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Molecular Genetic Analysis of the Adenomatous Polyposis Coli (APC)

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Molecular Genetic Analysis of the Adenomatous Polyposis Coli (APC) Molecular Genetic Analysis of the Adenomatous Polyposis Coli(APC) gene region by Garret Malcolm Hampton A thesis submitted in requirement for the degree of Doctor of Philosophy at the University of London May, 1992 Cancer Genetics Laboratory (formerly Director's Laboratory) Imperial Cancer Research Fund London and Department of Genetics and Biometry University College London London University Page -1- ProQuest Number: 10609182 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10609182 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 To my parents and my wife, Kate Page -2- Abstract Familial Adenomatous Polyposis (FAP) is a rare, autosomal dominant predisposition to colorectal cancer, affecting about one in ten thousand individuals in all populations studied. The gene responsible for this syndrome, designated APC (for Adenomatous Polyposis Coli) was mapped to 5q21-q22 by linkage analysis following a cytogenetic report of a male patient with polyposis and an interstitial deletion on 5q. The high incidence of allele loss at 5q21-q22 in carcinomas of sporadic patients suggests that mutation of the APC gene is a very frequent step in the tumorigenic pathway to nonfamilial colorectal carcinomas and emphasises the importance of isolating the gene and identifying its function. Attempts were made to identify this gene using a positional cloning strategy, on the basis of its genomic location rather than by a knowledge of its function. As a first step toward this goal, two approaches were taken to identify a large number of DNA probes mapping within the breakpoints of two, and later three independent deletions encompassing the APC gene. In the first approach, a novel method, termed 'alu-PCR’ was developed. By comparing the PCR patterns generated from normal and deleted chromosomes 5, a probe was identified mapping close to the APC gene. In the second approach, genomic libraries constructed from physically dissected DNA around chromosomal region 5q21-q22 were used to derive a large number of DNA probes. These probes facilitated the definition of a new minimally deleted region harbouring the gene and assisted with the construction of a physical map of this region. For the second phase of the cloning project, these DNA probes, in addition to others sub-localised to this region, were used to isolate a collection of yeast artificial chromosomes (YACs) which in total cover some 4-megabase-pairs (Mb) of DNA. Two of the YACs identified in this study, which cover a total distance of 1.1- Mb, were used indirectly to identify potential alterations, particularly small deletions, in homologs of chromosome 5 from FAP patients. One such deletion of 260-kb was identified and shown to be entirely overlapped by one of these YACs. This deletion, and the YAC used to identify it, contain the entire coding sequence of the APC gene. Page -3- Acknowledgements I am indebted to a great number of people who have helped, supported, listened and provided a thoroughly enjoyable three and a bit years at ICRF. Particularly, I am most grateful to Sir Walter Bodmer for taking me on board and for his support in writing fellowship applications, in addition to this thesis. To Dr. Sue Povey for her endless help and support and the sometimes unpleasant task of reading this thesis in its many forms. To Dr. David Markie, without whom, on many occasions I sincerely think I would still be in the dark. I thank him also for his incisive and honest comments on reading this thesis - may the Southern blot fairy soon visit. To Ms. Sally Cottrell for putting up with so much, and to all the many members of Walter's lab, both past and present, who have helped in so many different ways. I would also like to thank my many colleagues in the Solomon, Trowsdale, Frischauf, Sheer, Lehrach and Medical Oncology labs, for providing an excellent environment in which to work, in addition to providing some interesting Friday nights. Outside of ICRF, I owe a great dept to my parents, Marjorie and Archie Hampton, without whom, in all honesty, I know I would not be here now (post-conceptually, that is). I thank them for their continued and loving support. To my wife, Kate, who has suffered (on my behalf) a difficult and at times trying first year of marriage, but borne the scar with grace. Page -4- Contributions The experiments described in this thesis are entirely my own work unless otherwise stated. I am grateful for the following contributions: Dr. Wolfgang Balhaussen, Ms. Gabriele Leuteritz and Dr. Udo Trautmann for providing microdones and the photographs in figure 4.1. Ms. Tania Jones for the yeast in situ hybridisation experiments and for providing the photographs in figure 5.3. Ms. Sally Cottrell for many probe isolates, buffers and solutions that were always dependable. Mr. Tristan Ward for the physical mapping information depicted in figure A7.1, and for providing YAC-containing yeast strains. Ms. Kathy Howe for providing data and microclones. Dr. Huw Thomas for providing somatic cell hybrids. Drs. Mark Ross, Anthony Monaco and Hans Lehrach for providing the high density YAC filters. Dr. Anna-Maria Frischauf and Ms. Sian Searle for providing the high density cosmid filters. The ideas for the ’junction-trapping' experiments described in Chapter 7 of this thesis came equally from both myself and Dr. Ketan Patel. The experiments described in this chapter are, however, my own work. Page -5- Table of contents. Title. l Dedication. 2 Abstract. 3 Acknowledgements. 4 Contributions. 5 Table of contents. 6 List of figures. 13 List of tables. 14 Abbreviations. 15 1. Introduction. 18 Part I. Cancer genetics. 18 1.1. A genetic basis for cancer. 18 1.2. Congenital predisposition to cancer. 19 1.3. Germline and somatic mutations in cancer. 21 Part II. Colorectal cancer. 26 1.4. Common colorectal cancer. 26 1.5. Familial Adenomatous Polyposis (FAP). 27 1.6. Localisation of the APC gene. 31 1.7. Loss of chromosome 5 alleles and the relationship between inherited and sporadic colorectal cancer. 32 1.8. Other genetic alterations in the progression to colorectal neoplasia. 34 Part III. Cloning human genes. 37 1.9. Functional cloning. 38 1.10. Positional cloning. 40 1.10.1. Localisation of the gene: Linkage analysis. 40 1.10.2. Localisation of the gene: Chromosomal aberration. 41 1.10.3. Isolation of DNA probes. 43 Page -6- 1.10.4. Physical characterisation of defined chromosomal regions. 46 1.10.5. Yeast artificial chromosomes. 47 1.10.6. Identification of genes in cloned DNA. 50 1.11. Aims of this thesis. 52 2. Materials and Methods. 54 2.1. Materials. 54 2.1.1. Enzymes. 54 2.1.2. Chemicals. 54 2.1.3. Nucleic adds. 55 2.1.4. Solutions and buffers. 55 2.1.5. Media. 59 2.2. General DNA methods. 62 2.2.1. Preparation of plasmid DNA. 62 2.2.2. Extraction of genomic DNA. 63 2.2.3. Restriction endonuclease digestions. 64 2.2.4. Agarose gel electrophoresis. 65 2.2.5. Southern blotting. 65 2.2.6. Radiolabelling of DNA probes. 65 2.2.7. Hybridisation. 66 2.2.8. Competitive hybridisation. 66 2.2.9. Removal of radioactive probes. 67 2.2.10. Polymerase chain reaction (PCR). 67 2.3. Alu-PCR methods. 68 2.3.1. A lu-PCR. 68 2.3.2. Cloning of inter -alu PCR products. 68 2.3.3. Analysis of inter -alu product libraries. 69 2.4. Identification and isolation of cosmids. 70 2.4.1. Gridded cosmid library screening. 70 2.4.2. Isolation of unique cosmid sub-fragments. 70 2.5. Microclone methods. 70 Page - 1 - Yeast Artificial Chromosome (YAC) methods. 71 YAC library screening. 71 Secondary screening. 72 Yeast DNA preparations. 72 Restriction digestions of yeast DNA. 73 Pulsed field gel electrophoresis. 74 Derivation of YAC physical maps. 74 Comparative yeast and human Southern blot hybridisations. 75 Isolation of YAC insert termini. 75 A/w-vector PCR. 75 Vectorette PCR. 76 'Junction-trapping' of YAC insert termini. 77 Appendix 1. Oligonucleotide primers. 79 Appendix 2. Sources of DNA probes. 81 Appendix 3. Sources of human-hamster somatic cell hybrids. 82 Repetitive sequence polymerase chain reaction. 84 Part 1. Rapid generation of human chromosome- specific DNA probes from a somatic cell hybrid. 84 Introduction. 84 Results. 85 Alu-PCR from a somatic cell hybrid. 85 Cloning of inter -alu PCR products. 86 Analysis of the 'inter -alu' PCR libraries. 90 Hybridisation analysis of 'inter-a/w' PCR products. 90 Conclusions. 93 Part 2. Applications of the alu-FCR method: comparative analysis of somatic cell hybrids. 95 Introduction. 95 Page -8- 3.5. Results. 96 3.5.1. IRS-PCR of somatic cell hybrids: Primer IV. 96 3.5.2. IRS-PCR of somatic cell hybrids: Primers 517, 559 and LIHs. 96 3.5.3. Isolation and m apping of IRS-PCR 'difference' products. 99 3.5.4. Cloning of L5.4. 100 3.5.5. Isolation of a cosmid containing the L5.4 sequence. 100 3.6. Conclusions. 103 4. Mapping of microdissected genomic DNA probes in the APC gene-region.
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