A Screen for Drosophila Genes Relevant to the Nervous System

Tomlinson, Simon R (1994) A Screen for Drosophila Genes Relevant to the Nervous System. PhD thesis, University of Glasgow.

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Abstract

Understanding the molecular and genetic control of many brain functions remains one of the most challenging problems of modern biology. Although a number of brain structural and functional mutants have been isolated, their number and diversity is small compared to the complexity of the Drosophila nervous system. Using various differential screening methods in conjunction with a method of reverse genetics developed in the laboratory, the aim of the project was to gain molecular and eventually genetic access to this missing diversity. Following an assessment of differential and subtraction screening methods a differential screen was performed to isolate a set of about twenty cDNA clones representing genes expressed strongly in the head and weakly in the body. These clones were sifted further using partial sequence and approximate pattern of expression. Several clones were putatively identified as being derived from genes with homologues in other organisms including one previously known head specific gene, ninaE. Another clone from the screen, pST 123, was shown to be a cloned fragment of a message predicted to encode an unknown homologue of synaptobrevin, a family of molecules known to be involved in synaptic transmission. A third clone class, represented by the pST51 cDNA, was derived from a Na/K ATPase beta-subunit gene family member, a family of molecules involved in establishing membrane potentials in the nervous system, as well as in other processes such as cell-cell recognition. These two cDNAs were selected for further study, together with two other cDNA clones, pST162 and pST170, which defined genes with interesting patterns of transcription. A more detailed molecular characterisation of the four cDNA clones was performed to confirm the predicted gene expression pattern and to further investigate the molecular biology. For example, the beta-subunit cDNA insert from pST51, was sequenced and a full length predicted amino acid sequence identified. Phylogenetic analysis revealed that the encoded polypeptide was most closely related to the only other arthropod Na/K beta-subunit known, that of the brine shrimp. Together they define a new class of beta-subunit, clearly distinct from the three mammalian types. Expression analysis included head/body northerns and in situ hybridisation to head and head/body frozen sections using cDNA derived probes. This showed an expression pattern consistant with a gene expressed exclusively in the nervous system. A set of related cDNA clones identified using the pST51 cDNA insert reveal the likely presence of multiple transcripts generated with a variety of 5' and 3' untranslated regions. The analysis of the related cDNAs also identified the likely presence of a message encoding a second form of the beta-subunit. Genomic Southerns and in situ localisation to polytene chromosomes indicate a single copy gene, although lower stringency hybridisation experiments identify the presence of several related loci in the Drosophila genome. A similar analysis has been performed on the other selected cDNAs. Of these, the study of the pST 123 cDNA derived from a novel Drosophila synaptobrevin gene has proved to be the most informative. Interestingly, synaptobrevins have recently been identified as the target for tetanus toxin cleavage. From the predicted amino acid sequence it appears that the gene product should be cleaved by this toxin. A Drosophila synaptobrevin locus had previously been identified, but it appears that this locus produces proteins unlikely to cleave with the toxin, and has recently been reported as having a predominantly non-neuronal expression profile. In situ hybridisation experiments presented in this thesis suggest a neuronal role for the subunit isolated in this screen.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Gary Griffith
Keywords: Genetics, Neurosciences, Molecular biology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-75347
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 20:27
Last Modified: 19 Nov 2019 20:27
URI: http://theses.gla.ac.uk/id/eprint/75347

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