Panopoulou, Georgia; (1999) Construction of a catalogue of amphioxus (Branchiostoma floridae) genes expressed at neurula and gastrula stages by oligonucleotide fingerprinting. Doctoral thesis (Ph.D), UCL (University College London).

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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 preselection 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.

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