A Molecular Genetic Characterisation of Glutathione Synthetase in Drosophila melanogaster

Daniels, Joanne C. (2007). A Molecular Genetic Characterisation of Glutathione Synthetase in Drosophila melanogaster. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000fb1c

Abstract

Glutathione synthetase (GS) catalyses the final step of glutathione (GSH) synthesis. GSH is an important antioxidant, involved in detoxifying free radicals and maintaining the cellular redox balance. Oxidative stress is thought to be one of the causes of cellular senescence and organismal aging. The broad scope of this work is the analysis of oxidative stress resistance in Drosophila melanogaster, in relation to ageing, and specifically, this project aims to characterise GS in D. melanogaster.

The gene encoding GS in D. melanogaster (DmGS) is 8Kb long, and consists of up to 10 exons. Nine distinct potential transcripts, which differ due to alternative splicing, have been identified. In this study, cDNAs obtained from adult flies and S2 cells were sequenced, and confirmed the presence of six transcripts in vivo. Functional complementation in yeast corroborated the bioinformatic approach used to identify the two transcripts which are likely to produce an active GS protein.

A tandem duplication of GS in certain strains of D. melanogaster was identified using PCR techniques and southern blotting. Analysis of a small subset of sibling species indicated the duplication was not present in these closely related sibling species, or in any wild type D. melanogaster strains tested.

The effect of GS overexpression on oxidative stress resistance in adult flies was investigated, and the overall contribution of the protein to GSH production in relation to GCLC was also analysed. Increased levels of transcription of both genes raised GSH levels in adult flies, and also increased oxidative stress resistance.

After attempting to create a GS null mutant fly, GS knockdown in Drosophila S2 cells was studied using RNAi. GS RNAi was not found to have an effect on GSH levels, although in combination with GCLC knockdown, it did have a cumulative effect on lowering cellular GSH concentrations. Knockdown of either GSH synthesis genes did not have an effect on cellular viability, although upon exposure to DEM, GS and GCLC RNAi both significantly decreased cellular survival.