Construction of a catalogue of amphioxus (Branchiostoma Jîoridae) genes expressed at neurula and gastrula stages by oligonucleotide fingerprinting. Georgia Panopoulou a thesis submitted for the degree of Doctor of Philosophy in the University of London Windeyer Institute of Medical Sciences Department of Molecular Pathology and Clinical Biochemistry University College London Windeyer Building 46 Cleveland Street London W IP 6DB ENGLAND Max-Planck Institute fuer Molekulare Genetik Department Prof. Hans Lehrach Ihnestrasse 73 D-14195 Berlin GERMANY June 1999 ProQuest Number: U643583 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 U643583 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 To Dimitri, Vicky and Lena Abstract So far 9% of the human genome has been sequenced and for more than 50% of human genes there is a representative tag sequence (61,811 Unigene clusters, NCBI release statistics 1999). The major challenge of the next phase of the human genome project will be to try to understand the function of those genes. Cross-species comparison is an effective tool for providing clues based on sequence conservation combined with the possibility of genetic manipulations such as generation of mutants or crosses. However, the large evolutionary distance between the invertebrate and vertebrate model systems and human, from which the bulk of sequences deposited in the public databases is derived, limits the extent of such comparisons. I have chosen Amphioxus as a key organism for comparative study of vertebrate genes since it is phylogenetically placed at the transition point from invertebrates to vertebrates within the chordate phylum. Expression studies of mainly genes for which vertebrate homologues have already been identified showed conserved expression patterns strengthening its position as a close invertebrate relative to the vertebrates. Moreover, since most of the identified amphioxus genes have been found in fewer copies than their vertebrate counterparts, it is hypothesized that amphioxus branched off from the chordate lineage before two phases of genome enlargement, the first before the emergence of lampreys while the second close to gnathostome origins. As a result, amphioxus is expected to have a quarter of the number of vertebrate genes which makes it an attractive organism for identifying novel genes quickly. To construct an expression index of amphioxus genes, I have oligonucleotide fingerprinted a gastrula (BFLG) and a neurula (BFL26) stage cDNA libraries (110,592 clones in total). Oligonucleotide fingerprinting is based on the hybridisation of short oligonucleotides on high density grids carrying PCR products of cDNA libraries. Clones showing the same hybridisation pattern are clustered as representing the same gene. As a result, I have identified 1,783 clusters of different transcripts and 7,234 singletons in the gastrula stage while 4,674 clusters and 6,028 singletons in the neurula stage library. 5’ tag sequencing of 3,870 (4,079 sequences) distinct clones from both libraries and their subsequent sequence clustering confirmed the validity of oligonucleotide fingerprinting as a pre­ selection method able to achieve more than 3.5 fold normalisation and led to the identification of 1,811 from the BFLG and 1,516 from BFL26 unique transcripts. 38% of the BFL26 sequences and 20.7% of the BFLG sequences have known homologues in other organisms, among them some matching human disease genes. Table of contents ABSTRACT..............................................................................................................................................3 TABLE OF CONTENTS........................................................................................................................ 4 TABLE OF FIGURES............................................................................................................................ 7 LIST OF TABLES................................................................................................................................... 8 ABBREVIATIONS...................................................................................................................................9 ACKNOWLEDGEMENTS...................................................................................................................11 CHAPTER 1 ...........................................................................................................................................12 Introduction ...................................................................................................................................................................... 12 1.1. T he im por ta nc e of cross-species comparisons.......................................................................................12 1 .1 .1 .Finding genes faster and understanding their function ..........................................................12 1.2.2. Cross-species comparisons in gene identification and function analysis ......................... 14 1.1.3. Genome Duplications ................................................................................................................. 15 1.2. A m p h io x u s ................................................................................................................................................................... 19 1.2.1. Amphioxus phylogeny ................................................................................................................. 19 1.2.2. Amphioxus embryology ..............................................................................................................21 1.2.3. Amphioxus as an evolutionary model system ..........................................................................24 1.2.3.a. The anterior part o f the amphioxus nerve cord and its equivalence to the vertebrate brain .........................................................................................................................................................24 1.2.3.b. Insights into evolution of dors ov entrai axis specification through the study of amphioxus bone morphogenetic protein BMP2/4 .............................................................................25 1.3. O ligonucleotide fingerprinting ................................................................................................................... 29 1.3.1. Introduction ................................................................................................................................. 29 1.3.2. Sequencing by hybridisation (SBH ) ..........................................................................................30 1.3.3. Oligonucleotide Fingerprinting. Theoretical background. ...................................................32 AIM OF THIS THESIS........................................................................................................................ 37 CHAPTER 2 ...........................................................................................................................................39 M a ter ia ls a n d m e t h o d s............................................................................................................................................... 39 2.1. Re a g e n t su p p l ie r s...................................................................................................................................................39 2.2. S o l u t io n s......................................................................................................................................................................40 2.3. Experimental procedures ............................................................................................................................. 4 5 2.3.1 RNA isolation ................................................................................................................................4 5 2.3.2 cDNA library construction .......................................................................................................... 45 2.3.3 Introduction of Plasmid DNA into bacteria ..............................................................................50 2.3.4. Arraying o f libraries ...................................................................................................................57 2.3.5. Genomic DNA extraction .......................................................................................................... 57 2.3.6. Purification of plasmid and cosmid DNA (mini- preparation) ............................................ 5 7 2.3.7. Southern B lot ...............................................................................................................................55 2.3.8. Polymerase chain reaction ........................................................................................................ 58 2.3.8.a. Purification o f TAQpolymerase ............................................................................................58