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MOLECULAR G EN ETICS of the January, HUMAN X MOLECULAR GENETICS OF THE HUMAN X CHROMOSOME BY DAVID ANDREW HARTLEY a thesis submitted for the degree of Doctor of Philosophy in the University of London January, 1984 Department of Biochemistry St. Mary's Hospital Medical School London W2 IPG 1 Abstract The comparison of frequency of recombination with chromosomal position has been possible only in Drosophila melanogaster but it is known that chiasma distribution in the human male (and many other species) is not random. A number of cloned DNA sequences have been isolated from a library enriched for X chromosome DNA by flow cytometry. The chromosomal loci complementary to these cloned sequences have been investigated by hybridization to somatic cell hybrid DNAs and by direct hybridization 1 in situ' to human metaphase spreads. In this way a cytological map of the X chromosome defined by DNA sequence markers has been constructed. Genetic distances complementary to the physical separations determined have been estimated by determining the segregation of DNA sequence variants at each locus in three generation p e d i g r e e s . The sequence variation is manifested as altered restriction fragment migration in agarose gels. It is intimated from these analyses that the pattern of recombination frequency along the human X chromosome mirrors the non-random chiasma distribution seen along male autosomes. This supports the chiasmatype theory and has implications on the availability of genetic components of the X chromosome to recombination. 2 To ray father in the hope of convincing him of life outside o f E.coli 3 ...scientists getting their kicks when deadly disease can do as it pleases results ain't hard to predict Gil Scott-Heron 4 Ack n ov; ledgements To my supervisor Bob Williamson for support. To my other supervisor Kay Davies for tolerance and PR. To Graham Casey, Marion Stone, Teresa Davies and Paul Barton for teaching me the true meaning of cytogenetics. To Peter Goodfellow for hybrid cells. To Paul North for microchip technology. To Helen Kingston and Dennis Drayna for families and Lod scores. To all the Biochemistry Department and in particular the 'X group' for tolerating my singing and my socks. To Julian and Hark for endless attempts at the meaning of life and for going for it. To Jo, Sue, Teresa, /Auntie Janet, Sissel, Pauline and Susie for demonstrating the superiority of two X chromosomes. To Jack, Ali, Rob, Graham, Tim, Conrad, Ola and Simon for coming a close s ec o n d . To my family and friends. To my skin and blister for never letting up in giving me a hard time (no more than I deserved). To Jane. 5 Abbreviations dATP adenosine deoxyribonucleotide triphosphate AP RT adenosine phosphoribosyl transferase b p ba se pair 3 kb p 10 base pairs BSA bovine serum albumin o C degrees Centigrade u C i ,m C i ,Ci micro, milli, Curie dCTP cvtidine deoxyribonucleotide triphosphate DNA deoxyribonucleic acid DNAase deoxyribonuclease dpm disintegrations per minute E. coli Escherichia coli EDTA ethylene diamine tetraacetic acid G6 PD H glucose-6-phosphate dehydrogenase pg / ng , ug , mg , g pico, nano, micro, milli, gram dGTP guanosine deoxyribonucleotide triphosphate hrs hours HAT hypoxanthine adenine thymidine HGPRT hypoxanthine-guanosine phosphoribosyl transferase u l ,m l ,1 micro, milli, litre Lod logarithm of the odds nm,um,mm,cm nano, micro, milli, centimetre 2 cm squared centimetre mins minutes u M ,m M ,M micro, milli, molar cM centiMorgan PPO 2,5-diphenyloxazole PEG polyethylene glycol P probability RFLP restriction fragment length polymorphism RFLV restriction fragment length variant rpm revolutions per minute RNA ribonucleic acid RNAase ribonuclease RPMI-1640 Roswell Park Memorial Institute-1640 m e d i u m SSC saline sodium citrate secs seconds SDS sodium dodecyl sulphate SD standard deviation Tris 2-amino-2(hydroxymethyl)-propane-1,3-diol dTTP thymidine deoxyribonucleotide triphosphate UV ultraviolet V volts v / v volume per volume of water w / V weight per volume of water 6 F i g u r e s ge Secondary screenings of X-enriched library recombinants with human, Horl9X and mouse DMAs. 60 Screenings of X-enriched library with Horl9X and MCP-6 DMAs. 62 EcoRI digests of recombinants DX1-12. 64 Recombinants DX1-12 hybridized to human and lambda DMAs. 66 EcoRI digests of human, Horl9X, IV7I-5, MCP-6 and mouse DMAs. 71 Somatic cell hybrid DMAs hybridized to DX24 and RD6 . 73 Somatic cell hybrid DMAs hybridized to S21 and DX4 . 75 Somatic cell hybrid DMAs hybridized to DX7 and DX6 . 76 4C11 hybridized to a metaphase spread. 82 Distribution of grains over chromsome 6s after 'in situ' hybridization to 4C11. 85 Somatic cell hybrid DMAs hybridized to 4C11. 86 Repetitive seguences hybridized 'in situ' to metaphase spreads. 83 RC8 hybridized to a metaphase spread. 90 Distribution of grains over all chromosomes after 'in situ' hybridization to RC8. 91 7 15. Distribution of grains over the X chromosomes after 'in situ' hybridization to RC8. 93 16. LI.28 hybridized to a metaphase spread. 95 17. Distribution of grains over all chromosomes after 'in situ' hybridization to LI.28. 96 18. 52A hybridized to a metaphase spread. 99 19. Distribution of grains over all chromosomes after hybridization to 52A. 100 20. S21 hybridized to a metaphase spread. 102 21. Distribution of grains over all chromosomes after hybridization to S21. 103 22. DP31 hybridized to a metaphase spread. 106 23. Distribution of grains over all chromosomes after hybridization to DP31. 107 24. DX13 hybridized to a metaphase spread. 109 25. Distribution of grains over all chromosomes after hybridization to DX13. 110 26. Distribution of grains over the X chromosome after hybridization to RC8, LI.28, DP31, S21, 52A and DX13. 113 27. Cvtological map of the X chromosome. 114 28. The TaqI polymorphism detected by RC8. 117 29. The TaqI polymorphism detected by S21. 119 30. The TaqI variants detected by LI.28 and DP31 in the pedigree D3. 123 31. Pedigree D3. 124 32. Pedigree D2. 126 8 33. The TaqI and Bglll variants detected by DP31 and DX13 in the pedigree BD26. 128 34. Pedigree BD26. 129 35. The physical and genetic map of the human X chromosome. 133 9 Tables Page 1. Recombinants picked from the X-enriched library - insert sizes and hybridization to total human DliA. 69 2. Hybridization of recombinants to somatic cell hybrids - chromosomal localization. 78 3. Statistical analysis of 4C11 'in situ' hybridization. 84 4. Statistical analysis of RC8 'in situ' hybridization. 92 5. Statistical analysis of LI.28 'in situ' hybridization. 97 6. Statistical analysis of 52A 'in situ' hybridization. 101 7. Statistical analysis of S21 'in situ' hybridization. 104 8. Statistical analysis of DP31 'in situ' hybridization. 108 9. Statistical analysis of DX13 'in situ' hybridization. Ill 10. Restriction fragment variants in eight mothers detected by RC8, LI .28, DP31,S21, 52A and DX13. 121 11. Lod scores at different values of recombination fraction between the loci detected by LI.28 and DP31 and between RC8 and Ll.28. 131 1 0 Contents Page Abstract 2 Dedication 3 Quotation 4 Acknowledgements 5 Abbreviations 6 List of Figures 7 List of Tables 10 1 . Introduction 14 1 .1 . The eukaryotic chromosome 15 1 .2. Meiosis and the reassortment of genes 20 1.3. Recombination between linked genes and the construction of linkage maps 22 1.4. Crossing over and chiasmata 25 1 .5 . Localized recombination 27 1 .6 . Cytological and genetic markers in man 30 1 .7 . Flow cytometry and molecular cloning of the human X chromosome 33 1.3 . Summary and aims 35 2. Materials and Methods 37 2.1. Materials 38 2.2. Bacterial and phage strains 41 11 2.3. General methods 41 2.3.1. Restriction enzyme digestion 41 2.3.2. Ethanol precipitation of nucleic acids 42 2.3.3. Agarose gel electrophoresis 43 2.3.4. Autoradiography 43 2.3.5. Gel filtration 43 2.3.6. Determination of radioactivity 44 2.4. Specific methods 44 2.4.1. Preparing high molecular weight DHA from tissue culture cells 44 2.4.2. Small scale preparation of bacteriophage DMA 45 2.4.3. Large scale preparation of bacteriophage DNA 46 2.4.4. Labelling DMA by nick translation 47 2.4.5. Screening lambda recombinants 43 2.4.6. Southern transfers 50 2.4.7. Competing repetitive sequences in Southern blot hybridizations 51 2.4.8. Preparation of metaphase spreads 51 2.4.9. G-banding of metaphase chromosomes 52 2.4.10. 'In situ' hybridization 53 2.4.11. Scoring hybridized spreads 55 2.4.12. Pedigree analysis 56 12 3. Results 57 3.1. Screening recombinant bacteriophage 'in situ' 53 3.2. Isolation and characterization of individual recombinant bacteriophage S3 3.3. Chromosomal localization I. Somatic cell hybrids S3 3.4. Chromosomal localization II. 'in situ' hybridization 79 A. Introduction 79 B. Cytological mapping of the X chromosome 29 C. Summary of 'in situ' hybridization data 112 3.5. Restriction fragment length polymorphisms 115 A. RFLP and heterozygote detection 115 B. Inheritance studies 122 3.6. Genetic distances - a genetic map of the X 130 4. Discussion 135 4.1. DBA sequence organization of the human X chromosome 136 4.2. The construction of chromosome maps in r1 amma 1s 133 4.3. Localized recombination 143 5 . Summary and perspectives 154 6 • References 15 3 hrrata 16 6 13 C H AP TE R 1 Introduction 14 1.1. The Eukaryotic chromosome The chromosome, first observed by Waldeyer in 1888, can be defined as a linkage structure consisting of a specific linear sequence of genetic information. The eukaryotic chromosome has two main biological functions.
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