UBC Theses and Dissertations
Characterization of recombinant plasmids carrying Drosophila transfer RNA genes Rajput, Bhanu
The purpose of this study was to characterize recombinant plasraids carrying Drosophila melanogaster tRNA genes. The two groups of recombinant plasmids studied were those which carried tRNA₄Val genes and those with tRNA₄,₇Ser genes. pDt92 and pDt120, both tRNA₄Val gene-carrying plasmids, were characterized initially to determine the number of inserts they contained and the size of the inserts. For plasmids containing multiple inserts, the insert which carried the tRNA₄Val gene was also determined. These characteristics were studied by HindIII digestion of the plasmid DNA, agarose gel electrophoresis, Southern transfer onto nitrocellulose filters and hybridization to [¹²⁵I] tRNA₄Val. It was found that both, pDt92 and pDt120 contained two inserts each of sizes 0.5kb and 1.7kb,and 2.0kb and 5.*fkb respectively, with the 0.5kb and 2.0kb fragments carrying the tRNA₄Val genes. pDt92 and pDt120 then were recloned so as to contain only the fragments which carried the tRNA₄Val genes, namely the 0.5kb and 2.0kb fragment respectively. pDt92RC and pDt120RC plus three other tRNA₄,₇Ser gene containing plasmids, pDt16, pDt17RC and pDt27RC were further characterized by the technique of in situ hybridization to study the organization of these tRNA genes on the Drosophila genome. Four of these plasmids with the exception of pDt17RC hybridized to only one site on the Drosophila chromosome. Both, pDt92RC and pDt120RC hybridized to the 90BC site on the right arm of the third chromosome; pDt16 and pDt27RC hybridized to the 12DE site on the first or the X chromosome. pDt17RC on the other hand hybridized predominantly to the 12DE site and to a lesser extent to 2}E (2L), 56D (2R), 62D (3L) and 64D (3L) sites. These in situ hybridization results when studied together with those reported by Dunn et al. (1979b) show that genes for a single species of tRNA are located on more than one site on the Drosophila genome.
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