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Quantitation of tRNAVal/3b in Drosophila melanogaster by RNA-DNA hybridization

University of British Columbia

The purpose of this study was two-fold. The first objective was to determine the optimum hybridization conditions for recombinant plasmids carrying Drosophila tRNA genes. The plasmid DNA was bound to nitrocellulose filters and hybridized with a homologous tRNA probe. The second objective was to use these plasmids to quantitate levels of tRNAVal/3b in flies bearing deficiencies in the major tRNAVal/3b loci. Specific plasmids studied were those bearing genes for tRNAVal/3b, tRNA Ser/4,7, and tRNA Lys/5. pDt78R, pDt48, and pDt41R, all bearing tRNAVal/3b genes, were compared under identical hybridization conditions to determine which DNA annealed tRNAVal/3b most efficiently. pDt78R was found to anneal 95-100% of the input tRNAVal/3b under the widest range of hybridization conditions. A comparison of pDtl6, pDtl7R, pDt27R, and pDt73, all bearing tRNA Ser/4,7 genes, showed that pDtl7R annealed tRNA Ser/7most efficiently. Initial studies on pDtl7R demonstrated a low overall hybridization efficiency of 18%; however, it was found that adding 21% formamide to the hybridization buffer increased the efficiency of annealing to 85-90%. pDtl2 and pDt39, carrying tRNA Lys/5 genes, exhibited low hybridization efficiencies of 20 and 15%, respectively. Adding 15-20% formamide only increased the hybridization efficiency to 55 and 38%, and these plasmids were not studied further. Transfer RNA was extracted from 50-300 mg of adult flies and was specifically labeled in vitro. The levels of tRNAVal/3b and tRNA Ser/4,7 were quantitated by annealing to pDt78R and pDtl7R immobilized on nitrocellulose filters. The level of tRNAVal/3b in the tRNA isolated from flies deficient in the major tRNAVal/3b loci was examined. The results showed that deletion of half the major tRNAVal/3b loci resulted in a reduction of approximately 50% in the level of tRNAVal/3b did not produce the Minute phenotype; furthermore, the effects of deficiencies at two loci were approximately additive.

Text

2010-04-13

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http://hdl.handle.net/2429/23378

Genetics

University of British Columbia

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