Comparative vertebrate genomic sequence analysis studies based on Fugu rubripes Marc Robert Michel Botcherby M.Sc. A thesis submitted to the University of London for the degree of Doctor of Philosophy Department of Molecular Genetics Institute of Ophthalmology University College London University of London Bath Street London ECIV 9EL May 2002 ProQuest Number: U643460 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 U643460 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 This thesis explores the benefits of the comparative analysis of genomic DNA sequence in three vertebrate species: man, mouse and Fugu rubripes. The first region studied is the interval around Fibroblast Growth Factor 2 (FGF2). It falls within the candidate mapping interval for Insulin Dependent Diabetes 3 (Idd3) in mouse, in which Spermatogenesis Associated Factor (SPAF), Fibroblast Growth Factor 2 (FGF2) along with NUDT4 (formerly known as the antisense of FGF2, GFG) and a Centrin related sequence are known to be present. The Ensembl entry for the human draft sequence only contains FGF2. A Fugu cosmid was sequenced to 99.99% accuracy and is shown to contain orthologues to SPAF, FGF2, NUDT4 and a Centrin gene, as well as a novel gene prediction (vader), evolutionarily conserved in mouse and man. Comparison to the available mouse and human genomic sequences indicates the presence of SPAF, FGF2, NUDT4 and a Centrin gene in both the mouse and human FGF2 genomic regions. The second area of study is the conventional Protein Kinase C family (cPKC), which comprises 4 isoenzymes in man: a, p i, P2, and y. A number of approaches are taken to identify the Fugu cPKC homologues: genomic library screening by hybridisation, sequence scanning. Southern Blotting and degenerate PCR. A PKC a orthologue is identified and evidence for the existence of two further P homologues found. These genes are compared to the PKCs found in other species by means of phylogenetic trees. Two overlapping cosmids containing the PKCa orthologue are identified, sequenced to 99.99% accuracy and analysed. Potential regulatory elements are identified. A calcium channel subunit gene (CACNG5) is also found close to Fugu PRKCA, which reflects an association between all cPKCs and CACNG genes. This is interesting both from an evolutionary point of view and in the light of the association between cPKCs and Retinitis pigmentosa. Comparative sequence analysis is a useful tool for studying the evolution of gene families; the Fugu model provides a particularly effective means for the identification novel genes as well as regulatory elements. Declaration I declare that this thesis submitted for the degree of Doctor of Philosophy is my own composition and save as otherwise stated the data presented herein is my own original work. Marc RM Botcherby M.Sc. Acknowledgements I would like to thank the succession of people who have, in turn, acted as my supervisors, at the Institute of Ophthalmology, Prof. Shomi Bhattacharya, Dr Mai Al-Maghteth and eventually Dr Eranga Vithana, and at the HGMP Resource Centre, Dr Chris Mundy, then Dr Melody Clark and finally. Dr Greg Elgar. I would like to also thank Drs Sarah Smith and Melody Clark for their support and advice during my period of research and for their constructive criticism of the thesis. I would also like to thank my colleagues at the HGMP-RC for their support and understanding as, although this PhD was conducted in my spare time and it always came second to my work, it most certainly has impacted on the investment, both in terms of time and intellectual input, I have been able to made to my job. Finally and most importantly, I owe this to the supporting attitude of my wife, Sarah, and my young children, Francis, Nathalie, David and Patrick, the latter two having been bom during the timeframe of this PhD. Although I have tried always to put them first, I know the sheer amount of time I have had to devote to this work has caused them hardship, even though they have hardly ever mentioned it. L’homme n’est qu’un roseau, le plus faible de la nature; mais c’est un roseau pensant. Il ne faut pas que l’univers entier s’arme pour l’écraser: une vapeur, une goutte d’eau suffît pour le tuer. Mais quand l’univers l’écraserait, l’homme serait encore plus noble que ce qui le tue, puisqu’il sait qu’il meurt, et l’avantage que l’univers a sur lui, l’univers n’en sait rien. Man is only a reed, the weakest thing in nature, but he is a thinking reed. The entire universe need not take up arms to crush him: a humor, a drop of water is enough to kill him. But should the universe crush him, man would be even nobler than what kills him, since he knows he is dying and, of the advantage that the universe has over him, the universe knows nothing. Pascal, Pensée 347 TABLE OF CONTENTS List of figures and tables List of abbreviations INTRODUCTION................................................................................................. 1 1-The Human Genome Project (HGP) .................................................... 2 1-1 Mapping the genome ............................................................................ 2 1-1-1 The human genetic man ................................................................... 2 1-1-2 The human physical man .................................................................... 4 1-2 Sequencing the genome ........................................................................ 5 1-2-1 Four different sequencing approaches ................................................. 7 1-2-1-1 Sequencing cDNAs ........................................................................... 7 1-2-1-2 Sequence scanning of genomic clones ............................................ 8 1-2-1-3 Full length sequencing from genomic clones ................................ 9 1 -2-1 -4 Whole Genome Shotgun ................................................................ 10 1-3 The sequence of the human genome ................................................ 11 2- Making sense of the genome sequence ............................................. 15 2-1 The transcriptome ............................................................................ 15 2-1-2 Gene prediction by sequence analysis .......................................... 15 2-1-2-1 Exon predictions ........................................................................... 16 2-1 -2-2 Homology searching .................................................................... 17 2-1-3 Identifying genes by comparative sequence analysis ..................... 19 2-1-3-1 The Fugu model ............................................................................. 20 2-1-3-2 The mouse model .......................................................................... 24 2-2 Gene regulation .................................................................................. 26 2-2-1 Access to the genome ....................................................................... 26 2-2-2 Transcription ..................................................................................... 27 2-2-3 Identifying regulatory elements using bioinformatic tools 28 2-2-4 Identifying regulatory elements bv comparative sequence analysis 29 2-3 Understanding the function of genes ................................................ 31 3-What the future holds ............................................................................. 34 AIMS OF THE PROJECT....................................................................... 36 1 - Analysis of a highly conserved region in Fugu, Mouse and Man 36 2- Study of a gene family .............................................................................. 36 3- Comparative analysis of conventional PKCs in Fugu and man............. 37 4- Potential candidates for a disease gene .................................................... 37 Chapter 2: Materials and Methods .......................................................................... 38 2-1 : Isolation of cosmid DNA ...................................................................... 38 2-2: ProCipitate purification of cosmid DNA ............................................. 39 2-3 : Restriction digests .............................................................................. 39 2-4: Agarose gel electrophoresis .............................................................. 40 2-5: Alkali transfer Southern blot ............................................................. 40 2-6: DNA filter hybridisation .................................................................... 42 2-7: Preparation of random cosmid DNA fi-agments for ligation into Ml 3 or pUC vector ...................................................................................... 43 2-8: Ligation and transformation of somicated, end-repaired and size-selected cosmid fragments into vector ......................................... 44 2-9: DNA preparation from plasmids ......................................................... 45 2-10: