Characterization of purified poly(adenylic acid)-containing messenger ribonucleic acid from Saccharomyces cerevisiae

Abstract

Yeast poly(adenylic acid)-containing messenger RNA was isolated from total cellular RNA by affinity chromatography on poly(uridylic acid)-cellulose. The relative complexity of the isolated yeast mRNA was assessed by hybridization analysis with complementary DNA synthesized from the isolated messenger RNA (mRNA) with viral reverse transcriptase. Approximately 25% of the mRNA hybridized at an apparent Crt1/2 of 5 X 10(-3) mol sl.(-1), while the remainder hybridized at an average Crt1/2 of 10(-1) mol sl.-1. Poly(adenylic acid)-containing yeast mRNA was translated in vitro in a wheat germ cell-free extract, and the major polypeptides synthesized have the same molecular weight as the major proteins present in the cell. Four of these proteins were identified by coelectrophoresis and immune precipitation to be pyruvate kinase, enolase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase. These data demonstrate in agreement with the hybridization results that yeast contains major mRNA species and that some of the glycolytic enzyme mRNAs make up part of the major fraction. A procedure is outlined for the preparation of yeast mRNA which is essentially free of ribosomal RNA contamination and is further enriched in the major mRNAs present in the cell.