Molecular Analysis of the shaking-b Locus of Drosophila melanogaster

Crompton, Douglas Ewan (1995) Molecular Analysis of the shaking-b Locus of Drosophila melanogaster. PhD thesis, University of Glasgow.

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Abstract

One strategy for the isolation of molecules required for the establishment of specific synapses is to screen for mutations which disrupt identified neuronal connections and subsequently to clone and characterise the genes involved. The giant fibre system of Drosophila melanogaster is an ideal focus for such investigations. This system mediates the fly's jump-escape response, allowing neuronal connectivity mutants to be isolated as a subset of those flies which fail to jump in response to a light-off stimulus. The Passover mutation was isolated in this way, and was subsequently shown to be an allele of the shaking-B (shak-B) locus, thus implicating shaking-B in the establishment of neuronal connectivity, and inspiring the molecular analysis which is reported here. Genetic analysis reveals two distinct functions at the shaking-B locus, one (termed shak-B(neural)) is required for the normal development of the imaginal nervous system, while the other, shak-B(lethal) is an essential function, without which the animals die as embryos or first instar larvae. Most shaking-B alleles disrupt both of these genetic functions, although some (like shak-B passover) are specifically neural, while others (such as shak-B L41) affect only the essential function. The 19E3 polytene region in which shaking-B resides was cloned by chromosome walking from microcloned entry points and the breakpoints of deficiency chromosomes which encroach upon the shak-B region were used to define a 15 kb stretch of walk in which at least some of the gene must lie. Unique DNA fragments from this area were used to probe cDNA libraries and the embryonic cDNA KE2(1.8) was isolated. The KE2(1.8) cDNA was sequenced and found to contain no extensive regions of reading frame, though an internal 122 codon open reading frame (ORF) was implicated by computer algorithms as a likely coding region, and was found be highly homologous to the N terminus of the Drosophila Ogre protein and to part of the C. elegans Unc-7 protein, both of which are implicated in nervous system development. An asymmetric PCR strategy was used to sequence this small ORF from shak-B mutant chromosomes, and a 17 bp deletion which is predicted to abolish translation of the ORF was found to underlie the shak-BL41 allele. Due to the rarity of shak-B cDNAs, a library screening strategy based upon inverse PCR was devised. This technique enabled the isolation of cDNAs representing a further four shak-B transcript forms, while yet another two cDNAs were isolated by conventional means. Sequence analysis of these clones and of the genomic regions from which they were derived has provided a wealth of data regarding the putative products and genomic organisations of these transcripts. The SIPC8 cDNA contains an ORF of 372 residues, implying a protein of 44.4 kDa with extended homology to Ogre and Unc-7. In the neural and lethal shak-B alleles shak-B allelels and shak-B r-9-29 this reading frame was found to be disrupted by a mutation which introduces a stop codon in a downstream exon. This finding, together with the identification of the shak-BEC201 allele suggested that Shak-B(neural) and Shak'B(lethal) proteins have unique N terminal regions but converge upon common C terminal sequences. While the SIPC8 cDNA is disrupted by lesions causing lethal alleles, the P2.4 cDNA isolated by Krishnan and colleagues was found to contain an ORF with a unique N terminus, and a C terminus common to that of the SIPC8 reading frame. The unique N terminus of P2.4 was found (BCrishnan et aL, 1993) to be disrupted by lesions underlying shak-B(neural) mutations including shak-B passover go fulfilling the criteria demanded of a shak-B(neural) transcript. Shak-B proteins contain hydrophobic segments suggestive of transmembrane domains, and assessment of the likely transmembrane dispositions of all putative Shak-B proteins was carried out using optimal computer algorithms. Based on these structural predictions and on the phenotypes and expression patterns of shaking-B and its homologues, the possible functions of Shaking-B proteins are considered.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Jane Davies
Keywords: Genetics, Molecular biology, Neurosciences
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-75540
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:31
Last Modified: 19 Nov 2019 19:31
URI: http://theses.gla.ac.uk/id/eprint/75540

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