title: Cloning, characterisation and site-selected P-element mutagenesis of genes encoding v-ATPase in Drosophila
Creators: name: Guo, Yiquan
Publisher: name: University of Glasgow
Year: 1996
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Over the last few years, thousands of lines carrying lethal P-element insertions have been produced by the Drosophila community, which must presumably have inactivated a large number of essential genes. This thesis describes a fast and efficient approach to correlating cloned genes with mutant fly lines carrying P[lacW] insertions in the second chromosome (Torok et al., 1993). We have made use of the fact that P[lacW] contains a plasmid replicon to establish a collection of rescued plasmids containing genomic DNA flanking sites of transposon insertion. Plasmids representing a total of 1836 lines were individually rescued, and pooled in batches of 10 and 100. Pools of 100 plasmids were screened by hybridisation with cDNAs corresponding to cloned second chromosome loci. Hybridising pools were then narrowed down to single plasmids by a process of subdivision and rehybridisation, and corresponding mutant lines were obtained. Initial screening with 40 cDNAs has detected positive hybridisation for more than 10 genes. Mutations for 7 genes have been confirmed, of which insertions in genes encoding the A and c subunits of Drosophila V-ATPase are included. V-ATPase is a proton pump made of multiple subunits. The genes and cDNAs for A, E, and F subunits of V-ATPase have been cloned from Drosophila melanogaster via homology with the corresponding Manduca sexta genes. vha68-l and vha68-2, genes encoding two isoforms of V-ATPase A subunit, have also been isolated and sequenced. Both isoforms are composed of a polypeptide of 614 amino acids with a predicted molecular mass of 68.4 kDa and 68.3 kDa respectively. The vha68-2 gene is punctuated by four introns. The chromosomal location of both genes is at 34A on the second chromosome. Northern analysis of total RNA reveals that both isoforms are expressed in a similar pattern. They are ubiquitously expressed in head, thorax and abdomen of the adult fly. Developmental Northern blots of embryo, larvae, pupae and adult total RNA show general expression, but at a much reduced level during metamorphosis. A fly line (25/8) carrying a single P[lacW] insertion in vha68-2 was isolated by screening pools of rescued plasmids. The transposon is inserted into the first intron, in front of the translation start codon of vha68-2. The enhancer detector reporter gene carried by the P-element (beta-galactosidase) was generally activated, but particularly strongly in the gut and Malpighian tubes of both larvae and adults. The insertion largely reduces the transcript of the vha68-2 isoform which leads to a homozygous lethal phenotype at first instar larvae. The homozygous lethal phenotype can be reverted by 'jumping out' the insertion. Imprecise excision or internal deletion of the P-element created a set of novel hypomorphic or null alleles, with phenotypes which range from the first instar larvae lethal, as in the original P-element insertion line, to sub-lethals of different phenotype. A gene and a cDNA encoding the E subunit of V-ATPase have been characterised. The gene contains three small introns. Its deduced translation product has 226 amino acids and a molecular weight of 26.1 kDa. vha26 is present as a single copy at cytological position 83B1-4 on the third chromosome and gives rise to an mRNA species of 2.3 kb, with an expression pattern similar to that of vha68. A fly line carrying a single lethal P[lacW] insertion within vha26gene has been identified. The deduced translation product of the cDNA (vha14) for the F subunit is a 124 amino acid polypeptide with a molecular mass of 14 kDa. vha14 is present as a single copy at cytological position 52B on the second chromosome, and gives rise to an mRNA species of 0.65 kb. Unlike vha68 and vha26, the vha14 transcript shows relatively little variation during development and between adult head, thorax and abdomen, suggesting that the F subunit is a relatively ubiquitous component of the V-ATPase.