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UBC Theses and Dissertations

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UBC Theses and Dissertations

Aging and protein synthesis : serine and leucine transfer RNA genes in Drosophila melanogaster Dartnell, Vicki June

Abstract

This thesis consists of two distinct parts. Part I describes preliminary studies undertaken to investigate whether defective tRNA molecules may be at least partially responsible for the general decrease in protein synthesis ability seen with advancing age. Part II describes the cloning of three recombinant plasmids, each containing at least one putative gene for one of Drosophila melanogaster tRNA[sup Ser/sub 2b], tRNA[sup Ser/sub UCA], tRNA[sup Leu/sub CUG]. . To approach the first problem from an in vitro perspective, unfractionated Drosophila melanogaster tRNA was either degraded by approximately four nucleotides or elongated by one nucleoside 3',5'— diphosphate at the 3'-terminus, and these defective tRNAs were added to a rabbit reticulocyte lysate protein synthesis system in varying amounts. It was found that these molecules did not produce appreciable inhibition of protein synthesis in this cell-free system until they were present in quantities similar to the estimated amount of endogenous tRNA present. This finding suggests that such defective molecules would not play an appreciable role in the age-related protein synthesis decrease seen in vivo, as it is highly improbable that defective tRNA molecules would accumulate at levels approximating the levels of active tRNA in the cell. A search was conducted for partially 3'-degraded tRNA molecules among the entire tRNA population isolated from aging Drosophila, and among both ribosome-associated and non ribosome-associated tRNAs from such organisms. These tRNA samples were treated with alkaline phosphatase and 5'-labelled with [³²P]ATP and polynucleotide kinase. Differential labelling of tRNA bands from the aged population could indicate the presence of partially 3'-degraded tRNA in this population; this was not observed, however, other than for one band which appeared to label somewhat more darkly in the older population in two independent experiments. The significance of this band is not clear, but it does not appear abundant enough to affect protein synthesis, on the basis of the in vitro results described. Analysis of whole tRNA isolates from both young and aged Drosophila by two-dimensional polyacrylamide gel electrophoresis did not reveal consistent differences between these two tRNA populations. In Part II, two oligonucleotides complementary to the known tRNA[sup Ser/sub 2b] sequence were used to screen pUC 13 recombinant DNA libraries containing inserts of Drosophila genomic DNA purified by size. One of these oligonucleotides, GT8, hybridized to a 5.1 kb Hindlll restric- tion fragment containing tRNA[sup Ser/sub 2b], & 3.6 kb EcoRI fragment containing an apparent tRNA[sup Ser/sub UCA] gene, and a 3.6 kb EcoRI fragment containing a tRNA[sup Leu/sub CUG] gene. The entire structural genes for both tRNA[sup Ser/sub 2b] and tRNA[sup Leu/sub CUG] were sequenced, as well as the 56 3'-nucleotides of the putative tRNA[sup Ser/sub UCA] structural gene. The sequence data suggests that examples of genetic microheterogeneity are seen here for both tRNA[sup Ser/sub 2b] and tRNA[sup Ser/sub UCA]. The sequence of the tRNA[sup Leu/sub CUG] gene corresponded exactly to that of a previously cloned Drosophila tRNA[sup Leu/sub CUG]gene (Glew et al. (1986) Gene 44, 307-314), but the flanking sequences of these two clones were different. Thus, a second copy of this tRNA[sup Leu]gene was obtained m this work. In situ hybridization studies showed the three fragments to derive from chromosomal sites 88A (tRNA[sup Ser/sub 2b]), 58AB (tRNA[sup Ser/sub UCA])and 66B (tRNA[sup Leu/sub CUG]).

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