Quantitation and localization of globin messenger RNA in rabbit reticulocyte lysate

Abstract

A sensitive and quantitative method is described for the determination of globin mRNA distribution in rabbit reticulocyte lysate. The method uses high resolution sucrose density gradient centrifugation followed by [5'-3H]polyuridylate hybridization to poly(A)-mRNA in gradient fractions. Polyadenylate, purified globin mRNA, and ribonuclease-treated lysate are used to standardize the hybridization assay. It is demonstrated that changes of mRNA and ribosomal distribution do not affect quantitation of the total mRNA localization and Met-tRNAf which suggest that the monitoring of Met-tRNAf binding alone may not be sufficient to assess the mechanisms of control which affect the initiation of protein synthesis.