Characterization of a highly efficient protein synthesizing system derived from commercial wheat germ

Abstract

A crude extract of commercial wheat germ is capable of translating mRNAs from widely different sources with high efficiencies. Of six wheat germs analyzed only one was found capable of a high level or incorporation with natural mRNAs. Under optimum conditions at a saturating level of Tobacco Mosaic Virus (TMV) RNA (4.5 mug) and labeled amino acid, 68% of all the available (14)C leucine is incorporated in 70 min. at 30 degrees C with a stimulation of 425 fold above background (with an efficiency of 252 moles leucine/mole TMV RNA). Thus this system which is 30 fold more efficient for TMV translation than previous reported wheat germ cell free systems is capable of yielding 568 pmoles of (14)C leucine incorporated into protein in a 50 mul assay. 80% of the proteins produced have a molecular weight greater than TMV coat protein (17,400). This level of incorporation requires optimization of extract concentration, pH, Mg(+2), K(+) and spermine concentration as well as the method of extract preparation. Samples of crude polysomal RNA from hen oviducts (3% mRNA) and chorionating moth follicular cells (1% mRNA) are also translated in the wheat germ cell free system with high efficiency.

Transfer of in vitro synthesized barley endosperm proteins into the lumen of the endoplasmic reticulum

“…In vitro protein synthesis using the wheatgerm cell-free system has been found to show a sharp magnesium response (15,17). However, unlike the translation of mRNA associated with free or detached polysomes, the rough microsomes in barley ( Figure 3C) appear to be little sensitive to magnesium concentration.…”

“…An S-30 wheat-germ extract was prepared as described by MARCU and DUDOCK (15) and used for cell-free protein synthesis.…”

Phase-Specific Polypeptides and Poly(A)+ RNAs during the Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells

“…Wheat germ extract was prepared according to the method of Marcu and Dudock (25), and used to translate poly(A)+ RNA in vitro (40). One Ag of poly(A)+ RNA and 10 ,uCi of [35S]methionine (specific activity 34.6 TBq/mmol, New England Nuclear) were added per 10 zL ofwheat germ translation mixture and incubated at 30°C for 60 min.…”

In vivo and in vitro synthesis of CM-proteins (A-hordeins) from barley (Hordeum vulgare L.)

“…The wheat germ translation system was prepared and translation carried out as described by Marcu and Dudock (1974), with the following modification: K + and Mg 2+ were added as acetates, 2.5 vol of buffer were used instead of 2 vol., and 180 units/ml of pancreatic ribonuclease inhibitor (RNasin, Biotec Inc) were added to reaction mixtures. Incubations were carried out for 60 min at 29° C and 0.2-0.6 MBq of [ 35 S]cysteine (3.4-10 4 GBq mmol" 1 , New England Nuclear) were used per 25 ul of reaction mixture.…”