Abstract
Dry wheat embryos were allowed to imbibe water in the presence or absence of an inhibitor of mRNA synthesis (alpha-amanitin). At each of a series of times after the onset of imbibition, newly synthesized polypeptides were labeled, isolated, and compared with those made by cell-free translation of RNA prepared from the same embryos. In the absence of alpha-amanitin, characteristic time-dependent changes in the relative proportions of many polypeptides in the electrophoretic distributions of proteins synthesized in imbibing wheat embryos could be correlated with parallel changes in the cell-free translational capacity of bulk mRNA from the same embryos. These changes were virtually eliminated when alpha-amanitin was present in the imbibing medium. The findings are consistent with the possibility that transcription of new mRNA, which begins with the onset of imbibition, is responsible for change in the electrophoretic distributions of nascent polypeptides between 40 min and 5 h postimbibition of dry wheat embryos (Cuming, A. C. & Lane, B. G (1979) Eur. J. Biochem. 99, 217-224). Allied with the principal investigation, a useful modification has been developed in order to obtain an improved field of resolution (encompassing a range of Mr values between less than 5 X 10(5) and greater than 200 X 10(3), without using a gradient in sodium dodecyl sulfate/polyacrylamide gel.
Highlights
7 3 , 5 mM GTI’), 3 pl of “ion mix” (670 mM KAc, 5.4 mM MgAc2, 170 with basic proteins, most notably ribosomal proteins, being mM 2-mercaptoethanol), 2 pl of mRNA solution (approximatelv 0.01 conspicuous among the products of this early protein synthesis A d J ,0.5 pg), 8 pl of C.”S]methionine (approximately 75 pCi), and 10 [9, 10].The samestudies [10] have shown that soon after the period of early imbibition, and before 5 h postimbibition of dry wheat embryos, the electrophoretic distribution of newly made proteins in the 23,000 X g supernatant fraction of celifil of nuclease-treated lysate
Allied with the principal inves- particular, we have sought to ascertain the degree to which tigation, a useful modification has been developed in an increased proportion of these polypeptides might order to obtain an improvedfield of resolution
Ble RNA from imbibing wheat embryos directs synthesis of notdramaticallyaltered,the cell-free transla- the same array of polypeptides (Fig. 5B)seen in response to tional capacity of bulk mRNA is subjecto change between6 a supplement of thecorresponding bulk mRNA(Fig. 5A)
Summary
7 3 , 5 mM GTI’), 3 pl of “ion mix” (670 mM KAc, 5.4 mM MgAc2, 170 with basic proteins, most notably ribosomal proteins, being mM 2-mercaptoethanol), 2 pl of mRNA solution (approximatelv 0.01 conspicuous among the products of this early protein synthesis A d J ,0.5 pg), 8 pl of C.”S]methionine (approximately 75 pCi), and 10 [9, 10].The samestudies [10] have shown that soon after the period of early imbibition, and before 5 h postimbibition of dry wheat embryos, the electrophoretic distribution of newly made proteins in the 23,000 X g supernatant fraction of celifil of nuclease-treated lysate.
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