Construction of an integrated map of the genomic locus lq21 harboring the Human Epidermal Differentiation Complex as a platform for the identification of all genes in this complex, the study of their expression, regulation, function and evolution by Andrew P. South Thesis Submitted For The Degree of Doctor of Philosophy University of London 1999 Centre For Applied Molecular Biology School of Pharmacy ProQuest Number: 10104217 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 complete 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 10104217 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The human Epidermal Differentiation Complex (EDC) on chromosome lq21 consists of three structurally different, yet functionally related, gene families. Members of all three gene families have been shown to play important roles in epidermal differentiation. This thesis initially describes the assembly of a completely contiguous set of overlapping bacterial clones covering 2.45Mb of human genomic DNA from the lq21 locus, encompassing the entire EDC. All known genes (28) and eight DNA markers within the EDC are precisely localized to EcoRI restriction enzyme fragments that constitute a partial EcoRI and full Notl and Sail restriction enzyme map. The bacterial clones presented in this thesis have been accepted as the substrate for long range genomic sequencing of this region for the chromosome 1 sequencing project at the Sanger Centre, UK. In addition to providing a template for large-scale sequencing, the bacterial clones presented here will serve as a molecular resource for the elucidation of all transcripts within the EDC as well as the study of transcriptional regulation, function and evolution of the EDC. As an evaluation of this resource as a tool for the identification of transcribed sequences, exon trapping has been performed from three PAC clones. The exon trapping experiments described in this thesis have identified 13 putative exons that are shown to derive from the EDC. Searches of the publicly available databases with the sequences of these 13 putative exons have identified a novel cDNA clone that is shown to localize to the EDC. Two of the thirteen putative exons identified are homologous, but not identical, to this novel cDNA. These data coupled with Northern blot and RT-PCR analysis, suggests that yet another novel family of transcribed sequences has been identified within the EDC. Thirteen gene members of the SI00 family of calcium binding proteins constitute one of the three so-far identified multi-gene families residing in the EDC. By using the contiguous bacterial clone map towards the study of EDC evolution, two findings have been made. Firstly, evidence of an ancestral break-point inversion during the evolution of mammals is supported by the elucidation of the transcriptional orientation of four SI00 genes and by the identification of extensive alternative splicing of the 5’ untranslated region of one of these SlOO genes. Secondly, a similar clustering of SlOO genes to that seen in human and mouse is described for the first non­ mammalian vertebrate species, Gallus gallus. Acknowledgements Many thanks are due to my supervisor, Dean Nizetic, for his time, support and patience during the writing of this thesis. Respect and thanks also due to the members of Dean’s laboratory, past and present - Jurgen Groet, Rachel Flomen, Pedro Baptista, and Jane Ives. Appreciation to the lq21 consortium, especially Ghazala Mirza and Jiannis Ragoussis for fluorescent in situ hybridizations, is given. With much love and respect I thank my wife to be, Clare, for huge support and encouragement during the past years. This work has been funded by the Constance Bequest Fund, The School of Pharmacy, and the BMH4-CT96-0319 grant from the Commission of European Communities to the European lq21 consortium. Table of Contents Title 1 Abstract 2 Acknowledgements 3 Table of Contents 4 Figure finder 15 Table finder 17 Glossary of Abréviations 18 Aims of This Thesis 21 Chapter 1: Introduction 22 1.1. Towards a complete nucleotide sequence of human chromosome 1 22 1.1.1. Human genome project strategy 22 1.1.2. Genetic mapping 23 1.1.3. Somatic cell hybrid lines and radiation hybrid mapping 23 1.1.4. Long-range physical restriction enzyme mapping 24 1.1.5. Cloning genomic DNA with vectors capable of propagation within a host organism 24 1.1.5.1. Yeast artificial chromosomes 24 1.1.5.2. Bacterial vector/host systems 25 1.1.5.2.1. Cosmids 26 1.1.5.2.2. PI artificial chromosomes and bacterial artificial chromosomes 27 1.1.6. Sequencing 28 1.1.6.1. Clone end-sequencing approach 28 1.1.6.2. Whole genome “shotgun” approach 29 1.1.6.3. Clone mapping approach 30 1.1.7. Current status of publicly available genome sequencing 31 1.1.7.1 Global sequencing project 31 1.1.7.2. Chromosome 1 sequencing project 31 1.1.8. Global mapping and local mapping: parallel endeavors 32 1.2. Epidermal Differentiation 33 1.2.1. Epidermal Differentiation as a model for the study of cell differentiation in general 33 1.2.2. Biological markers of epidermal differentiation 34 1.2.2.1. The keratins 34 1.2.2.2. Profil aggrin 36 1.2.2.3. The Comified Envelope (CE) - protein content 38 1.2.2.4. The Comified Envelope (CE) - lipid content 43 1.2.3. Regulation of epidermal differentiation 44 1.3. The Epidermal Differentiation Complex 46 1.3.1. Gene families constituting the EDC 46 1.3.1.1. Loricrin, involucrin and the SPRR (CE precursor) gene family 46 1.3.1.2. Profilaggrin, trichohyalin, and repetin (intermediate filament association) gene family 48 1.3.1.3. S 100 gene family 49 1.3.2. Gene complexes and multi-gene families 50 1.3.3. Locus control regions 51 1.3.4. Clustered multi-gene families generally reside in ‘gene-rich’ regions of the genome 53 1.4. Genetic disorders associated with human chromosomal region lq21 55 1.4.1. Disorders of the skin associated with lq21 55 1.4.2. Other disorders linked to lq21 56 1.4.3. Cancer and lq21 56 Chapter 2: Materials and Methods 58 2.1. Materials 58 2.1.1. Specialized materials 58 2.1.2. Chemicals 59 2.1.3. Enzymes 59 2.1.4. DNA oligonucleotide primers 59 2.1.5. Nucleotides 62 2.1.6. DNA cloning vectors used 62 2.1.7. DNA size markers 63 2.1.8. Culture media 63 2.1.9. Solutions 64 2.1.10. Sources of genomic DNA 66 2.1.11. Sources of RNA 66 2.1.12. Northern blot 66 2.1.13. Host strains used 66 2.1.14. Bacterial clone libraries 67 2.1.15. IMAGE consortium cDNA clones used 67 2.1.16. COS7 cell line 67 2.2 Methods 67 2.2.1. Filter spotting and processing 67 2.2.2. Restriction enzyme digestion 69 2.2.3. Agarose gel purification of DNA 69 2.2.4. Retrieving single clones from microtitre plates 70 2.2.5. Agarose plug minipreps of S.cerevisiae containing YAC clones 71 2.2.6. Pulsed Field Gel Electrophoresis 71 2.2.7. Southern blotting 72 2.2.8. Radio-labeled probe preparation 72 2.2.9. Suppression of probe sequence over-represented within the genome 73 2.2.10. Hybridization of membranes (filters) containing Southern blotted DNA or high/low density gridded colony DNA 73 2.2.11. Polymerase Chain Reaction (PGR) amplification 74 2.2.12. Preparation of plasmid DNA 74 2.2.13. Glycerol stock production 76 2.2.14. Preparation of probes from bacterial clones 76 2.2.15. Dot blot construction 77 2.2.16. Competent cell preparation 78 2.2.17. Transformation of competent cells 78 2.2.18. Tissue culture of C0S7 cells 78 2.2.19. Exon Trapping 79 2.2.19.1. Preparation of pSPLB vector 79 2.2.19.2. Preparation of bacterial clone insert DNA 79 2.2.19.3. Ligation and transformation 80 2.2.19.4. Electroporation 80 2.2.19.5. Reverse transcription and PCR 81 2.2.19.6. Sub-cloning secondary PCR products 83 2.2.19.7. Generation of 3^^ PCR for library screening 83 2.2.20. Sequencing template preparation 83 2.2.21. Genomic DNA preparation 84 2.2.22. cDNA construction 86 2.2.23. Northern blot hybridization 87 2.2.24. Sub-cloning cosmid DNA fragments into pBluescript*^ vector 87 2.2.25. Oligonucleotide DNA primer design and annealing temperature calculation 88 Chapter 3: Construction of overlapping segments of human DNA cloned in bacteria representing the lq21 region, specifically the Epidermal Differentiation Complex 90 3.1. Summary 90 3.2. Introduction 91 3.2.1. Advantages of bacterial clone physical mapping 91 3.2.2. Mapping status of lq21 92 3.3. Production of a sub-library of recombinant bacterial clones enriched for 1 q21 originating DNA 95 3.3.1. Starting material 95 3.3.2. YAC isolation 95 3.3.3. Verifying the YAC STS/EST content 96 3.3.4. Screening the cosmid and PAC libraries 97 3.3.5.