Polyacrylamide gel mapping of chicken tRNA: comparison of polysome-bound and whole-cell tRNA from normal and avian sarcoma virus-infected chicken embryo fibroblasts.

Abstract

Analysis of the tRNA population from chicken cells was performed by means of polyacrylamide gel mapping. About 60 species were detected; most of these were positively identified by their acceptor specificity. The comparison of polysomebound and overall cellular tRNA gel patterns from normal and Rous sarcoma virus-infected chicken embryo fibroblasts led us to the following observations: some tRNA species were present in the same relative proportions in all the preparations, and within isoaccepting groups the same species was preponderant; however, although about 8% of whole-cell tRNA was recovered in polysomal preparations, amounts ranging from 3 to 30% were found for individual tRNA species. This points to the absence of a direct correlation between the amount of each mature tRNA species produced and the frequency with which it is used in this case of embryonic cells. No significant difference was observed between the whole-cell tRNA patterns from normal and infected cells. Thus, tRNA transcription appears unaltered when cells are transformed and virus producing. No change was observed in the extent of a post-transcriptional modification of tRNAPhe (the base Y). However, viral infection led to some changes in the relative proportions of individual species from polysomal preparations.Modifications of tRNA population in the cell induced by changes in its environment have been described in numerous reports (23,33). These studies have contributed to opening the question of the role of tRNA in the regulation of translation. Particular attention has been paid to the altered tRNAs in the process of cell differentiation and viral infection. In this last case, some variations in isoacceptor pattern were detected by changes in the chromatographic profiles of amino acid acceptor activity (5,14,31,34 variation in the frequency of codons being translated can occur, involving a concomitant change in the set of tRNAs selected by the polysomal machinery. A strict adjustment of the tRNA population to the amino acid composition of proteins being synthesized has been described for different tissues (6). In this case, the distribution of mature tRNAs in the cell would be the same as the distribution of tRNAs engaged in protein synthesis. We attempted to determine whether virusinduced transformation involves changes either in the amount of each mature tRNA species produced or in the frequency of their utilization or in both. To this end, we compared the distribution of overall cellular tRNAs and that of tRNAs bound to polysomes, assuming that those tRNAs are actively engaged in protein synthesis. Preparations of tRNA isolated from normal CEF and from RSV-infected CEF were analyzed by two-dimensional (2D) gel electrophoresis. The tRNA spots on the gel map were identified. To quantify each species, tRNA was labeled at the 3' end group with [a-32P]

A very poorly expressed tRNASeris highly concentrated together with replication primer initiator tRNAMetin the yeast Ty1 virus-like particles

“…The two dimensional polyacrylamide gel (2D gel) electrophoresis was performed at 5°C on 9.6% acrylamide in presence of 6M urea (first dimension) and on 20% acrylamide in presence of 4M urea (second dimension) both containing 20mM Tris-borate buffer, pH 8.3, 2.5mM EDTA. tRNA deacylation, 3' end specific labelling using tRNA nucleotidyl transferase and quantification of 32p tRNA from gel spots were performed as described (12).…”