Analysis of the in vitro translation products from RNAs of heat-shocked seedlings

We have reported previously (MGCNL 56:111-113, 1982; Can. J. Biochem. 60:569-579, 1982) that various tissues exhibit a rapid and reversible response to a brief temperature shift (heat shock) with the new and/or enhanced synthesis of a select set of heat shock polypeptides (HSPs), as detected by one- or two-dimensional PAGE separations and fluorographic analysis. Preliminary results from in vitro translations of total isolated RNA from 4- to 6-day-old heat-shocked seedlings indicated that the HSPs noted in vivo were also present among the in vitro translation products. Subsequent investigations have revealed that the majority of the products obtained following in vitro translation of total RNA from control or heat-shocked plumules are the same as those obtained from translation of poly(A)+ mRNAs purified by oligo(dT) cellulose chromatography. Similar results were obtained from translations in both the rabbit reticulocyte and the wheat germ extract in vitro translation systems, with the former being apparently more efficient in the extent of incorporation of labelled amino acid precursor into newly synthesized products. We have also noted that, while the high molecular weight HSPs from the in vivo lysates each resolve (by 2D-PAGE) into a number of polypeptides with different pIs, the HSPs with similar Mrs synthesized in vitro have fewer isoelectric variants. Equally striking is the observation that the 76,000 dalton HSP noted in vivo is absent among the in vitro translation products in either the heterologous rabbit reticulocyte, or in the more homologous wheat germ in vitro translation systems. This may be the result of a lack of stability of the mRNA for this polypeptide, or may indicate the requirement of "maize-specific" translational factors for its synthesis.

Our observations indicate that: (a) heat shock in maize induces new and/or enhanced translation of polypeptides (HSPs), (b) the heat shock polypeptides are synthesized from polyadenylated mRNAs which were not available for translation in control tissues, and (c) several levels of regulation (currently under investigation) appear to be operative in determining the final form(s) of these HSPs.

C. L. Baszczynski, D. B. Walden and B. G. Atkinson

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