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Cloning and expression of the Drosophila melanogaster CuZn superoxide dismutase gene

University of British Columbia

Aging and disease processes may be due to deleterious and irreversible changes produced by free radical reactions. The enzyme copper-zinc superoxide dismutase (CuZn SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1) performs a protective function by scavenging superoxide radicals. In order to determine whether additional SOD activity affects longevity and oxygen metabolism in Drosophila, our approach was to clone the Sod gene and introduce additional copies of the gene back into the genome via P element mediated transformation. The effects of increased SOD activity on Drosophila life span and oxygen free radical metabolism were investigated. The CuZn SOD cDNA and gene were cloned from Drosophila melanogaster. The sequence of the Sod cDNA and gene revealed an additional C-terminal triplet coding for valine not found in the mature SOD protein. The nucleotide sequence of the coding region has 56% and 57% identity when compared to the corresponding human and rat Sod genes, respectively. A probe of the cloned gene hybridizes to position 68A4-9 on Drosophila polytene chromosomes. In wild-type Drosophila the Sod cDNA hybridizes to a 0.7-0.8 kb transcript which is greatly diminished in a SOD 'null' mutant that produces only 3.5% of the SOD protein. A 1.8 kb EcoRI gene fragment containing the Sod gene was cloned into the P vector pUChsneo and microinjected into Drosophila embryos. Five transformed lines, each of which contain an additional copy of the Sod gene at different chromosomal sites were constructed. The chromosomal positions of the transposed Sod sequence were determined by in situ hybridization of the Sod gene to salivary gland polytene chromosomes. Analysis of RNA from the transformed flies revealed that the transposed Sod gene was expressed. The range of SOD activity for the five transformed lines was 131% to 170% of the value of wild-type. There was good correlation between the amount of Sod mRNA and the level of SOD activity in the transformed lines. Increased SOD levels in the transformed lines did not confer greater resistance to paraquat-generated superoxide radicals, nor increase their lifespan. The SOD 'null' mutant with 3.5% of the wild-type SOD activity was hypersensitive to paraquat when compared to wild-type, whereas the heterozygous SOD deficiency Df(3L)1xd⁹/TM3SbSer with 50% of the wild-type SOD activity was not. Mutants lacking SOD are dramatically impaired in oxygen metabolism and a few percent of wild-type activity appears to provide significant protection against superoxide, while 50% of the wild-type levels confers essentially the same resistance as wild-type. Despite the observation that the SOD activities found in a wide range of animals correlates directly with their longevity, Drosophila melanogaster appears to be well protected against the toxic effects of oxygen by its native levels of SOD.

Text

2011-01-28

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http://hdl.handle.net/2429/30971

Philosophy

University of British Columbia

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