Analysis of polysomes and polysomal RNA from control and heat-shocked maize seedlings

Previous studies have shown that 'the heat shock' response in maize involves new or enhanced synthesis of heat shock polypeptides (HSPs) and the increased transcription or availability of mRNAs for these HSPs (Baszczynski et al., 1982, 1983). In order to examine the intracellular distribution of RNAs prior to and following heat shock, polysomes were isolated from maize plumules grown at 25 C or following a shift from 25 C to 42 C. The polymers were separated into two classes: (a) free cytoplasmic, and (b) membrane-associated. From comparisons of sucrose gradient profiles, we estimate the distribution of polysomes in these two classes to be approximately 60-70% in the free cytoplasmic fraction and 30-40% in the membrane-associated fraction. Following heat shock, there is a decrease in the number and sizes of polysome peaks in both polysome classes.

Free and membrane-associated polysome preparations were used to direct the synthesis of polypeptides in a rabbit reticulocyte in vitro translation system (Baszczynski, 1983). A wide range of polysome concentrations was found to be suitable for efficient translation of polypeptides, ranging in size from less than 10 to greater than 100 kD. Fluorographic analysis of electrophoretic separations of the products obtained from translations of total polysomes extracted from heatshocked seedlings indicates that mRNAs for all molecular weight classes of the HSPs are present on polysomes following heat shock. This includes the 76 kD HSP class, which is not observed in translations of free RNA isolated from this same tissue. When the polysomes from heat-shocked seedlings are separated into free cytoplasmic and membrane associated fractions and translated in vitro independently, fluorographic analysis of the electrophoretically-separated products reveals that: (a) the high molecular weight HSPs are translated from mRNAs which are present on free as well as membrane-associated polysomes; and (b) the low molecular weight 18 kD class is translated almost exclusively from mRNAs which are present on membrane-associated polysomes. This latter finding suggests some form of cellular discrimination or compartmentalization of the various HSP classes in maize.

We are presently isolating the mRNA from these polysome preparations for analysis on agarose gels, for in vitro translational studies and for subsequent use in the production of cDNA probes.

Chris L. Baszczynski, Carol A. Rees-Farrell and N. Cathy Hogan
 
 


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