In vitro synthesis of barley storage proteins
Membrane-bound polysomes were isolated from developing endosperms of barley (Hordeum vulgare L.). The messenger RNA associated with the polysomes was separated from the ribosomal components by affinity chromatography on oligo-dT cellulose. The characteristics of the polysomes and of the poly-A+ RNA fraction are discussed. The poly-AT RNA fraction contained components of between 0.55 and 2.5 kilobases in length, and the degree of adenylation is 6.5%. Both polysomes and poly-A+ RNA were shown, by the use of radioactive precursors, to support the synthesis of trichloroacetic acid (TCA)-insoluble material in a wheat germ cell-free protein-synthesising system. The products of in vitro-protein synthesis resembled hordeins (the prolamin strorge proteins of the barley endosperm) in that theywere predominantly soluble in 55% propan-2-o1 plus 0.05% DTT, contained a low proportion of lysine as compared with leucine, exhibited similar immunological characteristics and had similar, but not identical, electrophoretic properties which were dependent on the barley variety used. Polysome products were the same size as native hordein but poly-A+ RNA products were approximately 2000 daltons larger. The larger size of the products of poly-A+ RNA translation is suggested to be due to presence of an extra leader sequence. The mechanism of processing and transport of the precursor polypeptides was investigated both in vitro, using stripped barley endoplasmic reticulum, and in vivo by injection of poly-A+ RNA into Xenopus oocytes. It is concluded that hordeins are synthesised as precursors by membrane-bound polysomes, and that the polypeptides ace co-translationally shortened and transported into the lumen of the endoplasmic reticulum in a manner consistent with the signal hypothesis. The difference in size between the products of polysome and poly-A+ RNA translation is discussed in relation to these observations. The poly-A+ RNA has been used to prepare clones of Escherichia coli (E. coli), some of which are identified as containing DNA sequences complementary to particular hordein mRNAs.