258 D8S282 D8S560 D8S298 D8S133 D8S136 D8S5 N E F L D8S137 c M 2.4 3.0 3.2 2.0 1.5 2.6 3.9 4.2 + 0.6 0.6 D8S552 + L P L + D8S87 -4-F G F R 1 _|_ D8S255 A N K 1 P L A T D8S22 5.4 8.6 4.9 1.1 1-1, * 0 . 7 0 4 1.0 1.8 4.7 2.8 2.8 9.3 4.3 4.3 0.9 2.1 2.1 82.4 cM Sex-Average Map Region of interest Figure 1: Cytogenetic Map of Chromosome 8p, with Corresponding Linkage Map Chapter 1. Introduction 4 The telomeric end of chromosome 8p was thought to be a particularly interesting area to study, as considerable data suggest that telomeres represent regions of high gene density (Saccone et al., 1992). Cytogenetically, 50% of thermal denaturation- resistant T bands, a subset of R bands, are located in telomeric areas, and correspond to the G+C richest isochore of the genome. This H3 isochore family comprises only 3% of the genome, yet has a gene concentration approximately eight times greater than in the G+C rich isochore families HI and H2, which make up 31% of the genome. Furthermore, gene concentration in the H3 isochore is at least sixteen times greater than in the G+C poor isochore families LI and L2, which comprise 62% of the genome (Mouchiroud et al., 1991). Analysis of the distribution of CpG islands throughout the genome provides further evidence for gene clustering in telomeric regions. CpG islands are non-methylated sequences between 100-600 bp in size, which surround the 5' transcription start site of 50-60% of human genes (Craig and Bickmore, 1994). They can be detected as clustered sites for certain rare-cutting restriction enzymes which contain CG in their recognition sequence (Lindsay and Bird, 1987). By cutting genomic D N A with the restriction enzyme Hpall (CCGG), and using fluorescent in situ hybridization (FISH) to hybridize these fragments onto metaphase chromosomes, Craig and Bickmore (1994) found the highest concentration of CpG islands to be located in T bands. Although hybridization to chromosome 8 was generally weak, the greatest concentration of signal occurred at 8qter, followed by 8pter. Interestingly, the 8q telomere consists of a T band, whereas the 8p telomere consists of a "mundane" R band (Holmquist, 1992). Chapter 1. Introduction 5 Only seven genes have been localized to 8p23. These include: EPMR, a locus which codes for a form of progressive childhood epilepsy with mental retardation (Tahvanainen et al., 1994; Ranta et al., 1996); arylamine N-acetyltransferase 1 (AAC1), an acetylating enzyme involved in the detoxification of arylamines (Hickman et al., 1994); GATA4, a transcription factor (Wood et al., 1994; White et al., 1995); defensin 1 (DEF1; Sparkes et a l , 1989; Wagner et al., 1991), and defensin 4 (DEF4; Leach et al., 1996), antimicrobial peptides that play a role in host defense mechanisms against infection; B L K , a tyrosine kinase (Drebin et a l , 1995; Islam et a l , 1995), and F7R, a regulator of coagulation factor VI1 (FaganetaL, 1988). 1.2 Human Chromosome 8 Interest and research on human chromosome 8 has been augmented by the recent localization of several additional disease loci to this chromosome. Most notably, the gene responsible for the rare autosomal recessive disorder known as Werner Syndrome was mapped to 8pl2 by positional cloning methods (Yu et al., 1996). Identification of mutations in a D N A helicase gene implies that defective D N A metabolism plays an important role in the premature aging process that is characteristic of these patients. Three putative tumour suppressor genes found in prostate cancer also map to the short arm of chromosome 8 (Macoska et a l , 1995). On the long arm of the chromosome are genes responsible for brachio-oto-renal syndrome (BOR; Kumar et a l , 1994), autosomal dominant retinitis pigmentosa (RP1; Sullivan et al., 1994), Zellweger syndrome (Masuno et al., 1994), Chapter 1. Introduction 6 Charcot-Marie-Tooth disease (CMT4A; Ben Othmane et al., 1994), multiple exostosis (EXT1; Cook et al., 1993), and Langer-Giedion syndrome (LGS; Ludecke et al., 1994). Despite these findings, the number of mapped genes on this chromosome is relatively small. Based on the rough estimate of 50,000-100,000 genes in the genome (Fields et al., 1994), chromosome 8 should contain between 2,500-5,000 genes as it comprises 5% of the genome. However, just over 100 loci have been mapped to date (Genome Data Base: 1.3 Linkage Mapping Genetic linkage refers to the tendency of two closely spaced loci to be transmitted together through meiosis. Linkage in humans was first demonstrated by Bell and Haldane (1937), who discovered that colour blindness and hemophilia were transmitted together on the X chromosome. The strength of linkage is inversely proportional to the amount of recombination between the two loci, and the unit of map distance, the Morgan (M), is a measure of the genetic length of a chromosome in which one recombination event occurs. One c M corresponds to a recombination frequency of 1%, and an average physical distance of one megabase (1 x 106 bp). However, since the frequency of recombination varies greatly along the length of a chromosome, the relationship between map units and physical distance is inconsistent. For example, the subtelomeric region of chromosome 21 represents a recombination "hot spot" where 7 c M corresponds to a distance of 350 kb (Gardiner et al., 1990; Burmeister et al., 1991). Chapter 1. Introduction 7 Genetic linkage analysis is a powerful tool that can be used to map genes based solely on their phenotypic effects. The inheritance pattern of an unmapped disease gene can be traced through a family by following the transmission of mapped, closely linked marker loci. Both the degree of polymorphism and the density of markers affect the ability to detect linkage between the disease and marker loci. A common measurement of marker polymorphism, or informativeness, is heterozygosity (H), which refers to the expected probability that any given individual in a population will be heterozygous at that particular locus. This is given by the equation: H=l- £ pi 2 , where pt represents the frequency of the i t h allele (Terwilliger and Ott, 1994). The polymorphism information content (PIC) takes into account the fact that a transmission from a heterozygous parent to an offspring may be uninformative if both parents are heterozygous for the same alleles. The PIC value is therefore calculated by subtracting this probability from the heterozygosity value: n n-1 n PIC= l - E p i 2 - I I 2 P i 2 P j 2 i=l i=l j=i+l where n=the number of alleles and p( = the frequency of the i t h allele (Botstein et al., 1980). The L O D score method (logarithm of the odds ratio for linkage) is the primary statistical test that is used to detect linkage. This method compares the likelihood of observing the segregation pattern of the two loci given that they are linked at a particular recombination fraction to the likelihood of the data given that they are not linked. The Chapter 1. Introduction 8 logarithm of the likelihood ratio is used so that data from different families can be added together. Development of this method by Morton (1955) helped to establish the first human linkage groups using blood group and protein polymorphisms. However, for the next few decades, linkage mapping was limited by the paucity of polymorphic markers. 1.3.1 DNA Polymorphisms In 1980, Botstein et al. proposed the use of restriction fragment length polymorphisms (RFLPs) as the basis for linkage map construction. RFLPs result from single base pair changes in D N A which create or destroy restriction enzyme cleavage sites (Kan and Dozy, 1978). However, these two-allele systems, based on the presence or absence of the restriction site, have a maximum heterozygosity of 0.5, limiting their usefulness in family linkage studies. Variable number of tandem repeat (VNTR) markers, or minisatellites, are polymorphisms which are based on multiple copies of tandemly repeated D N A (Wyman and White, 1980; Jeffreys et al., 1985; Nakamura et al., 1987). Allelic size variation is due to differences in repeat copy number. These loci are described as "hypervariable", as they are characterized by up to several dozen alleles (Thompson, 1991). A major drawback of VNTRs is that they are not evenly distributed within the genome, and appear to be clustered in subtelomeric regions (Jeffreys, 1987). Currently, the most favoured polymorphisms are microsatellite loci, or short tandem repeat polymorphisms (STRPs). These are repeat-length polymorphisms based on di-, Chapter 1. Introduction 9 tri-, or tetranucleotide repeats, and most commonly consist of a (CA) n motif (Weber et al., 1989). Variation in the length of the repeat is thought to arise from unequal crossing over during meiosis (Weber and May, 1989), or by strand slippage during replication (Levinson and Gutman, 1987). The (CA) n simple repetitive sequence is widely found in the genomes of eukaryotes, including yeast, fish, amphibians, insects, and mammals (Nordheim and Rich, 1983; Hamada and Kakunaga, 1982; Hamada et al., 1982). It is estimated that there are between 50,000-100,000 copies per haploid human genome, which places them on average every 30-60 kb (Hamada and Kakunaga, 1982; Weber and May, 1989). Although the function of these repeats has yet to be elucidated, it has been proposed that they serve as recombination hot spots (Pardue et al., 1987), or possess some transcriptional enhancer activity (Hamada et al., 1984). Informativeness of the (CA) n repeat is dependent on the length of the sequence, and on whether it is interrupted (imperfect) or not interrupted (perfect). Weber (1990) found that for perfect sequences with ten or fewer repeats, PIC values were very low or zero. For sequences with eleven to fifteen repeats, informativeness was variable, and consequently difficult to predict. On the other hand, > sixteen repeats consistently showed high PIC values, and values greater than 0.8 were obtained with a limited number of sequences that contained over twenty repeats. Additionally, PIC values for imperfect sequences were lower than expected based on the total number of repeats. The best predictor of informativeness for imperfect sequences was found to be the longest run of uninterrupted repeats. Chapter 1. Introduction 10 1.3.2 Current Status of the Human Genetic Linkage Map In 1994, a genetic map of the human genome was completed through the coordinated efforts of Genethon, the Cooperative Human Linkage Centre (CHLC), the University of Utah, and hundreds of collaborators at the Centre d'Etude du Polymorphisme Humain (CEPH). This comprehensive map spans 4,000 c M on the sex-averaged map, and has an average D N A marker density of 0.7 c M (Murray et al., 1994). 3,617 of the 5,840 loci are short tandem repeat polymorphisms, 427 are genes, and the remaining 1,796 mainly consist of RFLPs from anonymous D N A segments. This map has exceeded the initial goal of producing a map with 2-5 c M resolution, as set out by the National Centre for Human Genome Research at the National Institutes of Health (Collins and Galas, 1993). The final version of Genethon's human genetic linkage map is based solely on (CA) n repeat polymorphisms (Dib et al., 1996). The 5,264 (CA) n repeats comprising this map have a mean heterozygosity of 0.7, an average interval size of 1.6 c M , and span 3,699 c M on the sex-averaged map. Both maps are close to approaching the maximum attainable resolution for linkage mapping. A significant increase in resolution is limited by the number of highly polymorphic loci that are required. At Genethon, almost 20% of new markers are found to be duplicates of markers that have already been characterized. Secondly, increasing numbers of meioses would have to be tested over shorter genetic distances to resolve the new loci, which would also entail retyping the existing loci on the new meioses (Schmitt and Chapter 1. Introduction Goodfellow, 1994). Furthermore, both maps are of sufficient density to provide a framework upon which the physical map can be anchored. 11 1.4 Physical Mapping With the completion of the human genetic linkage map, current research activity is focused on the construction of integrated physical maps of progressively increasing resolution. At the lowest level of resolution is the cytogenetic map, based on the banding patterns of metaphase chromosomes. This is followed by a framework map, consisting of polymorphic markers positioned by genetic linkage analysis. This framework map is used to anchor a variety of clone based maps which make any D N A segment of interest easily accessible for analysis. Bacterial clones may in turn be used to determine the complete nucleotide sequence of the entire genome. These maps will be invaluable in identifying the exact chromosomal locations of genes, as lower resolution techniques such as genetic linkage analysis, mapping by gene dosage, fluorescent in situ hybridization, and somatic cell hybrid analysis localize genes to megabase regions of DNA. The advent of the polymerase chain reaction ( Saiki et al., 1985; Mullis and Faloona, 1987), and the subsequent use of STSs as the common language for physical mapping (Olson et al., 1989) have been instrumental in advancing physical mapping progress. The polymerase chain reaction is a powerful tool for exponentially amplifying short segments of D N A in vitro. This method is dependent upon a pair of oligonucleotide primers that flank the sequence of interest by hybridizing to opposite ends and strands of DNA. Chapter 1. Introduction 12 Synthesis of the intervening D N A is achieved by repeated cycles of heat denaturation, annealing of the primers to their complementary sequences, and extension of the annealed primers by D N A polymerase. Since many cycles of amplification are involved, large quantities of PCR product are synthesized that can be easily visualized on ethidium-bromide stained agarose gels, making radioactive labelling unnecessary. Although PCR was invented in 1985 (Saiki et al., 1985), it did not come into widespread use until 1989, when the procedure was refined by using a thermostable D N A polymerase (Taq polymerase) from the thermophilic bacterium, Thermus aquaticus (Saiki et al., 1988). This important modification eliminated the need to add fresh enzyme during each cycle, and significantly improved the specificity, yield, sensitivity and length of product that could be amplified as the reaction could be carried out at higher temperatures. STSs are short tracts of D N A sequence between 200-500 base pairs in length, which lack repetitive elements, and are therefore thought to represent unique sites in the genome (Olson et al., 1989). The major advantages of using STSs over other types of D N A markers are that they can be specifically detected by PCR in complex D N A samples, minute quantities of target D N A are needed, and primer sequences which define the STS can be stored as information in a database that can be accessed from any laboratory. By translating all types of mapping landmarks into STSs, data from a diverse range of physical mapping methods can be merged together into a consensus physical map. Furthermore, integration of the physical map with the genetic map is greatly facilitated by the use of common STSs that flank polymorphisms such as (CA) n repeats. Chapter 1. Introduction 13 1.4.1 Radiation Hybrid Mapping Radiation hybrid mapping is a somatic cell genetic technique that relies on the radiation-induced breakage of chromosomes to determine the order and distance between D N A markers along a chromosome (Cox et al., 1990). The basic premise of this technique is that the probability of breakage between two markers is proportional to the distance between them. Therefore, closely spaced markers will exhibit correlated retention patterns in clones containing the fragmented chromosomes, while distant markers will be retained independently. Cell death occurs as a result of chromosome fragmentation by high doses of radiation. These chromosomal fragments can be rescued by fusing them to nonirradiated hamster recipient cells that are deficient in the enzyme hypoxanthine phosphoribosyl transferase (HPRT). Growth in HAT medium (containing hypoxanthine, aminopterin, and thymidine) selects for donor-recipient hybrids that have retained the donor HPRT gene, as they can utilize hypoxanthine and guanine in D N A synthesis. Those lacking the enzyme must use a de novo purine synthesis pathway, which is inhibited by aminopterin. Thymidine is added to the media because its production is also blocked by aminopterin. It is unknown why nonselected human markers are each retained in approximately 30% of the hybrid cells. This technique was first developed by Goss and Harris in 1975, who measured the frequency of co-transferance of three unselected X-linked genes, phosphoglycerate kinase (PGK), glucose-6-phosphate dehydrogenase (G6PD) and oc-galactosidase (a-gal), with the selectable X-linked gene, HPRT. They found that PGK segregated from HPRT more Chapter 1. Introduction 14 frequently than either oc-gal or G6PD, so they concluded that PGK was more distant from HPRT than the other two markers. They also postulated that the segregation patterns of two unselected markers with HPRT would be independent of each other if they were located on opposites sides of the marker. Using this proposal, they determined that PGK and a-gal were situated on the one side of HPRT, while G6PD was on the other. They concluded that the order of the four genes was therefore PGK-a-gal-HPRT-G6PD. Fifteen years later, Cox et al., (1990) applied this technique to map fourteen D N A loci onto the long arm of chromosome 21. Using a panel of 103 radiation hybrid clones, they assayed for the retention of each marker in the clones by Southern blotting analysis. Their model assumed random chromosome breakage and independent fragment retention. However, measurements of breakage frequency between two markers were complicated by the fact that each hybrid could retain multiple chromosomal fragments. Therefore, to estimate the frequency of breakage (0) between two markers, A and B, Cox et al., (1990) took into account four possible outcomes: a) The fraction of hybrids retaining A but not B was represented as (A+B-)/T = 0R A(1-R B), where T= the total number of hybrids, and RA=the observed number of clones retaining A only, and R B= the observed number of clones retaining B only; b) Similarly, the fraction of hybrids retaining B but not A was represented as ( A - B + ) / T = 0 R b ( 1 - R A ) ; Chapter 1. Introduction 15 c) A hybrid retaining both markers A and B , may have resulted from a break between A and B , with the retention of both markers on separate fragments, or from no break between A and B , with the retention of both markers on a single fragment: (A+B+)/T = 0 R A R - B +(1-6)RAB d) Similarly, a hybrid not retaining either marker may have resulted from a break between A and B , with the loss of both markers on separate fragments, or from no break between A and B , with the loss of both markers on a single fragment: (A-B-)/T = 0(1-RA)(1-RB) + (1-6)(1-RA B) Solving for 0, the frequency of breakage was estimated from the equation: 0 = K A + B - ) + (A-B+)l [ T ( R A + R B - 2 R A R B ) ] 0 is analogous to a meiotic recombination fraction in linkage mapping, and ranges in value from 0 (two markers are never broken apart and are completely linked), to 1 (two markers are always broken apart and are unlinked). The likelihood of observing the given data, L0, is given by the product of the four equations. LOD score analysis can identify those marker pairs that exhibit linkage, and is defined as log [L0/(L0=1)]. The mapping function D = -ln(l-0), is used to estimate the distance between markers (D), with the unit of measurement being the centiRay (cR). Since higher doses of radiation induce a greater number of chromosome breaks, it is important to specify the dose when describing distance. For example, lcR8 0oo corresponds to a 1 % frequency of breakage between markers after Chapter 1. Introduction 16 8,000 rads of radiation. Cox et a l , (1990) found a linear relationship between centiRays and physical distance, with 1 cR approximating 50 kb. Additionally, 8,000 rads of radiation were sufficient for map construction at the 200-500 kb level of resolution. To order the fourteen loci along the chromosome, Cox et al., (1990) chose the order which minimized the sum of the distances between adjacent markers. They also calculated the odds against permutation of adjacent loci, by comparing the likelihood of the order of four adjacent markers against the likelihood of the order in which the two internal markers were inverted. Boehnke et al., (1991) developed two additional ordering methods taking into account data from many loci simultaneously. Minimum break analysis orders loci by finding the smallest number of obligate chromosome breaks that are required to explain the data. For example, i f a given hybrid (x) is tested for the presence of four markers, and the first two markers are retained while the last two are not (x = + + - -) the minimum number of breaks that must occur is one, between the second and third markers. This approach is analogous to minimizing the number of recombinants to infer order in genetic linkage mapping. The drawback of this method is that it does not provide relative likelihoods for other orders or distance estimates between loci. Based on the relative likelihood calculations of Cox et al., (1990), Boehnke et al., (1991) developed a maximum likelihood analysis that provides likelihood estimates of alternate orders under a variety of fragment retention models. Although radiation hybrids containing single haploid chromosomes are useful in mapping individual chromosomes, this approach would be impractical in efforts to map the Chapter 1. Introduction 17 entire genome, as approximately 4,000 hybrids would be required. Secondly, radiation hybrid panels are not available for every human chromosome. To overcome this problem, Walter et al., (1994) reverted back to the original protocol by Goss and Harris (1975), and used diploid human fibroblasts as donor cells. They calculated an average marker retention frequency of 23.7% for 40 chromosome 14 loci which were tested against a panel of 44 whole genome hybrids, which is similar to the marker retention frequencies reported for single chromosome hybrids. Walter et al., (1994) estimated that a whole-genome radiation hybrid map could be generated using a single set of 100 whole-genome radiation hybrid cell lines. In fact, a map of 500 kb average resolution has been constructed using the Stanford Genome Centre's G3 panel containing 83 R H whole-genome clones ( Currently, the Stanford Genome Centre is developing a higher resolution hybrid set, TNG3, consisting of 86 hybrids. Additionally, the Genebridge 4 panel of 93 hybrids is available from the Whitehead Institute ( 1.4.2 The Yeast Artificial Chromosome System Another important advance in physical mapping technology was the development of the Y A C cloning system (Burke et al., 1987). Due to the immense size of the human genome (approximately 3 billion base pairs, or 3 000 Mb), clones with large insert sizes are favoured in the construction of overlapping and contiguous regions of cloned D N A ("contigs"). Not only are fewer clones required, but single clones may then contain intact genes, complete with necessary regulatory elements like promoter sequences and enhancers. Y A C s have an Chapter 1. Introduction 18 insert capacity of approximately 1 Mb, which far exceeds the capability of any other cloning system. There are several Y A C vectors (pYAC2-pYAC5) which contain different restriction enzyme cloning sites within the SUP4 gene, but all are originally derived from the E.coli plasmid vector pBR322. The most widely used vector, pYAC4 (Figure 2), illustrates the basic characteristics essential for Y A C construction. An ARS sequence (autonomous replication sequence) is necessary for the insert D N A to be able to replicate autonomously; CEN4 confers mitotic and meiotic centromere function; TRP1 and URA3 are selectable markers for both vector arms, and the two TEL sequences initiate telomere formation in vivo. The SUP4 gene is an ochre-suppressing allele of a tRNA gene, and insertional inactivation by the cloned D N A produces red rather than white colonies. This vector, containing the D N A of interest, can be stably maintained as a linear, artificial chromosome when transformed into the yeast, Saccharomyces cerevisiae. The human Mega-YAC library constructed at CEPH consists of 34,560 clones representing approximately ten genome equivalents. These Mega-YACs have an average insert size of 1, 054 kb (Chumakov et al., 1995). The predominant method of establishing overlaps among Y A C s is by STS content mapping (Olson et a l , 1989). This approach uses PCR to detect whether a given STS is contained within a Y A C . Since STSs are unique sites within the genome, two different Y A C s that contain the same STS theoretically should overlap. An alternative method, known as cross hybridization, involves hybridizing the Y A C library with single-copy probes (Brownstein et a l , 1989). Lastly, a technique called fingerprinting may also be used Chapter 1. Introduction 1 (Bellanne-Chantelot et al., 1992). In this case, similarities in the restriction fragment length patterns of two different YACs, as detected by repetitive sequence probes, can infer potential overlapping regions. Chapter 1. Introduction 20 Figure 2 : The pYAC4 Yeast Artificial Chromosome Vector (Burke et a l , 1987; Burke and Olson, 1991) Chapter 1. Introduction 21 1.4.3 Current Status of the Human Physical Map An international collaboration involving Genethon, CEPH, and the Whitehead Institute genome centres has resulted in a landmark Y A C contig map of the human genome (Chumakov et al., 1995). Extensive analyses of 33,000 CEPH Y A C s produced a map of overlapping Y A C s covering approximately 75% of the genome in 225 contigs. STS content mapping in Y A C s was first performed on 2,066 mapped STSs (Gyapay et al., 1994), providing the backbone information upon which the rest of the map could be built. The Y A C library was screened by hybridization, using human-specific probes derived from individual YACs . This was followed by repetitive sequence fingerprinting using the repetitive sequence probes LINE-1 and THE-LTR (transposon-like human-element long terminal repeat). A parallel physical mapping objective is to construct a whole-genome map based on STSs spaced at 100 kb intervals, which will require approximately 30,000 STSs (Collins and Galas, 1993). The main advantage of this approach is that problems associated with clone-based maps will be eliminated, such as the high rate of chimaerism and instability in the case of Y A C s (Green et al., 1991). By having STSs at sufficiently high density across the genome, physical coverage of any region can be quickly regenerated by screening the appropriate clones via PCR. In another major international collaboration, an STS-based map of the human genome was published by Hudson et al. in 1995. This map incorporates over 15,000 STSs consisting of random single-copy sequences, expressed sequences, genetic markers and other loci from various sources. The average spacing between markers is 199 Chapter 1. Introduction 22 kb, with physical coverage of 94% of the genome. The results of STS content mapping of 10,850 loci in Y A C s were integrated with a radiation hybrid map of the genome containing 6,193 loci, as well as with a genetic linkage map containing 5,264 loci. 1.4.4 Sequencing Although Y A C s are useful in constructing low resolution maps with long range continuity, their large size, instability, and high level of chimaerism make them unsuitable candidates for large scale sequencing. Determining the complete D N A sequence of the human genome will have a major impact on studies of genome organization and structure, functional analyses, the identification of gene transcripts, promoter regions, and controlling elements, and the detection of mutations leading to hereditary diseases. YAC-based physical maps play an important intermediary role in producing "sequence-ready" maps, consisting of smaller and more stable clones. Many small-insert cloning systems are available, such as bacterial artificial chromosomes (BACs), based on the fertility-factor (F-factor) plasmid found in Escherichia coli (Shizuya et al., 1992), bacteriophage PI-derived artificial chromosomes (PACs) (Ioannou et al., 1994), and cosmids (Evans etal., 1989a). PACs are capable of stably maintaining human D N A fragments 100 kb in size, while the limit of BACs approaches 300 kb. The presence of two Pl-derived replication mechanisms in PACs allows for the stable propagation of clones under the single-copy replicon, while the multi-copy replicon is useful for preparing clone DNA. In contrast, Chapter 1. Introduction 23 recovery of D N A from BACs is relatively low due to the fact that only one or two copies of the F-factor replicate per cell. The advantages of both systems are that they exhibit no detectable chimaerism, and electroporation of ligation products into host cells eliminates the need for elaborate packaging extracts. Furthermore, the circular nature of both vectors facilitates the isolation and manipulation of cloned DNA, as it is less susceptible to shearing than linear Y A C DNA. However, both BACs and PACs have much smaller insert capacities than Y A C s . Although limited by their relatively small insert size of approximately 40 kb, cosmids represent one of the most characterized and widely used vector systems. They are derived from plasmids that contain bacteriophage X cos signals for in vitro packaging. The high efficiency of this packaging system (106-107 clones/|ig of DNA) allows chromosome-specific libraries to be constructed from small amounts of DNA, such as that obtained from flow-sorted chromosomes. In contrast, current Y A C , B A C and PAC libraries are derived from total genomic D N A , and must first be separated into sublibraries for chromosome specific studies. Additional features of cosmid vectors include bacteriophage T3 and T7 promoters for the synthesis of probes for chromosome walking, unique restriction sites to remove insert DNA, antibiotic resistance genes for selection, and a plasmid origin of replication for obtaining high yields of D N A (Figure 3). The "random shotgun" approach to D N A sequencing is the dominant sequencing strategy presently in use (Hunkapiller et al., 1991). This involves the identification of bins of overlapping cosmids, subcloning cosmid fragments into the single-stranded vector M l 3, Chapter 1. Introduction 24 and sequencing the subclones with six to eight fold redundancy, to increase the probability of obtaining overlapping sequence reads that can then be assembled into contigs. Chapter 1. Introduction 25 Figure 3: The sCos-1 Cosmid Cloning Vector (Evans etal., 1989a) Chapter 1. Introduction 26 1.5 Strategies for the Isolation of Sequence-Ready Cosmids Only a few isolated areas of the human genome are currently represented by high resolution maps based on contiguous, overlapping, and ordered cosmids. Of these, chromosome 16 is the most extensively characterized, with 35% coverage of the euchromatic arms (Doggett et al., 1995). A "bottom up" mapping strategy was adopted, where cosmids from a chromosome 16 specific library were first fingerprinted to detect overlaps. STSs were then developed from the ends of the contigs to provide a framework STS map upon which a corresponding Y A C contig was constructed. This type of approach requires a high level of redundancy and overlap between clones, precise restriction digestion measurements, and a comparison of fingerprinting data between each cosmid pair. In contrast, the "top-down" method uses prior low-resolution mapping information, such as STS content mapping in YACs , to localize cosmid clones into bins within already defined regions. Several alternate strategies have been developed for the isolation of sequence ready cosmids. Xie et al., (1994) used human telomeric repeat sequences and centromeric a repeats as probes to identify telomeric and centromeric cosmids from a chromosome 22 library. Chromosome walking from these cosmids further identified additional cosmids from the subtelomeric and pericentromeric areas. Vortkamp et a l , (1995) converted a Y A C contig of the Greig cephalopolysyndactyly syndrome (GCPS) region of chromosome 7pl3 into a cosmid library, by subcloning the Y A C s into the cosmid vector sCos-1 (Evans et al., 1989a). However, this approach is not Chapter 1. Introduction * 27 widely used, due to problems with chimaerism and rearrangements in the Y A C s that are perpetuated in the cosmid libraries. Baxendale et al., (1991) developed a procedure for directly screening cosmid libraries with Y A C clones that have been mapped to the region of interest. This involves separating the yeast chromosomes by pulsed field gel electrophoresis in low melting point agarose, excising the human artificial chromosome, and using the purified D N A as a hybridization probe against a chromosome specific cosmid library. The use of chromosome specific libraries eliminates the problem of inter-chromosomal Y A C chimaerism, since the chimaeric D N A will not be represented in the library. Sequence-ready cosmid contigs can then be built from the binned cosmids by repetitive sequence fingerprinting (Stallings et al., 1990), or by end labeling techniques (Evans and Lewis, 1989b; Haberhausen and Muller, 1995). Successful application of this technique has been reported for a 2 Mb region containing the Huntington's disease gene on 4pl6.3 (Baxendale et al., 1993; Zuo et a l , 1993), and for a 1.6 Mb region of Xp22 (Wapenaar et al., 1994). Nizetic et al., (1994) similarly obtained partially overlapping cosmids mapping to shared and non-shared intervals between overlapping Y A C s spanning large regions of chromosome 21. Holland et al., (1993) used Y A C probes against a whole genome cosmid library enriched for the X chromosome (49, X X X X X ) to build cosmid contigs in a 0.65 Mb region of Xq26. One difficulty associated with isolating Y A C D N A from a pulse field gel is that limited quantities of D N A are obtained for use as hybridization probes. A common way of circumventing this problem is to use PCR to amplify the target DNA. For example, Sutcliffe Chapter 1. Introduction 28 et al., (1992) ligated a linker-adapter onto restriction digested Y A C DNA, and used the known sequence of the linker as a primer for amplification. These PCR products were then used as probes for FISH analysis. Similarly, Dreyling et al., (1995) used sequence-independent amplification (Bohlander et al., 1992, 1994) of Y A C s to screen a gridded chromosome 9 cosmid library. Sequence independent amplification involves the use of a primer with a random pentanucleotide sequence at its 3' end and a defined 5' end in the initial rounds of synthesis directed by T7 D N A polymerase. A second primer containing the defined sequence is then used for amplification. Another PCR-based method for obtaining large amounts of Y A C D N A takes advantage of the ubiquitous Alu repeats that comprise the majority of short interspersed repeat sequences (SINES) in the human genome (Jelinek and Haynes, 1982). Human Alu sequences are approximately 300 bp long and consist of two directly repeating monomer units (Kariya et al., 1987). There are an estimated 900,000 copies of the repeat per haploid human genome, placing them on average every 4 kb. Although homologous Alu repeats are found in other mammalian genomes such as rodents, there is enough sequence divergence to reduce cross hybridization between species. This allows for the direct PCR amplification of human DNA, using primers derived from each end of the Alu consensus sequence, specifically from hybrid cells containing rodent backgrounds, as well as from various cloning systems such as yeast and bacteria (Nelson et a l , 1989). The main advantage of this technique is that Y A C D N A does not first need to be separated and excised from a pulse field gel, making its isolation less labour intensive and more efficient. However, the Chapter 1. Introduction 29 successfulness of Alu PCR is dependent upon both the orientation and spacing of adjacent Alu repeats in the target DNA. Jones et al., (1994) used Alu PCR products derived from Y A C s to isolate cosmids spanning a 400 kb interval in the BRCA1 region on chromosome 17. Fischer et al.,(1996) also adopted this technique to produce high-resolution cosmid contig maps of the BRCA2 region on chromosome 13, which will aid in future efforts to positionally clone this gene. Similarly, Y A C Alu PCR products were used as probes in this thesis to isolate cosmids mapping to the 8pter-8p23 region of human chromosome 8. The effectiveness of using this technique for obtaining cosmid subsets corresponding to Y A C S was also addressed by three experiments of progressively increasing specificity. Initially, Y A C Alu PCR products were hybridized onto a flow sorted chromosome 8 cosmid library contained on high density filters, and positive cosmids were scored. Secondly, a small subset of positive cosmids from each Y A C were picked for further analysis. D N A from this cosmid subset was digested with EcoRl and Southern blotted, and then reprobed with the same Y A C Alu PCR products used in the previous experiment. As a final verification of the identity of selected cosmids, individual cosmid EcoRl fragments were then hybridized back onto Southern blots containing Y A C D N A digested with EcoRl . 30 Chapter 2 Materials and Methods 2.1 Radiation Hybrid Mapping 2.1.1 Chromosome 8 Radiation Hybrid Panel A radiation hybrid panel was constructed in San Antonio, Texas by Bookstein et al., (1994) from a chromosome 8 specific human x hamster hybrid line, GM10156b, obtained from the National Institute of General Medical Sciences Mutant Cell Repository (Camden, NJ). The hybrid was y-irradiated with a dose of 5,000 rads and fused to the APRT and HPRT deficient Chinese hamster ovary cell line CHO-ATS-49tg following the method of Cox et al., 1990. After selection in adenine (10 mg/L), aminopterin (250 ng/L), and thymidine (10 mg/L), a panel of 95 hybrids was obtained. Hybrid D N A was received and stored in a 96-well microtitre plate at -70° C. 2.7.2 STS Content Mapping in Radiation Hybrids STS content mapping in the radiation hybrid panel was performed by PCR amplification using an automated thermal cycler (Perkin-Elmer Cetus). A somatic cell hybrid line, MGV269, containing 8pter-q22.1 (Drabkin et al., 1985, Sacchi et al., 1986) was used as a positive control. Reaction volumes of 40 uL were used, containing 100 ng of DNA, 50 mM Tris-Cl, pH 8.3, 0.05% Tween-20, 0.05% Nonidet-40, 2.0 m M of M g C l 2 , 0.2 mM of Chapter 2. Materials and Methods 31 each nucleotide (dATP, dCTP, dGTP and dTTP), 0.5 u of Taq polymerase, and 0.25 mM of each primer in a pair listed in Table 1. Samples were processed through 40 cycles of amplification, consisting of 1 min. at 94°C for denaturation, 30 seconds at the primer-specific annealing temperature (Table 1), and 1 min. at 72°C for extension. This was followed by a final extension of 10 min. at 72°C. S c o • P N S u .© u CZ5 H <u 3 H -a an CO IP 6fl CO Chapter 2. Materials and Methods 33 2.1.3 RHMapping Analysis R H mapping data were analysed with a computer program called R H M A P : Statistical Package for Multipoint Radiation Hybrid Mapping (Boehnke et a l , 1991), consisting of three separate programs: a) RH2PT provides two-point LOD scores for linkage of various marker pairs, and determines whether all markers fall into the same linkage group. LOD score values of 3.0 and 4.0 are specified, corresponding to 1000:1 and 10,000:1 odds for linkage, respectively. Locus retention probabilities and distance estimates are also given. Assumptions made by this program include random chromosome breakage with no interference, independent retention of chromosomal fragments, and equal fragment retention probabilities. b) R H M I N B R K provides marker orders by minimizing the number of obligate chromosome breaks. c) R H M A X L I K looks at all permutations of the data, and provides relative likelihood calculations for alternate orders For both R H M I N B R K and R H M A X L I K , four different ordering strategies are available: i) a list of user-specified locus orders can be inputted ii) stepwise locus ordering- this method builds orders one locus at a time. In the case of RHMINBRK, only those partial orders that are within K breaks of the current best partial order are kept, so when a partial order is eliminated, all complete orders descended Chapter 2. Materials and Methods 34 from it are also eliminated. Similarly, in RHMAXLIK, only those partial orders whose maximum likelihoods are similar to that of the current best partial order are kept. iii) simulated annealing- this approach starts with a locus order, and tests alternate orders by random block inversions of loci. iv) branch and bound- this strategy is similar to stepwise locus ordering, with the difference being that in RHMINBRK, partial locus orders are saved if they are within K breaks of the current best complete order, and in RHMAXLIK, partial orders whose maximum likelihoods are similar to that of the current best complete order are retained. This method guarantees that the optimal order is found; however, it may be impractical when many loci are present. There are also four different models of fragment retention that can be specified in both RHMINBRK and RHMAXLIK. The equal retention model is the simplest, and assumes that all fragments have the same retention probability. The centromeric model assumes a higher retention probability for fragments containing the centromere, while the telomeric model assumes a lower retention probability for fragments containing the telomere. In the left-endpoint retention model, fragment retention probability is dependent on the left-most locus. Lastly, the general retention model allows all fragments to have differing retention probabilities. In all cases, the equal retention model of fragment retention, and the branch and bound method of locus ordering were selected. Chapter 2. Materials and Methods 35 2.2 Yeast Artificial Chromosome Analysis 2.2.1 CEPH Mega-YACs A chromosome 8 subset of Y A C s from the Mega-YAC library (Bellanne-Chantelot et a l , 1992) was obtained by hybridization of Y A C Alu PCR products onto a chromosome 8 somatic cell hybrid panel (Chumakov et al., 1992) at CEPH. CEPH Mega-YAC D N A pools representing the entire library were also obtained from Research Genetics, Inc. These pools were screened by Jenny Barkman with chromosome 8p23 markers using a three-dimensional pooling scheme (Chumakov et al., 1995). Additional positive YACs from this screen, as well as Y A C s positive for markers D8S504, D8S264, D8S262, D8S201, D8S518 and D8S277 according to the Whitehead Institute database ( were individually purchased from Research Genetics, Inc. Y A C yRM2205 is a "half-YAC" containing the telomere of chromosome 8p, which acts as one functional telomere, while the other telomere is derived from Tetrahymena (Riethman et al., 1989). 2.2.2 Preparation of YAC DNA Y A C s were streaked out on A H C minimal media plates (Brownstein et al., 1989) and single pink colonies were innoculated in 3 mL of YPD + ampicillin solution for 2 days at 30°C, with shaking. 1 mL of the solution was centrifuged at 13,000 rpm for 2 min. The pellet was resuspended in 240 uX of lysis solution, and incubated at 37°C for 1 hour. After centrifugation for 2 min., the pellet was resuspended in 100 fiL of Y T E and 10 | iL of 10% SDS, and incubated at 65°C for 20 min. This was followed by the addition of 40 uL of Chapter 2. Materials and Methods 36 alkaline lysis solution III, and a 30 min. incubation on ice. The cell debris was pelleted by centrifugation at 13,000 rpm for 3 min. 150 uL of supernatant was transferred to a fresh eppendorf tube, and 300 uL of 95% EtOH was added to precipitate the DNA. The D N A was pelleted by centrifugation at 13,000 rpm for 15 min. and dried in a SpeedVac Concentrator for 5 min. The pellet was resuspended in 100 (iL of TE containing 25 (ig of RNAse and incubated at 37° C for 1 hour to degrade any contaminating RNA. 100 u l of isopropanol was added, and the D N A was centrifuged for 10 min., dried, and resuspended in 200 | l L of TE. The sample was stored at -20° C. Sorbitol Solution 0.9 M Sorbitol 0.1 M ethylene diamine tetra-acetic acid (EDTA), pH 8.0 » 100 m M tris (hydroxymethyl) aminomethane (Tris-Cl), pH 8.0 Lysis Solution 200 uL sorbitol solution 20 uX sorbitol solution with 1:25 (3-mercaptoethanol 20 uX sorbitol solution with a few grains of lyticase or zymolyase Y T E Solution 50 m M Tris-Cl, pH 8.0 10 m M EDTA, pH 8.0 Chapter 2. Materials and Methods 37 YPD + Ampicillin Solution 4.0 g tryptone (peptone) 2.0 g yeast extract 4.0 g dextrose 20 mg ampicillin 200 mL dH 20 A H C Minimal Media (ura-, trp-) Plates 6.7 g yeast nitrogen base w/o amino acids 10 g acid hydrolysed casein 20 g dextrose 20 mg adenine hemisulfate or adenine hydrochloride 12 g agar 1L dH 20 adjust to pH 5.8 with HC1 2.2.3 PCR Amplification of YACs STS Content Mapping GEPH Mega-YACs were tested for the presence or absence of each STS by PCR. 100 ng of Y A C D N A was amplified in a 25 | iL reaction volume containing 50 m M Tris-Cl, pH 8.3, 0.05% Tween-20, 0.05% Nonidet-40, 2.5 mM of M g C l 2 , 0.2 mM of each nucleotide (dATP, dCTP, dGTP and dTTP), 0.5 u of Taq polymerase, and 0.4 m M of each primer in a Chapter 2. Materials and Methods 38 pair listed in Table 1. The somatic cell hybrid line, MGV269, and milli-Q H 2 0 were used as positive and negative controls, respectively. The samples were covered with 25 | i L of mineral oil to prevent evaporation. 40 cycles of amplification each consisted of 1 min. at 94°C for denaturation, 30 seconds at the primer-specific annealing temperature (Table 1), and 1 min. at 72°C for extension. This was followed by a final extension of 10 min. at 72°C. Alu PCR Y A C Alu PCR products were obtained using primers derived from each end of the Alu consensus sequence. The primer names are A L E 3 (5 '^GCACTGCACTCCA GCCTGGG-3' ) and ALE1 (5 ' -GCCTCCCAAAGTGCTGGGATTA-3 ' ) and have an annealing temperature of 64°C (Cole et al., 1991). YAC Vector Arm PCR To check for the presence of Y A C D N A in the midst of total yeast DNA, PCR was performed using primers derived from the pYAC4 vector arm. The primer sequences are: 5 ' - C A C C C G T T C T C G G A G C A C T G T C C G A C C G C - 3 ' , and 5 ' - G G C T A C C G G A G T A T TTAATTG-3 ' , with an annealing temperature of 50°C. pYAC4 was used as a positive control. Chapter 2. Materials and Methods 39 2.3 Cosmid Analysis 2.3.1 Human Chromosome 8 Cosmid Library A human chromosome 8 cosmid library, LA08NC01, was constructed at the Los Alamos National Laboratory, New Mexico by Wood et al., (1992b). Chromosome 8 was isolated by fluorescence-activated flow sorting (Deaven et al., 1986) from a human x hamster hybrid cell line (UV20HL21-27) containing human chromosomes 4, 8 and 21 (Fuscoe et al., 1986). The D N A was extracted, partially digested with Sau3Al, and dephosphorylated. Ligation to the Bam HI cloning site of the vector sCos-1 (Evans et al., 1989a) was followed by transfection into E. coli D H a M C R cells. Kanamycin resistant E. coli colonies were transferred into 96-well microtitre plates containing L B broth, and were grown overnight at 37° C. Glycerol was added to a final concentration of 40%. A total of 20,160 colonies representing four genome equivalents were arranged in 210 microtitre plates, and were stored at -80°C. Hybridization with human D N A and Chinese hamster D N A showed that 85% of the clones were human specific, and cosmids were isolated in all cases when the library was screened with probes from nine loci mapping to different areas of the chromosome (Wood et al., 1992b). 2.3.2 Isolation of Cosmid DNA Cosmid D N A was prepared using an alkaline lysis miniprep procedure (Birnboim and Doly, 1979, Ish-Horowicz and Burke, 1981). Cosmid colonies were streaked out on agar plates containing kanamycin, and single colonies were innoculated in 5 mL of L B broth Chapter 2. Materials and Methods 40 at 37° C overnight. The cells in 1.5 mL of each culture were pelleted by centrifugation at 13,000 rpm for 2 min, and then were resuspended in 100 uX of Solution I for 5 min. at room temperature. This was followed by the addition of 200 | i L of freshly prepared Solution II, and a 5 min. incubation on ice. 150 uL of Solution III was added, with another 5 min. incubation on ice. The cell debris was pelleted by centrifugation at 13,000 rpm for 10 min. 400 pX of supernatant was transferred to a fresh eppendorf tube, and an equal volume of isopropanol was added to precipitate the DNA. The D N A was pelleted by centrifugation at 13,000 rpm for 15 min. The pellet was washed in 200 uL of 70% ethanol (EtOH), and dried in a SpeedVac Concentrator (Savant) for 5 min. It was resuspended in 50 uL of TE containing 25 ug of RNAse and incubated at 37° C for 30 min. to degrade any contaminating RNA. The sample was stored at -20° C. Alkaline Lysis Solution I 50 m M glucose 10 m M EDTA, pH 8.0 25 m M Tris-Cl, pH 8.0 4 mg/mL lysozyme Alkaline Lysis Solution II 0.2 N sodium hydroxide (NaOH) 1% (w/v) sodium dodecyl sulfate (SDS) Chapter 2. Materials and Methods Alkaline Lysis Solution HI 60 mL 5M potassium acetate (KOAc) 11.5 mL glacial acetic acid 28.5 mL dH 20 RNAse (lOmg/mL) 100 mL Tris-Cl, pH 7.5 30 mL 5 M N a C l 0.100 g RNAse lOmLdFLO L B Broth 1.0 g tryptone 0.5 g sodium chloride (NaCl) 0.5 g yeast extract 0.1 g dextrose dH 20 to 100 mL 1.2 g agar and 5 mg kanamycin for plates I X TE pH 8.0 10 m M Tris-Cl, pH 8.0 1 m M EDTA, pH 8.0 Chapter 2. Materials and Methods 42 2.3.3 Preparation of High Density Cosmid Library Filters High density cosmid library filters were prepared by Ashley Howard on Hybond N+ membranes (Amersham) which were overlaid on agar plates containing kanamycin. The filters were innoculated using a robotic 96-prong device (Biomek 1000, Beckman Instruments Inc.) which allowed for all of the clones from a single microtitre plate to be stamped on simultaneously. By staggering the placement of each stamping, 1,536 clones (96x 16 plates) could be placed on a single filter. The filters were grown overnight at 37° C, and the D N A was lysed, denatured, and fixed to the membrane according to the protocol of Grunstein and Hogness (1975). The entire library was gridded onto thirteen filters, labeled from A to M . 2.4 Restriction Enzyme Digestion Cosmid or Y A C D N A was digested with 1 unit/p:g D N A of restriction enzyme. A standard reaction volume of 20 pX was used, containing 1-5 (ig of DNA, I X restriction digest buffer, and 0.1 Ug/uX BSA. The reaction proceeded at 37° C for 1-2 hours, and was terminated by the addition of 5 pX of stop buffer. Chapter 2. Materials and Methods 43 Stop Buffer 0.25% bromophenol blue 0.25%) xylene cyanol 40%o (w/v) sucrose in dH 20 60 m M E D T A 2.5 Agarose Gel Electrophoresis Agarose gels were prepared by dissolving agarose in I X TBE running buffer, and allowing the solution to solidify in a casting tray at room temperature. The addition of 1 u.g/mL of ethidium bromide allowed visualisation of the D N A bands under ultraviolet light. Cosmid or Y A C restriction digests were electrophoresed on 0.8% agarose gels, at a constant voltage of 30 V overnight. 4 u.g of X D N A digested with Hindlll and SacII was used as a molecular weight marker. PCR products from Alu PCR, Y A C vector arm PCR, and STS content mapping in Y A C s and radiation hybrids were electrophoresed on 2-2.5%> agarose gels. 2.5 u.g of <])X174 D N A digested with Haelll (Gibco, BRL) was used as a molecular weight marker. The gels were photographed with a MP-4 camera. Chapter 2. Materials and Methods 44 1 0 X T B E 54 g Tris base 27.5 g boric acid 10 m M EDTA, pH 8.0 dH 20 to 500 mL 2.6 Southern Blotting EcoRl digested cosmid or Y A C D N A was transferred onto nylon membranes from agarose gels using the method of Southern (Southern, 1975). A ruler was placed alongside the gel to be photographed, so that each band could be identified on the blot by its migration distance. Excess agarose was cut away, and the gel was soaked in depurination solution (0.25 N HC1) for 10 min., denaturation solution (1.5M NaCl/0.5 M NaOH) for 30 min.,and neutralization solution (1 M Tris/1.5 M NaCl) for 30 min. The gel was rinsed briefly in dH 20. A glass dish containing 600 mL of 10 X SSC was overlaid with a glass plate. To create a "wick", two pieces of 3 M M Whatman paper were pre-wetted and positioned on the plate so that each end of the paper was immersed in solution. A piece of nylon membrane (Hybond N+, Amersham) and two pieces of 3 M M Whatman paper were cut to match the dimensions of the gel. After the gel was placed on the wick, it was covered by a piece of pre-wetted nylon membrane, one piece of pre-wetted Whatman paper, one piece of dry Whatman paper, and a stack of paper towels. The gel was surrounded by plastic strips Chapter 2. Materials and Methods 45 to prevent the buffer from flowing directly into the paper towels. The transfer was left at room temperature for 5 hours-overnight, and the D N A was fixed onto the membrane by microwaving the membrane between two pieces of Whatman paper for 2 min. 20 X SSC 175.3 g NaCl 88.2 g sodium citrate dH 20 to 1 L 2.7 Extraction of EcoRlfragments EcoRl fragments to be used as hybridization probes were extracted from agarose gels, and purified using a Qiaex II gel extraction kit (Qiagen). With this procedure, the agarose is solubilized using a high concentration of salt, which disrupts the hydrogen bonds between sugar molecules in the agarose. D N A is adsorbed onto silica particles in the presence of a high salt buffer and low pH (<7.5). It is then eluted in a low salt buffer ( IX TE) and high pH (>7.5). The D N A was diluted to a concentration of 1 ng/pL in I X TE, and 15 ng of D N A was oligolabeled, as described below. Chapter 2. Materials and Methods 46 2.8 Oligolabeling Y A C Alu PCR products, total human DNA, and EcoRl fragments were radioactively labeled using the random primer method of Feinberg and Vogelstein (1984a,b). The probe was denatured by boiling for 5 min, and cooling on ice for 5 min. A standard reaction contained 30 ng of DNA, 10 uL of 5X oligolabeling buffer-A (OLB-A), 0.1 mg/mL BSA, 50 p,Ci of oc-32P dATP, and 1 u of Klenow fragment. For half reactions containing 15 ng of DNA, all volumes were halved, except the Klenow fragment. This mixture was incubated at room temperature overnight, and the reaction was terminated by the addition of an equal volume of nick translation stop buffer (NTSB). Unincorporated nucleotides were separated from the labeled probe by exclusion chromatography using Sephadex G-25 beads. The mixture was added to the Sephadex column and centrifuged for 2 min. at 1,000 rpm. The labelled probe was collected, and the total volume was brought up to 100 uX with mQ H 20. Alu PCR probes and EcoRl fragments were preannealed prior to use to prevent hybridization of repetitive sequences. 300 |ig of human placental D N A and 11 uL of 25X SSC were added, boiled for 5 min, and incubated at 65° C for 1 hour. At the same time, cosmid filters or Southern blots were prehybridized with 5 mL of hybridization solution at 65°C for 1 hour. The probe was diluted in 5 mL of hybridization solution before being added to the filters, and the reaction proceeded overnight at 65°C. The filters were washed in 1 X SSC/1% SDS for 5 min. at Chapter 2. Materials and Methods 47 room temperature, and then in 0.2 X SSC/0.1% SDS for 1 hour at 65°C, with shaking. Hybridization was detected by autoradiography. If necessary, the filters were stripped by pouring 0.5% SDS at boiling temperature over them, and leaving them to cool to room temperature. Filters were used a maximum of three times. 5X O L B - A Mix solutions A:B:C in a ratio of 100:250:150 Solution O: 1.25 M Tris-Cl pH 8.0 0.125 M M g C l 2 , Solution A: 1 uL Solution O 18 u.L (3-mercaptoethanol 5 uL each dGTP, dCTP, dTTP (each dNTP is 0.1 M in 3 mM Tris pH 7.0, 0.2 mM EDTA) Solution B: 2 M Hepes pH 6.6 Solution C: Hexadeoxyribonucleotides suspended in 1 X TE to 90 OD/mL NTSB 50 m M E D T A 20 m M NaCl 0.1% SDS 500 mg/mL salmon sperm D N A Chapter 2. Materials and Methods Hybridization Solution 1 5 0 m L 2 0 X S S C 50 mL 5X Denhardt's Solution 7.5 mL 20% SDS 50 mL 100 mg/mL salmon sperm D N A 242.5 mL dH 20 100 X Denhardt's Solution 4% Ficoll type 400 4% polyvinyl pyrrolidone 4% (w/v) bovine serum albumin (BSA) Chapter 3 Results 3.1 Radiation Hybrid Mapping The 95 radiation hybrids in the chromosome 8 specific panel were tested twice for the presence or absence of each STS by PCR amplification, and ambiguous results were resolved by an additional PCR test (Appendix 1: R H Data). Hybrids typed with the marker D8S596 are shown in Figure 4. The retention frequencies for the loci in the hybrid panel ranged from 8.4 - 17.9%, with an average retention frequency of 13.5%) (Table 2). Table 2: Radiation Hybrid Retention Frequencies Locus # of Positive Hybrids Retention Frequency Scored (%) D8S596 8/95 8.4 D8S206 17/95 17.9 D8S264 16/95 16.8 D8S7 15/95 15.8 D8S504 9/95 9.5 D8S262 9/95 9.5 D8S201 12/95 12.6 D8S518 13/95 13.7 D8S439 14/95 14.7 D8S277 15/95 15.8 Chapter 3. Results 50 Figure 4: STS Content Mapping on the Radiation Hybrid Panel Using Marker D8S596 Note: Only four positives are shown here ( A l l , D8, F12, G10, H12 = positive control) although eight positives in total were found for this system. Chapter 3. Results 5 3.1.1 Two-Point Radiation Hybrid Analysis Two-point radiation hybrid analysis was used to establish linkage groups based on pairwise L O D scores of at least 3.0, and 4.0, corresponding to 1,000:1 and 10,000:1 odds, respectively. This analysis demonstrated that all ten loci could be placed into the same linkage group at 10,000:1 odds, meaning that at least one locus order exists where the maximum LOD scores between loci #1 and #2, #2 and #3 #9 and #10 are >_4.0. Two-point LOD scores, breakage frequencies and distance estimates are given in Table 3. Chapter 3. Results Table 3: Two-Point LOD Scores, Breakage Frequencies and Distance Estimates Locus 1 Locus 2 Breakage Freq. (%) Distance (cR5,ooo) LOD Score D8S596 D8S206 41.5 53.5 4.93 D8S596 D8S264 47.7 64.8 3.91 D8S596 D8S7 34.6 42.5 5.85 D8S596 D8S504 6.5 6.7 10.27 D8S596 D8S262 6.5 6.7 10.27 D8S596 D8S201 22.4 25.3 7.59 D8S596 D8S518 26.8 31.2 6.95 D8S596 D8S439 41.1 53.0 4.67 D8S596 D8S277 44.5 58.9 4.27 D8S206 D8S264 47.7 64.8 4.51 D8S206 D8S7 52.6 74.7 3.67 D8S206 D8S504 44.6 59.0 4.53 D8S206 D8S262 44.6 59.0 4.53 D8S206 D8S201 52.9 75.3 3.49 D8S206 D8S518 55.4 80.8 3.19 D8S206 D8S439 57.8 86.3 2.90 D8S206 D8S277 60.1 91.9 2.64 D8S264 D8S7 34.7 42.6 6.79 D8S264 D8S504 41.5 53.5 4.93 D8S264 D8S262 41.5 53.5 4.93 D8S264 D8S201 25.1 28.9 8.52 D8S264 D8S518 36.6 45.6 6.19 D8S264 D8S439 55.4 80.8 3.19 D8S264 D8S277 57.8 86.3 2.90 D8S7 D8S504 28.6 33.7 7.11 D8S7 D8S262 38.2 48.1 5.37 D8S7 D8S201 21.6 24.3 9.20 D8S7 D8S518 25.1 28.9 8.52 D8S7 D8S439 44.8 59.4 4.73 D8S7 D8S277 55.4 80.8 3.19 D8S504 D8S262 12.3 13.1 9.20 D8S504 D8S201 16.1 17.5 9.20 D8S504 D8S518 20.6 23.0 8.42 D8S504 D8S439 44.5 58.9 4.27 D8S504 D8S277 47.7 64.8 3.91 D8S262 D8S201 16.1 17.5 9.20 D8S262 D8S518 20.6 23.0 8.42 D8S262 D8S439 34.6 42.5 5.85 D8S262 D8S277 38.2 48.1 5.37 D8S201 D8S518 13.8 14.9 10.82 D8S201 D8S439 35.6 44.1 6.04 D8S201 D8S277 38.9 49.2 5.57 D8S518 D8S439 21.6 24.3 9.20 D8S518 D8S277 33.5 40.8 6.69 D8S439 D8S277 12.2 13.0 12.29 Chapter 3. Results 53 3.1.2 Multipoint Radiation Hybrid Analysis Initially, the four loci D8S264, D8S201, D8S518 and D8S277 were chosen for multipoint radiation hybrid analysis, on the basis of previous positioning by genetic linkage mapping in the order: telomere - D8S264 - D8S201 - D8S518 - D8S277 - centromere at 1000:1 odds against order inversion (CEPH database, version 7). With the radiation hybrid data, both minimum break and maximum likelihood analyses supported the same order at 1000:1 odds. Five loci, consisting of the four core markers plus one additional marker, were simultaneously analyzed by both minimum break and maximum likelihood analyses. Locus orders derived from minimum break analysis were compared to those derived from maximum likelihood analysis. In all cases, minimum break orders were included in the maximum likelihood orders, and thus minimum break analysis was concluded at this point. Maximum likelihood analyses of five loci simultaneously failed to produce statistically significant placements for the remaining six markers. However, D8S206 appears to be positioned on either the distal or proximal end, while D8S439 seems to be placed on either side of D8S277 (Table 4). Chapter 3. Results 54 Table 4: Maximum Likelihood Placements of Six Loci With Respect to Four Core Loci Telomere Core Order Centromere Locus D8S264 D8S201 D8S518 D8S277 D8S206 ** * *(74.9) D8S262 •(190.6) *(27.6) *0 .5) *** *(68.7) D8S439 *** *(335.9) D8S504 •(130.5) *(18.9) ** * *(20.3) D8S596 *(5.6) *(31.9) *(4.4) * ** *(2.4) D8S7 *(15.0) * * * *(4.4) *(305.9) * m o s t l i k e l y pos i t i on * o ther poss ib l e pos i t i ons , w i t h numbers in parentheses i nd i ca t i ng the l i k e l i h o o d o f the less l i k e l y pos i t i on c o m p a r e d to the mos t l i k e l y pos i t i on Similarly, maximum likelihood analysis of ten loci simultaneously produced a total of 63 different orders with odds against order inversion < 1,000:1. The top five locus orders derived from this analysis are shown in Table 5. Table 5: Top Five Locus Orders Obtained from Maximum Likelihood Analysis of Ten Loci Simultaneously Odds Marker Order 1 D8S206 D8S264 D8S7 D8S504 D8S596 D8S262 D8S201 D8S518 D8S439 D8S277 1.8 D8S206 D8S264 D8S7 D8S201 D8S504 D8S596 D8S262 D8S518 D8S439 D8S277 1.8 D8S206 D8S264 D8S7 D8S201 D8S262 D8S596 D8S504 D8S518 D8S439 D8S277 4.4 D8S206 D8S264 D8S201 D8S262 D8S596 D8S504 D8S7 D8S518 D8S439 D8S277 6.4 D8S206 D8S264 D8S201 D8S7 D8S504 D8S596 D8S262 D8S518 D8S439 D8S277 Chapter 3. Results 55 Despite the fact that a definitive order for the ten loci could not be determined, it appears that D8S518-D8S439-D8S277 are contiguous, and that D8S206 lies distal to D8S264 (shown in bold in Table 5). D8S596 is a telomeric marker, and can therefore be anchored at the telomeric end. Excluding D8S262, D8S504 and D8S7, a likely order derived from the R H mapping data is: D8S596 D8S206 D8S264 D8S201 D8S518 D8S439 D8S277 This order can be refined by the integration of genetic linkage data. Genethon's linkage map places D8S504 distal to D8S264, either distal or proximal to D8S206, and D8S262 between D8S264 and D8S518 (Gyapay et al., 1994). Furthermore, D8S262 is linked between D8S201 and D8S518 at odds exceeding 1000:1 for placement (CEPH database, version 7). Therefore, the previous order can be modified to include nine loci, excluding D8S7: D8S596 D8S206/D8S504 D8S264 D8S201 D8S262 D8S518 D8S439 D8S277 Chapter 3. Results 56 3.2 STS Content Mapping in YACs 43 individual Y A C s representing an 8p23 enriched subset (Section 2.2.1) were tested for the presence or absence of each STS by PCR amplification (Figure 5). Using the nine-locus order previously derived from the R H and linkage mapping data, a preliminary Y A C contig map was generated (Figure 6). Double linkage between D8S504/D8S7, D8S264/D8S201, D8S201/D8S262/D8S518, and D8S439/D8S277 was exhibited, meaning that the STSs are each positive on two different YACs . Therefore, D8S7, which was not previously assigned a position, is shown next to D8S504, although its precise location either distal or proximal to D8S504 cannot be determined. A comparison of linkage, RH, and STS content maps is shown in Figure 7. Despite the fact that many gaps exist between markers (e.g. D8S596/D8S206; D8S206/D8S504; D8S7/D8S264; D8S518/D8S439), six YACs were chosen to comprise a minimum tiling path. These include the telomeric Y A C yRM2205, 693dl 1, 741h4, 787cl 1, 810f8, and 967c 11. In cases where two or more YACs were positive for a given marker, preference was given to the Y A C which was positive for a greater number of markers, and/or had STS content data verified by the Whitehead Institute. Chapter 3. Results 57 Figure 5: STS Content Mapping in YACs Using Marker D8S201 Note: Only 31/43 YACs are shown (+) control = somatic cell hybrid MGV269, (-) control = mQ H20 a as Q 11 mm ill j Lie II IP ft. 0 0 V E o O E o cu OS cu E _© H 01 © a .2° s o U U a CU u Cu < cu 3 M 5 Ss H «n o CN — CN — V] U JV1 3d YA K O N YA * * O N I T ) 3 < 6 >• 5 .e 2 <x I 1 h O N 82 O 50 I g oo •& 00 a c c § > 8 N O OO OO 0 0 Q c N o b O N o N O m <N <N m OO OO OO OO o o 0 0 o o 0 0 Q Q Q Q oo 0 0 Q 1 2 8 a: cd X ) •si —1 ^ to a o WN OO o o Q o © O N CQ N O N O O m oo 0 0 Q 0 0 0 0 Q oo 0 0 Q N O ( N OO 0 0 Q 0 0 N O f— •<* t N t N N O N O 0 0 o t N — t N oo oo OO oo 0 0 0 0 0 0 0 0 Q Q Q Q O N ac N ( N <N m CQ < r-t N oo 0 0 Q N O p -v i t N oo oo 0 0 0 0 o t N V N O V > t -O N O N < O N t N t N OO a . o U OO H oo a . 3 en CO U " oo £ H £ oo w » 92 o N O O N Chapter 3. Results 60 3.3 Identification of Cosmids Contained Within Minimum Tiling Path YACs 3.3.1 YAC Alu PCR Products Onto High Density Cosmid Colony Filters In an effort to isolate bins of cosmids defined by the minimum tiling path YACs, Alu PCR products were used as hybridization probes onto the chromosome 8 cosmid library. Alu PCR products derived from Y A C 787c 11 are shown in Figure 8, and an example of a probed high density cosmid filter is shown in Figure 9. An average of 327 cosmids were obtained per Y A C (Table 6). These data were entered into Octobase, a chromosome 8 specific database maintained in the Wood lab (Appendix 2). Figure 8: Ethidium Bromide Stained Agarose Gel of Alu PCR Products Derived from YAC 787cll (+) control = somatic cell hybrid line MGV269; (-) control = mQ H 20 Chapter 3. Results 61 10 11 12 f g b • • • a • ••a • • • a n a n a • • • • n a n a • a n a • • • • • n a n • • • • • • • • • a n a n a n a • • • • auaa • O D D • • • • • • • • • D Q D • • • • • • • • n a n a • • • • • • • • • • • • • • • • • D D D • • a n D D D D • • • • • • • • • • • • • • • • • • n o • • • • • • • • • • cc • • • • • • • • • • • • • • • • • D D D • D I D D D D D D a a n • • • • • D D D • • • • • • • • • a • • • • • • D D D D • • • • • • • • • • • D • • • B • D Q D • • • • a a a a • • • • • • • • • • • a • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • D D D D • • • • n o n n • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • a D O • a o n • • • • • • • • • • • • • • • • D D D D • a n o D a D O D D D D D D D D n a n a D D D D D D D D • D D D • • • • • • a n • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • D D D D D D D D a a a a 7 i ) 10 11 12 • • • • • • • • • • • • • • • • • D D • • • • I D D D • • • • • • • • • • • • • • • • • naa 1 D DD • • • • • • • • • • • • • • • • • • • • i a a a • • • • • • • • • • a nan o • • • I D D D • • • • • • • • • • • • o • • • • • • • I D D D • • • • • • • • • • • • a • • • • • • • I D D D • • • • • nan • • • • • • • • • • • • I D B D • • • • • • • • • • • • • • • • • • • • • an • • • • • • • • • • • • • • • • • • • • n a n • • • • • • • • a • • • a • • • a • • • • • • • D DO • • • • • • • • • • • • • • • • ! • • • • • • • • • • • • • • • • • • • • • • • ) • • • • • • • o D D D a • • • • • D D • • • • ! • • • • • D D • • • • • • • • n • • • • • • • • • • • • • • • • • • • • • • • • • • o • • • ! • • • • • B D • • • • • • • • • • • • • • c m ! • • • • • • • • • • • • • • • • • • • • • • • ! • • • o a n a n nun • • • • a • • • D • • • ! • • • • • • • • • • • • • • • • • • • • • • • • • • a • • • • n a a • • • • • • • • • • • • ! • • • a • • • n • • • • P P P a • • • a • • • ! • • • • • • • • • • • • • • • • • • • • • • • 1 • • • • • • • • • • • • • • • • • • • • • P P 1 • • • • • • • • • • • • • • • • • • • • • • • 1 • • • • • • • • • • • • • • • • • • • • • • • ! • • • • • D D • • • • • • • • • • • • • • • • 1 • • • • • • • • • • • • • • • • • a n • • • • 1 • • • • • • • • • • • a • • • • • • • • • • • ! • • • • • • • a • • • p • • • • • • • p • • • i a o n a una a • • • D a • • a • • • p • a n 1 o o o • • • • • • • • • • • • • • • • • • • • I D D D • • • • • • • • • • • • • • • • p • • • • strong positive Q weak positive Figure 9: Example of a High Density Colony Cosmid Filter ("L") Probed with Alu PCR Products from YAC 967cll, and Corresponding Positives Chapter 3. Results 63 3.3.2 YAC Alu PCR Products onto EcoRl Digested Cosmid DNA Following the isolation of an average of 300 cosmids per Y A C , a small subset of three cosmids per Y A C were chosen for further analysis, on the basis of specificity to only one probe, and on strong hybridization signals to the high density cosmid filters (Table 7). The objective of this second experiment was to confirm whether these cosmids represented true positive results, by hybridizing the same Y A C Alu PCR products against a more specific target consisting of EcoRl digested cosmid DNA. Total human D N A was also used as a probe to identify those EcoRl fragments which were hybridizing to the Alu PCR probes due to the presence of incompletely suppressed repetitive elements. Since seven hybridization reactions were required (six Y A C s + total human DNA), four identical Southern blots were prepared, three of which were stripped and re-used. An ethidium bromide stained agarose gel of the eighteen cosmids digested with EcoRl is shown in Figure 10. It is apparent from the similarities in restriction digestion patterns that the cosmid pairs 76G9/151H12, 125E5/197E7 and 23D5/77A3 share many EcoRl fragments in common and may represent overlapping clones. Chapter 3. Results 64 Table 7: Subset of Eighteen YAC Specific Cosmids Chosen for Further Analysis Minimum Tiling Path YAC Cosmids 6 F 8 y R M 2 2 0 5 7 6 G 9 151H12 4 2 B 8 6 9 3 d l l 186D1 2 0 1 G 2 6 8 E 9 741h4 187F3 189G10 7 5 C 2 7 8 7 c l l 106B3 136G1 16H1 810f8 125E5 197E7 2 3 D 5 9 6 7 c l l 7 7 A 3 183F12 Chapter 3. Results 62 Table 6 : YAC Chimaerism, Sizes and Numbers of Positive Cosmids Identified per YAC YAC Chimaerism* Size (kb) ** # of positive cosmids yRM2205 not available 240*** 313 693dll no not available 389 741h4 yes-chromosome 14 not available 177 787cll no 1740 317 810f8 yes-chromosome 9 1180 289 967cll yes-chromosome 17 1110 476 * Chimaerism determined at MIT ( by STS content mapping ** YAC sizes determined at CEPH ( by pulsed field gel electrophoresis followed by hybridization to a probe containing a mixture of labeled pBR322 and total human DNA ** Riethman, 1993, GDB ( Chapter 3. Results 65 Figure 10: Ethidium Bromide Stained Agarose Gel of Eighteen Y A C Specific Cosmids Digested with EcoRl Chapter 3. Results 66 It was predicted that a given Y A C Alu PCR probe would hybridize specifically to the three out of eighteen cosmids that were originally identified by that Y A C in the preceding experiment. However, from the Southern blotting results, it is evident that a large amount of non-specific background hybridization is occurring, despite preannealing of each Alu PCR probe with total human DNA. Hybridization of the EcoRl digested cosmids to the total human D N A probe is shown in Figure 1 la. With Y A C 967c 11, the cosmids 23D5 and 77A3 clearly hybridized preferentially to the Alu PCR probe in comparison to the fifteen additional cosmids, while 183F12 only showed a background amount of hybridization (Figure 1 lb). It is likely that this cosmid represents a false positive result. A similar pattern was observed with Y A C yRM2205, where the cosmids 76G9 and 151H12 hybridized strongly to the probe while 6F8 did not (Figure 11c). Although not as striking as the first two results, the cosmids 106B3 and 136G1 hybridized preferentially to Y A C 787cl 1, and 75C2 did not (Figure l i d ) . Only cosmid 197E7 hybridized preferentially to Y A C 810f8 (Figure l ie) . Both Alu PCR products from YACs 693dl 1 and 741h4 exhibited similar hybridization intensities for the majority of cosmids, and each set of three specific cosmids/YAC could not be distinguished (Figures 1 If and 1 lg). In total, this experiment identified seven true positive cosmids out of eighteen (Table 8). Chapter 3. Results 67 2 CO -a 1—1 i— r—I -a -a — CJ m i — i o o "3- oo 1 — oo tN S3 oo O S 00 pq in (N Q sC oc W r- CTN U Co ffi o o o o o o o i ^ o m ^ (N o m h ^ (N r- oo 187F3E20.0 151H12E4.0 201G2E3.2 197E7E5.0 -136G2E2.9 23D5E1.7 Figure 11a: EcoRl Digested Cosmids Probed with Total Human DNA Arrows in Figs. 1 la-g show the positions o f fragments in the corresponding cosmid lanes which were used as probes in the following experiment (Exp. 3.3.3). Fragment sizes (in kb) are indicated by the number following " E " . Chapter 3. Results Figure lib: EcoRl Digested Cosmids Probed with Alu PCR Products from YAC 967cll Chapter 3. Results o 151H12E4.0 Figure 11c: EcoRl Digested Cosmids Probed with Alu PCR Products from YAC yRM2205 Chapter 3. Results 70 o 0 0 [> U < 2$ X 2° Q d f t £ o r* S © a £3 w «2 oo m vo C N m r - oo ( N t> 1 3 6 G 1 E 2 . 9 Figure l id: EcoRl Digested Cosmids Probed with Alu PCR Products from YAC 787cll Chapter 3. Results 71 as o oo 197E7E5.0 Figure lie: EcoRl Digested Cosmids Probed -with Alu PCR Products from YAC 810f8 Chapter 3. Results Figure 1 If: EcoRl Digested Cosmids Probed with Alu PCR Products from YAC 693dll Chapter 3. Results Figure 1 lg: EcoRl Digested Cosmids Probed with Alu PCR Products from YAC 741h4 Chapter 3. Results 74 3.3.3 Individual Cosmid EcoRl Fragments as Probes Onto YAC EcoRl Southern Blots A final test was designed to confirm the origin of select cosmids. Six cosmids (one per Y A C ) from the preceding experiment (Exp. 3.3.2) were chosen, and one strongly positive EcoRl fragment/cosmid was isolated. To avoid using EcoRl fragments containing repetitive sequences, an attempt was made to select fragments which did not hybridize to the total human D N A probe (Figure 11a). However, in the case of cosmids isolated with YACs 810f8, 741h4 and 693dl 1, this was not possible due to the lack of strongly hybridizing cosmid EcoRl fragments (Figures 1 le-g, respectively). The names of the six isolated fragments (Figures 1 la-g) were derived from the original cosmid number, followed by an " E " for EcoRl , and the fragment size in kb. These fragments were oligo-labeled, and probed onto Southern blots containing Y A C D N A digested with EcoRl (Figure 12). This ensured that the correct signal could be easily visualized, as it would be exactly the same size as the cosmid EcoRl probe. "if — — — JN = —• r- o r-- ^ o\ -<r oo — NO CL + Figure 12: Ethidium Bromide Stained Agarose Gel of YAC DNA Digested with EcoRl (+) control = genomic CEPH DNA Chapter 3. Results 75 Positive results were obtained for Y A C yRM2205 using probe 151H12E4.0 (Figure 13a); Y A C 967cl 1 using probe 23D5E1.7 (Figure 13b), and Y A C 787cl 1 using probe 136G1E2.9 (Figure 13c). Interestingly, the probe 136G1E2.9, which was originally isolated from Y A C 787c 11, also hybridized to Y A C 741h4, confirming the overlap between the two Y A C s (Figure 13c). Since cosmid 136G1 was not positive with the Alu PCR probe derived from Y A C 741h4 in either experiments 3.3.1 or 3.3.2, this represents a false negative result for this Y A C . Negative results were obtained for Y A C 741h4 using probe 187F3E20.0; Y A C 810f8 using probe 197E7E5.0, and Y A C 693dl 1 using probe 201G2E3.2. Therefore, experiments 3.3.2 and 3.3.3 were repeated with six additional cosmids for each of the three YACs (data not shown). A further positive result for Y A C 810f8 with probe 168A2E6.0 was observed (Figure 13d). Numerous hybridizations with different cosmid probes failed to produce positive results for Y A C s 693dl 1 and 741h4 (data not shown), excluding the false negative result obtained for Y A C 741h4 with probe 136G9E2.9, as mentioned above. In total, one cosmid was isolated for five out of the six minimum tiling path YACs. A summary of these results is found in Table 8. Chapter 3. Results 76 « _ 1 . 7 k b Figure 13a: Southern Blot of YAC EcoRl Digests Probed with 151H12E4.0 b: Probe 23D5E1.7 (Positive control not shown) Chapter 3. Results 11 2.9 kb *~6.0 kb Figure 13c: Probe 136G1E2.9, d: Probe 168A2E6.0 Showing Overlap Between YACs (+) control = CEPH genomic DNA 787cll and 741h4 (Positive control not shown) Chapter 3. Results 78 Table 8: Summary of Results of Individual Cosmid EcoRl Fragments Probed Onto YAC EcoRl Southern Blots EcoRl Probe Name Alu PCR Total YAC YAC Cosmid size (kb) human blot DNA 967c11 23D5 1.7 23D5E1.7 strong + - + yRM2205 151H12 4.0 151H12E4.0 strong + - + 787cll 136G1 2.9 136G1E2.9 strong + - + 810f8 168A2 6.0 168A2E6.0 ' strong + weak + + 197E7 5.0 197E7E5.0 strong + + -693dll 201G2 3.2 201G2E3.2 weak + weak + -178H9 3.9 178H9E3.9 weak + - -178H9 7.0 178H9E7.0 weak + weak + -27A1 16.0 27A1E16.0 weak + + -67H5 12.0 67H5E12.0 weak + + -127H12 8.0 127H12E8.0 weak + + -127H12 6.0 127H12E6.0 weak + + -741h4 187F3 20.0 187F3E20.0 weak + + -70A8 10.0 70A8E10.0 weak + weak + -88H6 7.0 88H6E7.0 weak + weak + -72E10 16.0 72E10E16.0 weak + - -136G1 2.9 136G1E2.9 - - + 79 Chapter 4 Discussion 4.1 Radiation Hybrid Mapping R H mapping was used to establish the chromosomal order of ten STSs mapping to the telomeric region of human chromosome 8. This method involves inducing chromosome breaks by irradiation, rescuing the fragments via fusion to a HPRT deficient rodent cell line, selecting hybrid cells in HAT medium, and typing hybrids for the markers of interest. Marker order and pairwise distance estimates are calculated based on the principle that the probability of breakage between two loci is proportional to the distance between them. R H mapping complements yet confers many advantages over other mapping techniques, such as meiotic linkage analysis, in situ hybridization, and pulsed field gel electrophoresis. For example, linkage mapping requires the use of polymorphic markers to distinguish between paternally and maternally derived alleles, while non-polymorphic markers are informative in R H mapping because haploid data can be used. Additionally, the assumption of zero interference appears to be valid in RH mapping since chromosome breaks are induced essentially at random, whereas in linkage mapping recombination frequencies are affected by interference and increase progressively from centromere to telomere (Boehnke et al., 1991). Similarly, the nonrandom distribution of rare cutting restriction enzymes sites used in pulsed field gel electrophoresis makes it difficult to order long stretches of D N A with this technique alone (Cox et al., 1990). A further advantage of Chapter 4. Discussion R H mapping is that the required level of resolution can be optimized by increasing or decreasing the radiation dose. However, it cannot be used to map disease loci. 80 4.1.1 Two-Point Radiation Hybrid Analysis Since the inclusion of unlinked markers complicates interpretation of R H mapping data, two-point analysis was initially used to detect linkage between marker pairs based on LOD scores_> 3. This criterion was exceeded, and all markers could be placed in a single linkage group at LOD scores_> 4 (Table 3). Cox et a l , (1990) calculated a direct relationship between R H map distance and physical distance with l cR g0oo ~ 53 kb, by measuring the physical distance between two markers by pulsed field gel electrophoresis, and comparing this to the distance estimate provided by R H mapping. A similar approach was taken to estimate the relationship between cR 5 0oo and kilobases in the chromosome 8 specific R H mapping panel. The physical distance between three STSs contained in Y A C 787c 11 was roughly estimated to be equal to the size of the Y A C , which spans 1740 kb (Table 6). This particular Y A C was chosen because it does not appear to be chimaeric or contain gaps, and the three STSs contained within it were also found to be positive according to the Whitehead Institute (Table 6; Figure 6). Chapter 4. Discussion 81 From Table 3: D8S201-D8S262 17.5 cR •5000 D8S262-D8S518 23.0 cR--5000-40.5 cR« •5000 1740kb 1 cR. •5000 43 kb It would be interesting to calculate the relationship between cR 5 0oo and physical distance for other regions of the chromosome tested with this hybrid panel, to determine whether the observed correlation of 1 cR 5 0oo ~ 43 kb fluctuates between different chromosomal locations. 4.1.2 Multipoint Radiation Hybrid Analysis Minimum break analysis is a useful supplement to maximum likelihood analysis. Due to its nonparametric nature, assumptions about fragment retention and chromosome breakage are unnecessary; however, likelihood calculations of alternate orders cannot be made. Since all minimum break orders were included in the maximum likelihood orders, minimum break analysis was not looked at further. Multipoint maximum likelihood analysis uses a complex set of algorithms to ensure that the optimal locus order is found. Four loci were placed in the order tel-D8S264-D8S201-D8S518-D8S277-cen at 1,000:1 odds against order inversion, which confirms the linkage data (Figure 7). Statistically significant placements for the remaining six loci with respect to the four core loci could not be established, and consequently, a conclusive order involving all ten loci could not be determined (Tables 4 and 5). Chapter 4. Discussion ° 2 Possible reasons for the failure of RH mapping to establish a definitive locus order include the mistyping of hybrids, and the amount of radiation used in the construction of the hybrid panel. It was observed with D8S439 (data not shown) that the mistyping of one hybrid had a significant impact on the outcome of the analysis, where a single change from negative to positive resulted in the difference between being able to place D8S439 in one position with respect to the four core loci at 1,000:1 odds, vs. the two positions that it currently occupies (Table 4). The amount of radiation used in the construction of the hybrid panel affects the frequency of breakage between loci, as well as their retention frequencies. Breakage frequencies between linked loci were found to range between 6.5-55.4% (Table 3). A critical balance between insufficient and excessive breakage must be maintained, as significant linkage between two markers could not be established at breakage frequencies_> 55.4%, presumably because most or all loci lie on separate fragments. Conversely, a lack of breakage would imply that most or all loci lie on the same fragments, making the determination of locus order difficult. The average retention frequency for the ten STSs was 13.5% (Table 2), which is significantly lower (p<0.001) than the average frequency of 18.7% observed for 35 additional loci typed on the same hybrid panel. In some R H data sets, retention frequencies for markers have been shown to vary directly with their proximity to the centromere, which may be due to an intrinsic centromeric effect that promotes retention (Benham et a l , 1989). Given that centromeres play a vital role in chromosome segregation, it is possible that the Chapter 4. Discussion °3 centromeric fragments are selectively retained during cell division, prior to the stage when the fragments are incorporated into the rodent cells. With the exception of D8S7, loci which had many placements with respect to the four core loci exhibited the lowest retention frequencies. These included D8S596, D8S504, and D8S262, with corresponding frequencies of 8.4%, 9.5% and 9.5%, respectively. Given the low retention rates for these markers, they must be physically close to the other markers in order to obtain overlapping retentions. However, marker density could not be accurately calculated, as the physical size of the region remains unknown. As R H mapping did not establish a conclusive order for the ten loci, physical distance could not be approximated by the corresponding cR 50oo distance estimates. Y A C sizes also could not be used because of the gaps in the Y A C contig. 4.2 YAC Contig Construction The cloning of megabase-sized pieces of D N A into YACs has bridged the resolution gap between traditional linkage mapping and standard physical mapping techniques using small insert clones such as cosmids. The use of STSs as the common language for physical mapping is well established, and STS content mapping via PCR to detect overlap relationships among Y A C s is widely employed. This strategy was used to build a preliminary Y A C contig of the telomeric region. Existing gaps in the current contig may be filled in by testing additional YACs , and by developing more closely spaced STSs. Double linkage was exhibited by markers Chapter 4. Discussion 84 D8S504/D8S7, D8S264/D8S201, D8S201/D8S262/D8S518, and D8S439/D8S277 (Figure 6). Given the high rate of chimaerism and rearrangements in YACs , as well as the possibility of laboratory error, double linkage is clearly a more reliable criterion for building contigs than is single linkage. The Whitehead Institute independently confirmed the STS content data for selected Y A C s and STSs. One problem associated with information collected at the Whitehead Institute is that negative results are not reported, so i f a Y A C is not positive with a particular STS, it may not have been tested, or may have tested negative. Conversely, not all of the Whitehead Institute data could be confirmed by data presented in this thesis despite the fact that all Y A C s were tested for each STS. Although Y A C s were screened prior to STS content mapping with vector-arm PCR, this tests for the presence of the Y A C vector rather than the human insert DNA, and may be the reason why negative results were observed. Six Y A C s comprising a minimum tiling path were chosen. Apparent gaps in these YACs may represent deletions or intrachromosomal chimaerism, but gaps are also dependent on the given marker order. Changes in the marker order result in the closure of existing gaps, as well as in the creation of additional ones. This particular marker order was chosen based on R H and linkage mapping data, as well as on double Y A C linkage. Chapter 4. Discussion 85 4.3 Effectiveness of Using YAC Alu PCR Products as Probes for the Isolation of Cosmid Subsets 4.3.1 Initial Screening The initial screen to isolate cosmid subsets was performed by hybridizing Alu PCR products from each of the six minimum tiling path Y A C s onto gridded high density filters containing the chromosome 8 cosmid library. The number of Alu PCR products derived from a given Y A C was maximized by using primers which directed amplification from the left and right ends of the Alu consensus sequence. This allowed for the amplification of sequences irrespective of the orientation of the flanking Alu repeats. The use of a single primer would limit the amplification to those sequences which were flanked by Alu repeats that were oriented in opposite directions. However, there may be a tendency for Alu repeats to lie in the same orientation (Nelson et al., 1989; Cole et al., 1991), which would necessitate using both primers simultaneously. Eight discrete Alu PCR products were produced from Y A C 787c 11 (Figure 8). Given that the size of this Y A C is 1740 kb, this corresponds to an average amplification of one inter-Alu fragment per 200 kb. Cole et al., (1991) reported generating one inter-Alu fragment per 75-100 kb, using primers both singly and in combination. Since Alu elements occur on average every 3-6 kb, and the largest inter-Alu PCR product is approximately 1 kb in size, it appears that only a small fraction of the sequences between Alu repeats are being amplified. This may be attributed in part to the fact that not all repeats will have exactly the same sequence as the Alu consensus sequence, from which the primers are derived. As a result, Chapter 4. Discussion 86 mismatches will not be amplified as efficiently as perfect matches. Secondly, PCR will preferentially amplify shorter molecules, as longer molecules may not be made in their entirety in cases where the reaction does not go to completion. However, it is also possible that the amplification of additional fragments may be occurring below a threshold level that permits their visualisation on an agarose gel. Additionally, the discrete bands seen in Figure 8 may represent complex mixtures of similarly sized products. PCR amplification of the somatic cell hybrid line MGV269, used as a positive control, resulted in a smear of DNA, presumably because individual products were obscured by the large number of products that were made. The negative control showed a small amount of contamination which was not observed in the products from Y A C 787c 11. It is questionable how representative the Alu PCR products are of their respective YACs . Undoubtedly, these probes are somewhat biased upon the spacing and distribution of Alu repeats. However, on average over 300 cosmids were isolated per Y A C . If the average Y A C size is 1068 kb (Table 6), the average cosmid size is 35 kb, and the cosmid library represents four genome equivalents, one would expect to identify only 122 cosmids per Y A C . The fact that an excess of cosmids is being picked up by these Alu PCR probes may suggest that the probes cover large regions of their respective YACs . The possibility that excessive numbers of cosmids are being picked up due to false positives is discussed in the following section. Considerable variation in signal intensity can be seen on the cosmid filters (Figure 9), making scoring of the data difficult and somewhat subjective. This initial screen does not Chapter 4. Discussion 87 represent a very specific test to isolate cosmids, and could be improved by double spotting cosmids, or by using duplicate screenings, where only cosmids positive on both accounts are scored. 4.3.2 Secondary Screening A more specific test was designed where Y A C Alu PCR products were hybridized onto Southern blots containing EcoRl digested cosmid DNA, rather than onto cosmid colony blots. For this purpose, three cosmids per Y A C were chosen from Experiment 3.3.1 which hybridized specifically to one Y A C . Preference was given to strongly positive cosmids, but many of the cosmids with very intense signals were found to be positive with the majority of Alu PCR probes screened against the library. These cosmids may represent a small subclass of the library which contain Alu elements oriented in such a way as to promote the hybridization of primer sequences derived from both ends of the Alu repeat. For example, two Alu elements, A and B, could be spaced together in a cosmid so that A hybridizes to one primer end, while the second primer end hybridizes to B due to its close proximity to A, in a bimolecular type of reaction. Perhaps the binding of the second primer to B has a synergistic and stabilizing effect on the binding of the first primer to A, resulting in a strong hybridization signal from any Alu PCR derived probe. Southern blots of EcoRl digested cosmid D N A were probed individually with the same Alu PCR products from each minimum tiling path Y A C used in the initial screening. Chapter 4. Discussion °° The purpose of using total human D N A as a probe was to identify EcoRl fragments which were positive for repetitive sequences. As can be seen in Figure 1 la, numerous fragments hybridized to this probe, which is not unexpected given the abundance of repetitive elements such as satellite DNAs, short interspersed nuclear elements (SINES), and long interspersed nuclear elements (LINES) in human D N A (Thompson et al., 1991). Cosmids 23D5 and 77A3, isolated with Y A C 967cl 1, were thought to represent overlapping cosmids based on similarities in restriction digestion patterns. This was further corroborated by the finding that many shared fragments displayed similar hybridization patterns (Figure 1 lb). With Y A C yRM2205, the potentially overlapping cosmids 76G9 and 151H12 also had many positive fragments in common (Figure 11c). This demonstrates that the chances of obtaining true positives are greater when two potentially overlapping cosmids are chosen, although this is not the case with Y A C 810f8, where only cosmid 197E7 hybridized to the probe and 125E7 did not (Figure 1 le). The background hybridization observed with each Y A C probe despite suppression of repetitive sequences by total human D N A maybe indicative of the inability of total human D N A to block out low to moderately repetitive sequences such as T H E (transposon-like human-element repeat) (Paulson et al., 1985), VNTRs (Wyman and White, 1980; Jeffreys et al., 1985; Nakamura et al., 1987), M E R sequences (medium-reiteration frequency sequences) (Jurka, 1990), and others (La Mantia et al., 1989; Moyzis et al., 1989; Kaplan and Duncan, 1989). A possible way of alleviating this problem would be to use cloned copies of specific repeats for suppression, rather than total human DNA. Chapter 4. Discussion 89 Starting from a vast library collection consisting of 20,000 clones, the number of cosmids identified for each Y A C was significantly reduced to an average of 300. However, only seven out of eighteen selected cosmids were found to be true positives upon further analysis. When experiment 3.3.2 was repeated with additional cosmids for Y A C s 693dl 1, 741h4 and 810f8, nine out of eighteen cosmids were found to be strongly positive. Due to the large amount of background hybridization observed in this experiment, it is important to include additional cosmids in the analysis, in order to gauge the strength of hybridization of the cosmids that are expected to be positive. These findings suggest a false positive rate of approximately 55%. Zuo et al., (1993) found a false positive rate of 35% for strongly hybridizing cosmids using Y A C probes isolated from pulse field gels. Thus, this technique may be more effective than using Y A C Alu PCR products as probes for identifying cosmid subsets. 4.3.3 Confirmation of the Identity of Selected Cosmids The importance of using additional methods of analysis to confirm the identity of the isolated cosmids has been well demonstrated. Therefore, cosmid to Y A C hybridizations were undertaken, where single EcoRl fragments which were preferably strongly Alu PCR positive and total human D N A negative were used as probes against Southern blots containing EcoRl digested Y A C DNA. Based on Table 8, it is clear that the strength of hybridization in Experiment 3.3.2 is indicative of whether a cosmid represents a true or false positive. The cosmid probes which Chapter 4. Discussion 90 gave clear evidence of being contained within their respective YACs (151H12E4.0, 136G1E2.9, 168A2E6.0 and 23D5E1.7) were all strongly positive with their corresponding Y A C Alu PCR probes, and either negative or weakly positive with the total human D N A probe, indicating that unique sequences were being isolated. Probe 197E7E5.0 was strongly Alu PCR positive as well as strongly total human D N A positive, but appeared negative on the Y A C blot. This suggests that the strong positive result was due to repetitive sequences, perhaps contained within the Alu PCR probe, which were subsequently suppressed in the less complex EcoRl probe. Probes that were only weakly positive with the Alu PCR probe in addition to being negative or weakly positive with the total human D N A probe gave negative results in all cases. Zuo et al., (1993) reported similar findings, where only 9.5% of weak cosmid signals identified on primary screening were subsequently positive on a secondary screening. Data presented in this thesis support the recommendation made by Zuo et a l , (1993) to exclude weak signal clones from further analysis. Probe 136G1E2.9, originally isolated by Y A C 787cl 1, was unexpectedly also positive with Y A C 741h4, confirming the overlap between the two YACs . It is conceivable that Y A C 741h4 did not pick up this cosmid in either experiments 3.3.1 or 3.3.2 because the shared EcoRl fragment is situated at the end of Y A C 741h4, and the miex-Alu PCR product cannot be made (Figure 14). Overall, the chances of having an EcoRl fragment located at the end of a Y A C , and of obtaining a false negative result in this manner are exceedingly small. Chapter 4. Discussion 4- 2.9 k b ^ YAC787cll EcoRl EcoRl Alu p r imer #2 Alu pr imer #1 no template Alu pr imer #1 available for primer#2 YAC 741h4 Figure 14: Possible Explanation For the Overlap Relationship Between YACs 741h4 and 787cll Chapter 4. Discussion 92 4.4 Conclusions: Initial results generated in this thesis contributed to the physical map construction of human chromosome 8p, from 8pter-8p23. Since this telomeric region is potentially gene dense, refinement of the physical map will assist in the future localization of genes, cDNAs and polymorphic markers that map to this interval. R H mapping corroborated previous linkage data for four out of ten STSs at odds exceeding 1,000:1 for order placement. It is an effective method for providing independent ordering information, but is most beneficial when combined with existing mapping techniques such as pulsed field gel electrophoresis. STS content mapping was useful in establishing possible Y A C overlaps, and aided in the identification of minimum tiling path YACs. As cosmids are invaluable reagents in high resolution mapping, positional cloning, and genomic sequencing strategies, a technique to isolate cosmid subsets from this region was employed. The use of Alu PCR products derived from the six minimum tiling path YACs as probes onto the arrayed cosmid library seems to represent at best a moderately useful screen. Subsequent analyses of 36 strongly positive and Y A C specific cosmids led to a false positive rate of approximately 55%. This is partly reflective of the difficulty encountered in scoring the data, a problem which may be partially resolved by double spotting cosmids or using duplicate filters, and by only scoring strongly positive clones. Results presented in this thesis suggest that effort should not be spent on weakly positive clones. As well, it is recommended that clones be isolated which are unique to a particular probe, or at least positive for a minimal number of probes. Chapter 4. Discussion 93 The best predictor of whether a given cosmid represents a true positive lies in the strength of hybridization of the Y A C Alu PCR and total human D N A probes to EcoRl digested cosmid D N A (Experiment 3.3.2). If an EcoRl fragment is strongly positive with the Y A C Alu PCR product, but negative or weakly positive with total human DNA, the cosmid most likely signifies a true positive, presumably because the EcoRl fragment consists of unique sequence that is complementary to the probe sequence. This is even more likely i f two cosmids appear to be overlapping based on similarities in restriction digestion fragments, and share similar hybridization patterns. Conversely, if an EcoRl fragment is strongly positive with both Alu PCR and total human D N A probes, it is most likely hybridizing to repetitive sequences, and thus represents a false positive result. Given this information, the subsequent corroborative experiment may be unnecessary. However, hybridizing cosmid EcoRl fragments back onto Y A C EcoRl Southern blots is a very specific test which definitively establishes whether the cosmid shares sequence homology to the corresponding Y A C . In this case, a false negative cosmid was fortuitously discovered which confirmed the overlap originally suggested by STS content mapping between the two Y A C s 787c 11 and741h4. Chapter 4. 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Appendix 1: R H Mapping Data 108 Appendix 2: Positive Cosmids Identified With Minimum Tiling Path YACs * indicates cosmids specific to YAC probe (at time of experiment) YAC: yRM2205 1D1 23A1 61E3 103F5 130F7 162A5 182C10 192A5 1D9 23B8 61F1 103G2 130F8 162F7 182C11 192B6 1F1 24A5 61F9 104C3* 133F2 162F9 182D10 192D5 1H1* 25A1 62G4 104C6 133F3 163C6 182F1 192E8 2F10* 26A1 63A9 104C7 134F10 164A11 183D3 193D3 2H1 27D7 63C1 104C8 135F4 164C4 183F3 193E8 3F9* 28A8 63F4 104F12 137C4 165A6 184B7 193F12 4F11* 28C2 63F8 104G2 137D10 166A6 184F8* 194D7 4G12* 28C9 64E8 106A4 141A3 166F7 184H7 195G4 5D9* 29A9 70F12* 106A5 141F6 166H7 185B7 195H7 5F11* 29B1 72C11 108H6 145A3 167A11* 185C4 196B5 5G4 30B3 73C5 109C12 145A8 167B12 185D3 197C6 6D11 30B4 73D12 112E11 145C9 167C6 185D10 198D11 6E11 31D2 73F12 113E8 145G3 167C10 185E9 199B1 6F8* 32A2 76G9* 116C10 147A5 167D11 185E10 199C11 6F12 32B1 80D11 116C12 147B3 167H5 185H7 199F12 6G12 33A4 80F7 116E12 148A2 168C8 186B8 201A6 7E4 33B1 80F12 116G12 148A11 169B6 186F12 201C12 9G3 36H6 83B11 117C1 148D8 169G7 187D7 201G4 12D9* 37B1 83F6 121E6* 149G1 170A11* 187E9 202A11 12G1 38E3 86F7 121F6 150A4* 171C12 187F1 202B5 13F1 40H6 89B12 123D7 150A5* 173A11 188A3 202C3 16C12* 43A5 89F6 124C12 150C9* 175D11 188F3 204H6 16E11* 50B12* 90E1 124D12 151B7* 176B2 189H5 206B6 16F6* 51C1 91C9 124G12 15 IB 11 * 176H7 189H7 206G7 17A7 51H6 92E2 125A5 151H12* 177B9 190A8 208H6 18B3 52D1 93G9 125E10 152A5* 178B3 190B3 18C1 52D4 95C9 125F6 152B9* 178C9 190B5 18D10 53A8 96C3 125G4 153H1 178F3 190B11 19A2 54F8 96C9 127F5* 154B3 178F12 190C5 19A5 55F1* 99E11 128F5* 154C9 179A10 190D1 19B1 56A7 99F11 129B2 155A3 179B6 190D3 20A9 56C1 100G2 129B8 155C12 179D11 190E3 20B2 56D1 100H3 129E7 156D3 180A8 190F12 22A1 57B1 101F10 129F10 157A3 180B3 190G4 22A5 58A4 101H12 130D9 157A4 180B6 191B7 22B5 59B12* 102H11 130D10 157A9 180D3 191C7 22B9* 59F1 103C12 130E2 157A10 181C9 191C9 22C1 59F12 103D8 130E4 158A5 181F12* 191D12 22C5 60C8 103D12 130E9 159B12 182A3 191F11* 22C6 61D3 103E3 130E10 160A3 182C3 191H10 109 YAC: 693dll 2A11 40H11 61B2 82C3 107E12 125A5 144E2 167C10 3C2 42B8* 61D1 82C5 108E3* 125B4 145A8 168C8 4C3 46B9 61D6 82F7* 108E11 125G9* 145C9 168E8 4 H 6 46F4 61D10 84D5 109A1 127H12* 145C12* 169B6 6D4* 47F9 61F1 85A5 109C12 128A9 145E11 169C2 7 A 9 49B1 61F3 86A8 109E4 128A11 145G3 169D9 7D3* 4 9 E 1 2 61G3 86D5 110D2 129A9 146G12 169D10 7E8 49F1 62G2 86F2 110D6 129B2 148A11 170B12 7G12 5 0 A 1 2 6 2 H 2 86F7 110H12* 129D6 148C11 171B6 8A5 50G1 63A2 87D12 111E12 129F10 148E5 172C6 9B3 51C1 64C9 87F11 111G2 130D10 148H11 172D9 10G1 52D1 6 4 E 6 88B1 112C4 130E9 149C5 172H9 11A9 52G12* 64H5 88C3 112E11 130F7 149G1 176C9 11H12 53B8 65D10* 88E12 113C3 131H4 150A10 176H7 12H3 53C1 65F10* 89A3 113C9 132F5 151G12 177B5 18B7 5 3 D 1 * 65G3 89A4 114B3 134F7 153C5 178H9* 18C1 53D6 67H5* 89B2 114H3 134F9 153H1 178H10 18E11 5 4 A 9 69D6 90A2 115A5 134H5 154D12 179A4 19A5 54A10* 69D10* 90B1 115A10 134H12* 156B12 179B2 20D5 54A11 6 9 H 7 90D8 115F9 135D4 156G3 179E3 21A5 54B4 70E11 90E12 116F8 135D6 156G11 180D11 21B5 54C12 70G4 91C4 116G12 135D9 157A4 180H10 21C9 54D10 72B2 92D6 117C1 135F4 157A9 181F12 22C1 5 4 E 6 73B2* 92E11 117C8 136D1 157D11 181H10 2 2 E 6 56B6 73B7 92G1 117C10 137C8 157H9 182A1 22H3 56C1 73C5 93A8 117G8 137E12 158A5 182B5 23F10* 56D10 73F2* 93B3 118A2 137F12 159E5 182C10 23G12 56D12 73H5 93C6 118C11 139F7 159H3 182H8 2 5 E 8 56G1 76B2* 9 3 D 9 119A11 139G8 160A3 183A4 2 7 A 1 * 56G12 76D6 93E12 119A12 140E1 160H9 184D2 28C9 57B1 77C5* 94B10 119H3 140E11 161D4 185D6 28F11 57B4 78D10 100A3 119H4* 141E10 161E7 185D10 30B3 5 7 D 1 * 79B1* 100G2 121A9 141F12 162A10* 185G11 30B4 57D11 79G5 100H3 121A12 142C7 162C1 186A11 31B4 57H1 79H6 101D9 121G12 142C10 162D2 186D1* 32B1 58A4 80F2* 101F10 123A5 142E9 163E7 189D4 33F1 58B5 80F4 103E3 123B3* 142E12 164E11* 189D5 33F6 58D12 80H10* 103F6 123G12 142F9 165A10 189H7 34C9 5 8 E 6 81A8 103G2 124B2 143B6 165D1 189H8 36H6 59H1 81B10 104C8 124C9 143D6 165H1 190A11 37H3 59H3 81E2 104G2 124F9 143G12 166A4 190B5 37H4 59H4 81E8 105E6 124G12 144D8 166H7 190C12 39H9 60C8 81F12 106H3 124H4 144D9* 167B12 190D10 191C7 191H10 192D5 192D12 193D3 193E8 193F12 194D7 194H8 195A11 196A10 196B10 196D10 196F11 197C5 197E6 197E10 197H7 198E8 199D12 199G5 199H11 200D3 200E5 200G3 201C1 201C9 201C12 201E10 201F2 210G2* 202A11 202B5 202D12 202H8 203D10 203E10 203G12 205H7 206B6 207C11 207C12 207D10 208C8 208E1 YAC: 2A11 2E6 3C2 3E8 4H6 5D12 11C5 15C12 15D5 17C11 18B7 18C1 18E11 19A5 19B1 19C5 20C1 21A5 21C9 22C1 22H3 23B8 23C5 24B7 24C12 26F3 28A11 28C9 28F1 28F6 29A6 29C7 29C11 30B3 30B4 31A10 31B4 31D11 31H3 32A2 32B1 33F1 34A6 741h4 34C9 82G12 40H11 84G8 41A6 86D2 42B8* 86F2 45A12 86F7 46B9 86F9 47F9 86G11 50A12 87F11 52D1 88D3 52H10 88E12 53C1 88H6* 56A7 89B4 56A11 89D4 56C1 89F6 56C2 89F12 57A1 90E1 57D11 90F3 58A4 91F9 61B2 91H10 61E3 92F1 61F3 92G11* 61H1* 93E1 63C1 93F8 65D1 93H7 67A5* 95D4 68A3 96D3 68A9* 96G11* 70A8* 99G1 70B11 101F1 71C11* 101F10 72C11 106H3 72E10* 110D2 73C5 111F1 73D12 111G2 74C6* 112F8 75D12 129A9 79A9 129B2 81D3 132C9 81E2 140E1 81F1 143A12 81F12 144A4 81G12 145A8 82D1 145E11 145G3 201G2* 148A11 202H8 148D8 203E3 148E4 206B6 148E5 208E1 149C5 149G1 150A10 152H7 153C5 153F8* 153H1 156D3 157A10 157H9 158A5 158C3 158C4 159E5 160H9 179C6 181G8 181H10 182C10 183H12 185E10 186A11 186D1* 190A8 191C7 191D12 191H10 193D3 193E8 194D7 195H7 196B10 196C7 197E10 200G3 201A6 201B7 201C9 112 YAC: 787cll 2B8 40B7 76A8* 103G3 2D4 40B8 76H3* 104A4 2E6 40E7* 77E6 104B4 4B7 40H6 81D8 104C7 5B2* 40H8 81D10 104C8 8A5 40H11 81E6 104G2 11C1 41A6 82C5 105G3 11H12 41A8 82C7 106A2 12H11 41A9* 82C8 106A4 13C1 41 A l 1 82D1 106B3* 17H9 42A9 82H7 106H3 18B7 42D3 83F6 107A5 18C10* 43B8 85C8 109A1 18D10 43D3 85C9 109E4 18E11 43E2 86E7 111G2 19E12 43E4 86F7 113A7 22G4* 43H8 88C9 113B5 23C12 44A12 88E7 113C3 24C2* 44C9 89F6 116C10 24C12 44C10 90D8 116D8 28A8 45A12 91C4* 117A7 28C9 45H5 92B5 117C10 28F11 46B7 95C9 117G3* 29E6 46F4 95F6 118C11 31A10 47E4 96B8 119E3 33A4 48E2 96C9 121G5 34A5 52D4 96F6 122C12 36C10 53C3 99F1 123D4 36H6 54D10 99F11 125A5 37A6 54H6 100A3 126C3 37A9 56C1 100B3 126H9 37E9* 57B1 100G2 129A9 37H4 59F12 101A5* 130E7* 38A9 61B2 101D2 130E10 38B7 61D3 101D11 131A5* 38D3 62G4 101F1 131H3 38E2 63G9 101F10 131H4 38E3 64H5 101H12 132D2 38H3* 67B7* 102F3 132H3 39C5 67E4* 102H11 134A5 39D3 71B8* 103D8 134H3 39E4 75B8* 103F5 134H5 40A8 75C2* 103G2 136G1* 137A9 154D12 165A6 198B1 137C4 154F12 165A10 198D11 137C6* 154G11 166A6 199D11 137F6* 155B10* 166B10 199G5 138B11 155B12 166H7 199H11 138B12 155C6 167B12 200G3 139F7 155C12 167C10 201A6 139F8 155D2 167F11* 201B1 142B9 155G11 167H5 201C1 142C7 155H12* 172F11 202A11 143A12 156B12 172H9 204G1 145B2 156D3 173B2 205A2 145C9 156F12 173F12* 206B6 145D2 156G3 174B2 206H3 145G3 156G11 175H5 207H5 146A9 156H12* 176C9 208H6 146G11 157A2 179D11 146G12 157A4 180B5* 147A5 157A10 180D11 147B4 157B10 181C12 147B11* 157B11 181G8 147F11* 157C5 181H10 147H12* 157C6 182C3 148A6* 157H9 182C10 148A12 158A1 182C12 148C6 158A5 185A1* 148D8 158C3 185D10 148H11 158C4 185G10 148H12* 158C12 187F1 149C5 158G2 187F6 150B11 159B12 189D4 151F11 * 159C10 189H7 . 152A6 159E5 189H8 152H7 159G11 190C12 153B5 159G12 191E6 153B11* 160A3 191F10* 153C5 160D10 193D3 153C11 160G12* 195A11 153G3 160H9 196A10 154A6 162A5 196D10 154A8 162C1 196F6 154A9* 162C10 197C6 154C6 162F11 198A10 YAC: 810f8 2E6 72C11 93E1 118A2 147B3 170H1 194C10 4H6 78D10 93F8 120A2 148D7 170H8 194G11 6H7 79A4 94D5 120A7 148D8 173A12 195D2 7D2 79D2* 94F3 120C4* 149B1 173B2 195H12* 7E8 79H3 94G5 121 A3 149C5 174B5 196D1 7H9 80H1 96D5* 121A12 149G1 174D6 196D10 7H11* 81D3 96H9 121B2 150F11 176B1* 196G12 10H9 81D12 98E9 122E9 152B4* 176H7 197E7* 12H11 81E2 98E11 123B2 153B5 177B9 197E10 13B2 81E8 98F12 123D4 153C5 178B3 197F2 13H9 81F1 98H11 123G8* 153G4 178B8 199C11 16H1* 81H8 99F1 124B2 154E6 179G1 199G11* 16H2* 82A2 101D11 125A5 155D6 180B3 199H11 18B7 82A6 101F11 125E5* 156D6 180B8 200E2 18C1 82A10 101G9 126C3 157A4 180D1 200G3 22C1 82D1 103C12 127A2 157C10 180E1 200G12 22C5 82G4 103D8 128C1 157D6 181A12 201B1 22C6 82H7 103D10 129E7 157F6 181B8* 201B7 22E6 83A9 103F5 129F10 157H9 181B9 201C1 22H3 84H8 103H9 130G1 158A1 181B11 201C9 26B1 85D10 104F10 130H1 158E7 181D1 201C12 29E6 86D2 104H5 131D11 159A1 182F1 201F2 29H2 86D5 105F11 131D12 159C10 183A4 202H12 30G2* 86E7 106G5 133D10 160A3 184D1* 203E3 30H6 86F7 106H3 133F2 160E7* 184D2 203G9 31H3 86H8 108E11 134F10 160G3* 184E2 203G12 31H6 87D9 109C12 136F10* 160H3* 185B7 204E1 36G5 87H9 110C10 137D10 161A3* 186B8 205G11 41G6 88A2 110D2 137D12 162C1 187F1 206B6 52B4 88C2* 110D8 137F12 163B1 189G2 208E1 52D1 88D3 110D9 138C8 164A11 190B11 208G11 52D4 88H7 110D10 141 A l 1 164A12 190C1 53B8 89D4 110G5 141E1 165D1 190C12 56C2 89D5 111E12 141G1 165E7 190D1 58B5 89H10 112E11 141H12 165G8 191B7 58E6 90E9 113C3 142D5 165H1 191D2 59A12 90F3 114B2 142E1 165H4 192D2 61D3 91H10 114B3 142E12 166F3 192F4 61F3 92D9* 114C7 144F2 166H8 192F6* 62A2 92G1 115A5 145H12* 168A2* 193C11 63A10 92H8 115B2 146A7 169A4 193D3 69C9* 93B8* 116B1 147A5 169B1* 193H5 69D11 93D9 116D3 147B1 170G8 193H6 114 YAC: 967cll 1F6 23D5* 61D4 77F7 100H3 113H1* 141B12 157F6 1F8 23E1 61F1 78G3 101F1 119E3 141C8 157H9 1F9 25A4 61F3 79A4 101F4 119G2 141D11 158B2 1G2 25A12 61H12* 79H6 101H12 119G10 141E8 158B5 2B8 25C1 62D4 80D3 102B10 120A7 141F12 158D9 2D4 25E2 62E2 80D5 102F3 120E6 142E12 159B12 2D6 25G7 62E3 80F4 102H11 121G5 143A12 159D1 3B10 26A4 62F7 80F7 103B2 121H1* 144E2 159E5 3F4 26C1 62G4 81D10 103D12 122G2 144H3 159F2 4B8 26F3 63C1 82C8 103F5 122G5 146E2 159F3 4C12 26G3 63C7 82H7 103H9 123A5 147E8 159G1 4E3 27A4 63C9 84B3 104A4 123E9 148A11 159H3 6C10 28F1 63D4* 84H8 104B4 123G12 148A12 160A3 6E8 29C1 63E3* 85A5 104C6 123H9 148B2 160D8 6G1 30B4 63F4 85C8 104C8 124G5 148D8 160H9 7A5 30E8 63G5 85G11* 105D12* 124H3 148E5 161D4 7D2 31A12 63G9 85H4* 105H8 124H4 149B2 161F1 7D4 31B4 64C9 86G11 106B2 124H5 149E8 161H12 7E8 31D2 64F2 88G3* 106B8 125A5 149F3 162A5 8A5 32D2 64H5 89B4 106B11 126D11 150B11 162B7 8A8 32D5 64H8 90D8 106H2 126G9 151D1* 162C1 8D2 33H6 65D1 90F3 108A4 126G10* 151F1* 162D11 8E4 36H6 66H3* 91A5 108E4* 127B1* 151F2 162F10 8F3 39H1 67F8 91H1 109A1 128A7 151G1* 162G11 9E9 40H6 67G11 92B5 109C12 128H3 152E5 162G12 10G1 40H11 68A3* 92G1 109E4 129F10 152E6 163B1 11A5 46E11 69H5* 92G9 110B10 129H2 152H7 164A11 11C1 46F4 70B11 92H10* 110D2 130D12 152H9 164A12 14D12 47D12 71G11 94F3 110D6 130H1 153B5 165A6 15C11 50F4 72C11 94G1 110D7 131D11 153C5 165D1 17B4 50G4 72D3* 95G8 110D8 131D12 153H1 165F1 17C4* 50H6* 73A5 95D8 111B7 132C5 154E5 165G8 17E2 52H10 73B7 95G11 111D12 132C11 154E6 165H4 17H9 53H7* 73G11 96A5 111E12 133H2 154E8 165H6* 18G2 58D12 73H5 96B2 112A2 134F9 154F12 166A4 19A2 58E6 74A5 96B8 112A3 134F10 155B3 166A6 20C1 58G9 74F4* 96C3 112B11 137A9 155B4 166B10 21H9 59C7* 75B11 97B5 112C3* 137C8 155B5 166G7 22C1 60C8 75G11 98B5 112E4* 137F12 155H9 166H7 22H4 60F2* 76D4 98E4 112F8 138B12 156F4 167C10 22H8 60G5* 76D6 98H11 112H12 139F8 156F12 167D3 22H9 60G11 76G4* 99F1 113A7 139H12 157B10 167D4 23A5* 61C8 77A3* 100A3 113G6 140E1 157D11 167D11 168E12 185D10 201A12 169A4 185G4 201B1 169G7 186F12 201B7 170G8 187D7 201C9 170H1 187D8 201C12 171F10 187F1 202A11 172E2 188A3 202A12* 172E11 189D1 202B5 172F8 189G2 202E2 172H1 189G3 202F7 173A3 190B5 202G11 173A12 190B11 202H7 173H6* 190C1 203B4 175G8 190C12 203B6 176A9 190D1 203B12 176C10 190G4 203E3 176H2 191A11 203E10 176H7 191B7 203G9 177B5 191C1* 204D7* 177B6 191D2 205B3 177B9 191D8 205E8 177C10 191D12 205G11 178A5 192A5 206B3 178B3 192D11 206B4* 178F12 193C11 206B6 179B6 193C12 206C7* 179D9* 193E8 206E3* 179D11 193G11 206E9 180A8 194B12 206G7 180D3 194C10 206H2* 180D11 194C12 207B5* 181B9 194E9 207B12* 181H10 195A11 207C7 182A3 195G11 207D7 182A11 195H7 208B5 182C10 197E9 208B9 182D10 197E10 208C8 182H3 197F7 208C11 183A1 198D11 208D8 183A12* 198E8 208E1 183D3 199E8 208F1 183D8 199F2 208G11 183F12* 199H11 185B9 200D11 185D3 201A6 116 Appendix 3: Published Work Done Prior to the Completion of this Thesis Additional laboratory work was performed during the course of this thesis which resulted in the publication of the following article [Cytogenet. Cell Genet. 73:331-333 (1996)]. My contribution to this paper involved the isolation and linkage mapping of a simple tandem repeat polymorphism gene marker for the heregulin (HGL) locus on human chromosome 8p. 117 Cytogenet Cell Genet 73:331-333 (1996) Cytogenetics and Cell Genetics Analysis of CA repeat polymorphisms places three human gene loci on the 8p linkage map R. Bruskiewich, 1 T. Everson, 1 L. M a , 1 L. Chan, 1 M. Schertzer, 1 J.-P. Giacobino, 2 P. Muzzin, 2 and S. W o o d 1 1 Department of Medical Genetics, University of British Columbia, Vancouver, BC (Canada), and 2Departement de Biochimie medicale, Centre Medical Universitaire, Geneva (Switzerland) Abstract. The gene loci for luteinizing hormone-releasing hormone ( L H R H ) , the beta-3 adrenergic receptor ( A D R B 3 ) , and heregulin ( H G L ) have been assigned to the short arm of human chromosome 8, but the positions of these loci on the human genetic linkage map have not been previously reported. We have isolated simple tandem repeat polymorphisms (STRPs) for these loci. These S T R P s enabled us to determine the genetic map locations for these genes. Luteiniz ing hormone-releasing hormone ( L H R H ) is a key neuroendocrine molecule in the hypothalamic-pituitary-gonad-al hormonal system controll ing human reproduction. Impaired function of this hormone may underlie such reproductive phe-notypes as hypogonadism and precocious puberty (Cattanach et a l , 1977; Mason et a l , 1986). L H R H may also inhibit tumor-cell proliferation (Harris et a l , 1991; Szende et a l , 1991; L imonta et a l , 1993; Irmer et a l , 1994, 1995). It is interesting to note that a tumor suppressor inactivated in certain reproduc-tive cancers is postulated to reside on chromosome 8p (Keran-gueven et a l , 1995). Yang-Feng et al. (1985) assigned the L H R H locus to 8p21—>pll.2 by in situ hybridization and somatic cell hybrid analysis using a cloned c D N A probe (See-burg and Adelman, 1984; Adelman et a l , 1986). Osh ima et al. (1994) have also recently placed L H R H on a radiation hybrid map. The beta-3 adrenergic receptor ( A D R B 3 ) is a member of a family of adrenergic receptors involved in the signal transduc-tion of the hormones epinephrine and norepinephrine. These Supported by the Canadian Genome Analysis and Technology Program of the Medi-cal Research Council of Canada (GO 12753). Received 26 October 1995;revision accepted 22 March 1996. Request reprints from Dr. Stephen Wood, Department of Medical Genetics, 6174 University Boulevard, Vancouver, British Columbia (Canada) V6T 1Z3; telephone: 604-822-6830; fax: 604-822-5348; , e-mail: adrenergic receptors are G-protein-coupled catecholamine re-ceptors with seven membrane-spanning domains (Emorine et a l , 1987). A D R B 3 is expressed in a variety of tissues, including adipocytes (Granneman et a l , 1993). Its high expression in murine adipose tissue suggests a possible involvement of A D R B 3 in obesity and diabetes (Muzz in et a l , 1991; Nahmias et a l , 1991). A D R B 3 is located within a chromosomal region that is consistently ampli f ied in human breast cancer (Dib et a l , 1995). H u m a n ADRJB3 has been shown to map to 8pl2—> 8p 11.1, and its murine homolog to mouse chromosome 8, by in situ hybridization (Nahmias et a l , 1991). Heregulin ( H G L ) , or neu differentiation factor (NDF) , is a ligand which interacts with the Neu/ErbB-2 receptor tyrosine kinase (Holmes et a l , 1992). It is a 44-kDa glycoprotein that is similar in amino acid sequence to epidermal growth factor (EGF) . Alternative splicing produces at least 10 isoforms, clas-sified into two groups, a and p, which differ in their E G F - l i k e domains (Peles and Yarden, 1993). The receptors for H G L and E G F are encoded by related protooncogenes that are associated with a variety of human malignancies (Yarden and Ul l r ich , 1988). Lee and Wood (1993) mapped H G L to 8 p 2 2 - > p l l using somatic cell hybrids. Orr-Urtreger et al. (1993) localized the H G L gene to 8p21 ->p l2 by in situ hybridization. Thomas et al. (1993) excluded H G L as a candidate for the Werner syn-drome locus ( W R N ) , posit ioning H G L on the linkage map rela-tive to W R N with a max imum lod of 5.32 at a recombination fraction of 0.017. KARGER E-mail © 1996 S. Karger A G , Basel U Fax + 4I 61 306 12 34 0301-0171/96/0734-0331$ 10.00/0 ttp://www. kargcr. ch 118 n.i D 8 S 1 3 6 D 8 S 5 D 8 S 1 3 7 D 8 S 8 7 F G F R 1 D 8 S 2 5 5 cM 4 . 7 8p Sex-Average Map HGL LHRH ADRB3 Fig. 1. Chromosome 8p linkage map with three new simple tandem repeat polymorphisms (STRPs). The thin horizontal lines connect each of the three novel gene-associated STRP markers with the corresponding reference map markers exhibiting no recombination with the gene marker; the bold vertical lines indicate the 1:1,000 likelihood intervals containing the novel gene markers. TCCAGTGGTGCC-3'; the final concentration of MgCl 2, 2.5 mM; and the T m , 58 °C. For HGL, the CA strand primer was 5'-CATTGATTATGGAA-TGCC-3'; the GT strand primer, 5'-GTTGAAAAAAATTGTGTTCA-3'; the final concentration of MgCl2, 2.5 mM; and the T m , 46°C. STRP genotyping Amplification reactions of 40 cycles consisted of 1 min denaturation at 95 °C, 30 s annealing at the specified T m , and extension for up to 2 min at 72 ° C. A 40-ng sample of genomic DNA was used with 10 pmol of each prim-er in a 25-ul reaction mixture. The reaction buffer contained 50 mM Tris-Cl (pH 8.3), 0.02% NP 40, 0.02% Tween, and the MgCl2concentration indi-cated above. Each dNTP was present in a concentration of 200 mM. One selected primer in each system was 5' end-labeled with [y-32P]ATP, and 0.25 pmol (0.125 mCi) was added to each reaction with one unit of Taq poly-merase (BRL). PCR products were run out on 5 % Long Ranger modified polyacrylamide denaturing gels (AT Biochemicals) and detected by autoradi-ography on Kodak X-OMAT RP film. To determine STRP allele sizes, a radiolabeled M13mpl8 plasmid sequence ladder was run on the gel along-side the genotyping reactions. Linkage analysis The three STRP loci were positioned on the linkage map by two-point and multipoint linkage analysis using the CRIMAP linkage program and genotypes in Version 7.1 of the CEPH database. In this paper, we report the identification of simple tandem repeat polymorphism (STRP) gene markers for L H R H , H G L , and ADRB3 and the results of genotyping eight C E P H families to place these loci on the 8p linkage map. Materials and methods Families Eight CEPH reference families (102, 884, 1331, 1332, 1347,1362, 1413, and 1416) were genotyped for each of the three markers (LHRH, HGL, and ADRB3). Heterozygosities were estimated from parents and grandparents of the pedigrees. STRP locus identification Derived or published primer sequences with minor modifications were used as gene-specific sequence tagged site (STS) reagents for identifying cos-mids containing the target loci from a genomic library, LA08NC01, of flow-sorted chromosome 8 DNA (Wood et al., 1992). The STS primers were as follows: LHRH: 5'-CCTTGTCTGGATCTAATTTGATTG-3' and 5'-TCA-CCTGGAGCATCTAGGGTACA-3 ' (exon #2 primers, Nakayama et al., 1990); HGL: 5'-CCTTTTCAGGATGTGGTCATTG-3' and 5'-CTGTCT-GCCTGAATAGGAGC-3' (primers oher21.11.5 and oher21.11.1; Lee and Wood, 1992); ADRB3: 5'-AGCACGTTGGCCAGAAAGAAG-3' and 5'-TCCTCCGTCTCCTTCTACCTT-3' (derived from a sequence reported by Emorine et al., 1989). Cosmids were screened for (dC-dA)n simple tandem repeat sequences (STRs) using poly-GT oligonucleotide hybridization. Posi-tive restriction fragments were subcloned into a Bluescript IIKS vector (Stra-tagene) by either "shotgun" subcloning or band isolation from agarose gels onto DEAE membranes. Dideoxynucleotide sequencing reactions on double-stranded templates were carried out using vector primers and Sequenase (US Biochemical) to identify the STR sequences. Primers for STR detection by PCR amplification were designed from unique flanking sequences. The STR primers and specific PCR amplification conditions were as fol-lows. For LHRH, the CA strand primer used was 5'-GACTTATCCTC-CTTGTTTCCC-3'; the GT strand primer, 5'-ATAAAGGACAGTCATTC-TGGAG-3'; the final concentration of MgCl2, 2.0 mM; and the annealing temperature (Tm), 58 °C. For ADRB3, the CA strand primer was 5'-GCA-ATGCTTTGTGCCTGTGC-3' ; the GT strand primer, 5'-ATGCTATAA-Results LHRH Cosmids 37F12 and 145G1 were identified as containing a portion of the L H R H gene. A polymorphic STR with a struc-ture of (CA)23TTA3(CT)n was subcloned and characterized from cosmid 145G1. The cloned allele yielded a 237-nucleotide product upon amplification. All eight C E P H families were informative, giving 10 additional alleles ranging in size from 219 to 243 nucleotides and one smaller allele of 187 nucleo-tides. Heterozygosity for this system was 0.86. Pairwise lod scores for L H R H against the reference set of 8p markers were calculated, with no recombination observed between L H R H and D8S5, with a Z r a a x of 9.33. Multipoint linkage analysis placed L H R H with equal likelihood in either of the genetic intervals flanking D8S5, bounded proximally by D8S137 and distallybyD8S136 (Fig. 1). ADRB3 Cosmid 92G1 was found to contain both the ADRB3 gene and a C A dinucleotide STR. This STR, whose structure is (AC)nGC(AC)9, resides in the 3' untranslated region of the gene. Labeling the C A strand primer in the specified PCR sys-tem yields a cloned STR product 96 nucleotides in length. Three C E P H pedigrees were informative, giving a total of 48 genotyped individuals available for linkage analysis. Two al-leles were noted, 96 and 94 nucleotides in size, with estimated frequencies of 0.814 and 0.186, respectively. The calculated heterozygosity for this system is 0.303. Pairwise lod scores for ADRB3 against the reference set of 8p markers were calculated, with zero recombination observed with D8S87 ( Z m a x = 3.61) and FGFR1 ( Z m a x = 8.43). Multipoint analysis placed ADRB3 in the genetic interval bounded proximally by D8S255 and dis-tallybyD8S137(Fig. 1). 332 Cytogenet Cell Genet 73:331-333 (1996) 119 HGL P C R screening identified cosmids 45A2, 67D8, and 91C2 containing the published heregulin STS. Cosmid 91C2 contained a polymorphic S T R with a structure of ( G T ) 1 2 T C ( G T ) 5 N 9 (TG) 4 . Labeling the C A strand primer gave a 118-nucleotide cloned S T R amplif ication product. Six of eight C E P H pedigrees were informative for the S T R P . A sec-ond allele, 104 nucleotides in length, was also observed. The frequencies of the first and second alleles were 0.24 and 0.76, respectively. The heterozygote frequency was 0.365. Pairwise lod scores for H G L against the reference set of 8p markers were calculated, with zero recombination observed between H G L and D8S87 ( Z m a x = 9.03), whereas 6% recombination was observed between H G L and F G F R 1 ( Z m a x = 9.77). Mul t ipoint linkage analysis placed H G L in the genetic interval bounded proximally by F G F R 1 and distally by D8S137 (Fig. 1). Discussion We have placed three genes on the chromosome 8p linkage map by analyzing S T R P s in C E P H families. The order of H G L and A D R B 3 is uncertain, since the 1,000:1 l ikel ihood intervals for these loci overlap. The order c e n - A D R B 3 - H G L - t e l is more likely by a factor of 2.3:1. Whi le no recombination was observed between D8S87 and either H G L or A D R B 3 , the more proximal locus F G F R 1 recombines with H G L but not A D R B 3 . The proximity of F G F R 1 and A D R B 3 is supported by physical map data placing them within 900 kb on a single Y A C (Dib et a l , 1995). L H R H maps close to D8S5, with which it shows no recombinants, while its map interval places it distal to both H G L and A D R B 3 . Thus, the order of these three loci is cen -A D R B 3 - H G L - L H R H - t e l . Current linkage maps are largely based on anonymous D N A markers, whereas it is the genes that are the elements of biologi-cal interest and whose map locations are of primary interest. This study adds three genes to the 8p linkage map and provides a highly polymorphic marker for the L H R H locus. References Adelman JP, Mason AJ, Hayflick JS, Seeburg PH: Iso-lation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hor-mone and prolactin release-inhibiting factor in hu-man and rat. Proc natl Acad Sci, USA 83:179-183 (1986) . Cattanach BM, Iddon CA, Charlton HM, Chiappa SA, Fink G: Gonadotrophin-releasing hormone defi-ciency in a mutant mouse with hypogonadism. Nature 269:338-340 (1977). Dib A, Adelaide J, Chaffanet M, Imbert A, Lepaslier D, Jacquemier J, Gaudray P, Theillet C, Birnbaum D, Pebusque MJ: Characterization of the region of the short arm of chromsome 8 amplified in breast car-cinoma. Oncogene 10:995-1001 (1995). Emorine LJ, Marullo S, Briend-Sutien MM, Patey G, Taka K, Delavier-Klutchko C, Strosberg AD: Mo-lecular characterization of the human beta 3-adrenergic receptor. Science 245:1118-1121 (1987) . Granneman JG, Lahners KN, Chaudhry A: Character-ization of the human beta-3 adrenergic receptor gene. Molec Pharm 44:264-270 (1993). Harris N, Dutlow C, Eidne K, Dong KW, Roberts J, Millar R: Gonadotropin-releasing hormone gene expression in MDA-MB-231 and ZR-75-1 breast carcinoma cell lines. Cancer Res 51:2577-2581 (1991). Holmes WE, Sliwkowski, MX, Akita RW, Henzel WJ, Lee J, Park JW, Yansura D, Abadi N, Raab H, Lewis GD, Shepard HM, Kuang W-J, Wood WI, Goeddel DV, Vandlen RL: Identification of hereg-ulin, a specific activator of pl85erbB2. Science 256:1205-1210(1992). Inner G, Burger C, Muller R, Ortmann O, Peter U, Kakar SS, Neill JD, Schulz KD, Emons G: Expres-sion of the messenger RNAs for luteinizing hor-mone-releasing hormone (LHRH) and its receptor in human ovarian epithelial carcinoma. Cancer Res 55:817-822 (1995). -Irmer G, Burger C, Ortmann O, Schulz KD, Emons G: Expression of luteinizing hormone releasing hor-mone and its mRNA in human endometrial cancer cell lines. J clin Endocrin Metab 79:916-919 (1994) . Kerangueven F, Essioux L, Dib A, Noguchi T, Allione F, Geneix J, Longy M, Lidereau R, Eisinger F, Pebusque MJ, Jacquemier J, Bonaiti-Pellie C, So-bol H, Birnbaum D: Loss of heterozygosity and linkage analysis in breast carcinoma: indication for a putative third susceptibility gene on the short arm of chromosome 8. Oncogene 10:1023-1026 (1995) . Lee J, Wood WI: Assignment of heregulin (HGL) to human chromosome 8p22->pl 1 by PCR analysis of somatic cell hybrid DNA. Genomics 16:790-791 (1993). Limonta P, Dondi D, Moretti RM, Fermo D, Garattini E, Motta M: Expression of luteinizing hormone-releasing hormone mRNA in the human prostatic cancer cell line LNCaP. J clin Endocrin Metab 76:797-800(1993). Mason AJ, Hayflick JS, Zoeller RT, Young WS, III, Phillips HS, Nikolics K, Seeburg PH: A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the "hpg" mouse. Science 234:1366-1371(1986). Muzzin P, Revelli J-P, Kuhne F, Gocayne JD, McCom-bie WR, Venter JC, Giacobino JP, Fraser CM: An adipose tissue-specific |3-adrenergic receptor: mo-lecular cloning and down-regulation in obesity. J biol Chem 266: 24053-24058 (1991). Nahmias C, Blin N, Elalouf J-M, Mattei MG, Strosberg AD, Emorine U : Molecular characterization of the mouse beta-3 adrenergic receptor: relationship with the atypical receptor of adipocytes. EMBO J 10:3721-3727(1991). Orr-Urtreger A, Trakhtenbrot L, Ben-Levy R, Wen D, Rechavi G, Lonai P, Yarden Y: Neural expression and chromosomal mapping of Neu differentiation factor to 8pl2->p21. Proc natl Acad Sci, USA 90:1867-1871 (1993). Oshima J, Yu C, Boehnke M, Weber J, Edelhoff S, Wagner M, Wells DE, Wood S, Disteche C, Martin G, Schellenberg G: Integrated mapping analysis of the Werner syndrome region of chromosome 8. Genomics 23:100-113 (1994). Peles E, Yarden Y: Neu and its ligands: from oncogene to neural factors. Bioessays 15:815-824 (1993). Seeburg PH, Adelman JP: Characterization of cDNA for precursor of human luteinizing hormone releas-ing hormone. Nature 311:666-668 (1984). Szende B, Srkalovic G, Timar J, Mulchahey JJ, Neill JD, Lapis K, Csikos A, Szepeshazi K, Schally AV: Localization of receptors for luteinizing hormone-releasing hormone in pancreatic and mammary cancer cells. Proc natl Acad Sci, USA 88:4153-4156(1991). Thomas W, Rubenstein M, Goto M, Drayna D: A genetic analysis of the Werner Syndrome region on chromosome 8p. Genomics 16:685-690 (1993). Wood S, Schertzer M, Drabkin H. Patterson D, Long-mire JL, Deaven LL: Characterization of a human chromosome 8 cosmid library constructed from flow-sorted chromosomes. Cytogenet Cell Genet 59:243-247(1992). Yang-Feng TL, Seeburg PH, Francke U: Human lutein-izing hormone-releasing hormone gene (LHRH) is located on short arm of chromosome 8 (region 8pll.2->p21). Somat Cell molec Genet 12:95-100(1986). . Yarden Y, Ullrich A: Growth factor receptor tyro-sine kinases. A Rev Biochem 57:443-478 (1988). Cytogenet Cell Genet 73:331-333 (1996) 333 


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