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Title: Identification and the characterisation of a novel gene required for the development of the larval head of Drosophila melanogaster
Author: Bubb, Gillian
ISNI:       0000 0001 3506 8089
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1993
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The aim of this study was to identify novel genes required during early embryonic development, of Drosophila melanogaster. This thesis describes the identification of a gene required for development of the larval head This gene has not been detected previously in conventional mutagenesis screens and was detected using the technique of 'enhancer trapping' This gene was selected for further investigation on the basis of the β-galactosidase expression pattern from the P[1ArB]enhancer detector transposon insertion, A3 50.1M2. The expression in this line was considered to be interesting since it occurs very early in a spatially restricted manner. The insertion was mapped to 59F1-3 on chromosome 2R The P-element insertion was shown to be hemizygous lethal over Df(bwS46) and this lethality can be reverted by excision of the P-element This showed that the insertion must have disrupted a gene that is essential for development The P[C1rB] insertion D26, is homozygous viable, and viable when hemizygous over Dft2R)bwS46 It had been shown to have a similar expression pattern and map position to the A350.1 M2 insertion, and it was therefore likely that it had inserted near to the same essential gene disrupted by A350.1M2. This was later confirmed by non complementation of ΔD26 excision chromosomes with A3 50 1 M2 The β-galactosidase expression pattern of A3 50 1M2 in early gastrulating embryos, corresponds to cells that are the precursors of the midgut, Malpighian tubules and the proctodeum in the posterior, and in the anterior, the anterior midgut and head ectoderm In germband extended embryos expression corresponds to cells of the anterior and posterior gut, gnathal and hypopharyngeal segments of the head and a large number of cells of the nervous system Genomic clones from the region flanking the insertion were obtained from a previously isolated plasmid rescue clone and a cosmid clone from the brown gene walk Two fragments from this region known to contain only unique sequences were used to screen a cDNA library Three cDNA clones were isolated cDNA clone 19 and cDNA clone 3a3 were shown to be overlapping, cDNA 19 being a 5' trunkated form of 3a3 cDNA clone 6 seemed to be a different gene which may be from a different genomic location Fragments from each of these three clones were used to perform in situ hybridisation to whole mount Drosophila embryos cDNA clones 19 and 3a3 gave expression patterns strongly reminiscent of the β-galactosidase pattern of A3 50 I M2 The expression of these cDNA clones occurs slightly earlier than in A350.1 M2, in two broad stripes in the anterior and posterior of the embryo These cells correspond to the same cells that express β-galactosidase slightly later in line A3 50.1 M2 By germband extension the expression is very similar to that of A3 50 1 M2 cDNA clone 6 is not expressed during embryogenesis. All three cDNA clones map back to the genomic walk cDNA 3a3 is 6 kb to the right of the A3 50.1M2 insertion, and approximately 50 kb to the right of the brown gene cDNA 3a3 was sequenced in both strands and the protein sequence was predicted in all three frames The frames program on GCG showed there was no long open reading frame in any of the frames The two longest frames occur in the same frame ORF1 and ORF2, which encode 92 and 88 amino acids respectively. The best homologies to known protein sequences are a sodium channel protein from rat cardiac muscle which shares 28 1% homology over 32 amino acids for ORF1, and a minor core protein V which has 35.1% identity over 37 amino acids, for ORF2. The sequence has provided little information about the function of the gene The viable D26 insertion was excised using transposase mediated excision and imprecise excision events were selected by lethality over Df(2R)bwS46 Ten imprecise excision events were selected and were all shown to be embryonic lethal when hemizygous. These excision events were placed into two complementation groups The cuticular structure of dead first instar larvae showed that some of the excision events had defects in the head skeleton. These defects include reduced lateralgrate and disintegrated dorsal bridge. There were no obvious homeotic transformations accompanying these defects. There were no obvious defects in the corresponding earlier embryos. The morphological defects observed in mutant larvae correspond to a small subset of the total expression domain of the 3a3 transcript.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH Natural history ; QL Zoology