In vitro translation of polyadenylate-containing RNAs from dormant and germinating spores of the fungus Botryodiplodia theobromae

Abstract

Polyadenylated RNA isolated by oligodeoxythymidylate-cellulose chromatography from spores of the fungus Botryodiplodia theobromae was translated in a cell-free protein-synthesizing system derived from wheat embryo. Reaction conditions which would yield efficient and accurate in vitro translation of the spore RNA were established. Dual isotopically labeled mixtures of in vitro translation products from germinated and dormant spore polyadenylated RNA, as well as polyadenylated RNAs from intermediate stages of germination, produced qualitatively similar gel electrophoresis patterns, with polypeptides of 10,000 to 55,000 molecular weight. Proteins synthesized in vivo and extracted from germinating spores at three different stages possessed a greater size range, with molecular weights up to 85,000, although the in vitro synthesis apparently did yield the lower-molecular-weight proteins which were synthesized in vivo. Tryptic digest patterns of proteins translated in vitro from polyadenylated RNA of dormant and germinated spores were found to be identical in positions in only 40% of the spots. Furthermore, a dual-label comparison by isoelectric focusing of proteins translated from polyadenylated RNA of germinated and dormant spores also showed qualitative and quantitative differences among the in vitro translation products. We conclude that there are differences between the in vitro translation products of mRNA from dormant and germinated spores and that the mRNA preserved in the dormant spores contains genetic information which is qualitatively different from that of the germinated spores.