Extracts from wheat embryos have been used to study the incorporation of amino acids into TCA insoluble products using hepatic mRNA fractions. The properties of this system are described and compared to the incorporation obtained with polyU and rabbit globin mRNA. SDS-acrylamide gel analysis showed that the major polypeptide synthesized with globin mRNA co-migrates with rabbit globin (15 500 daltons). Rat liver products were numerous, with molecular weights from less than 10000 to greater than 65000 daltons. The KC1 concentration for maximum incorporation into TCA precipitable polypeptides with hepatic mRNA was not the optimum KC1 concentration for synthesis of complete products.
I. INTRODUCTION
Recently a number of eukaryotic in vitro protein synthesizing systems have been described which are stimulated by the addition of exogenous polysomal RNA fractions. Using convenient sources of eukaryotic mRNAs, either animal virus RNAs [1-3] or mRNAs isolated in fairly pure form because of tll~ir predominance in specialized cells (e.g., globin, ovalbumin, ~-crystallin, and immunoglobUiin mRNAs [4-8]), or unusual size (e.g., myosin and histone mRNAs [9, 10]), many reports have described in vitro translation of these mRNAs into correct polypeptide products. The availability of such systems permits a study of the control of gene expression at the translational level.
Because of the increasing number of reports of faithful translation of mRNAs in heterologous systems, many believe there is no specificity of translation factors for eukaryotic mRNAs. However Wigle and Smith have reported isolation of a protein factor in ascites cells which specifically stimulates translation of EMC RNA [11]. Nudel et al. [12] have identified another factor from reticulocytes which stimulates synthesis of ~-globin, and not fl-globin. In addition, Uenoyama and Ono have shown that two interacting factors, one inhibitory and the other stimulatory, regulate the synthesis of catalase on rat liver polyribosome preparations [13,14]. With the exception of the latter example (in which case initiation of new polypeptide chains is not required), these reports of translation modulation factors are restricted to systems which use either viral mRNAs or mRNAs from cells undergoing terminal differentiation.
To extend studies of modulation of protein synthesis to systems in which many different proteins are synthesized, we have chosen to examine translation of hepatic products, a group of well-characterized polypeptides. We selected wheat germ extracts because of their reproducibility. This report describes the translation properties of this system, including an improved method for preparing mRNA fractions.