Die translasie van reovirus-RNA in oosiete van xenopus laevis - 'n vergelykende studie

Abstract

An approach commonly employed in the study of molecular biology is initially to study a simple system as a model in order to comprehend more complex systems. An example of this principle is the elucidation of the mechanism of protein synthesis ·mainly by studying prokaryotes. Likewise, research in molecular virology has to a great extent contributed to the current knowledge of gene expression in eukaryotes. Extensive studies of the molecular biology of reovirus have already been done. The reovirus genome consists of ten double-stranded RNA segments which are transcribed to ten messenger RNA species by a conservative mechanism. The enzyme activities catalyzing this viral mRNA synthesis reside in the inner capsid core proteins. The double-stranded RNA genome segments are surrounded by an inner capsid core and an outer capsid shell. During multiplication of the reovirus the host cell translation apparatus is used to translate the reovi rus mRNA. In this study the translation of reovirus mRNA and denatured dsRNA is investigated in Xenopus laevis oocytes. The primary aim of this study is to evaluate the fidelity in the translation of reovirus RNA in the oocyte system. In this way a contribution can be made in the development of a suitable whole-cell system to elucidate aspects of the molecular reovi rology. In the first instance adequate quantities of reovi rus mRNA were synthesized by in vitro-transcription and dsRNA was isolated from reovi rions. In a comparative study the translation of above-mentioned RNA was investigated in both a cell-free wheat germ system and the oocyte system. The denatured dsRNA was successfully translated in the wheat germ system, but it was established that this procedure is not as such suitable for translation in the oocyte system . This phenomenon may be the result of the renaturation of plus- and minus-stranded RNA. The reovirus specific translation products synthesized in the oocytes were then analyzed by polyacrylamide gel electrophoresis after immunoisolation . It was found that the ratio between the reovi rus translation products formed in the oocytes was comparable to the translation efficiency of the reovi rus mRNA in vivo. The peptide profile of the putative a3 translation producs formed in the oocytes is identical to that of the authentic reovi rus protein. In contrast with results obtained from the wheat germ system protein µl C was formed in the oocyte system by the correct post-translational cleavage of the primary gene product µl. These observations indicate a high degree of fidelity in the translation of reovirus mRNA in the oocyte system, which suggest that this system may provide a means for studying aspects in molecular biology.