Xenopus laevis oösiete as translasiesisteem vir reovirus-mRNA

Abstract

Several methods of approximation can be applied in the study of the translation process. In this dissertation a micro injection of Xenopus laevis oocytes was used in conjunction with reovirus mRNA as model in the study of eucariotic messenger translation. Firstly the oocyte system was standardized with the use of globin 9S mRNA of rabbit reticulocytes. The translation patterns of different groups of oocytes were compared after the injection of 9S mRNA or 9S mRNA in the presence of haemin or polysomal material. The results are i) the biosynthesis of haemoglobin in the oocyte as a result of globin 9S mRNA injection ii) the stimulation of haemoglobin synthesis, as mentioned in i), with the addition of haemin iii) an increase in total translation as a result of the injection of polysomal material. These are in agreement with the existing literature. In the second phase of the experiment&l approach the in ovo translation of reovirus mRNA was investigated. To make this possible reovirus particles were isolated from infected mouse-L- fibroblasts. The ten reovirus messenger species were prepared in vitro with the use of chymotrypsin. The unfractionated messengers (ten species which were identified in their three classes) were subsequently injected into the oocytes whereupon the translation process was followed. Five virus-specific polypeptides were tentatively demonstrated with the aid of poly-acrylamide gel electrophoresis in a Na-phosphate-SOS system. Immunoprecipitat i on and fluo rography

were used as subsidiary aids. The sensitivity as well as the applicability of the oocyte system is prominent in the discussion of the post-translational modifications performed by this system.