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Molecular Biology and Gene Regulation

Isolation and in Vitro Translation of Polysomes from Mature Rye Leaves ¹

Department of Plant Sciences, University of Western Ontario, London, Ontario Canada N6A 5B7

Cytoplasmic polysomes have been prepared from mature leaves of winter rye (Secale cereale L. cv Puma). This is the first time a method has been developed for isolation of highly polymerized polysomes from mature leaves. The degree of intactness of isolated plant polysomes has been determined by two independent but complementary methods: size class distribution by sucrose gradient centrifugation and in vitro translation. The polymerization of isolated polysomes was estimated by the ratio of the proportion of large polysomes to the proportion of small polysomes obtained from the profiles. Our results show that the composition of the optimal polysome isolation buffer for mature rye leaves is different from that reported for young tobacco and pea leaves. Polysomes were translated in vitro with the S-105 wheat germ fraction. The degree of polysome polymerization has a significant effect on their in vitro translation since both the incorporation of amino acid and the presence of high molecular weight polypeptides are proportional to the large polysomes/small polysomes ratio. This study emphasizes the need to evaluate isolation conditions carefully before proceeding with polysome studies in any particular tissue or in tissues under different physiological status.