Last year (MGCNL 59:76, 1985), we reported on the isolation of a poly (A+) RNA size fraction (630-850 nucleotides) from shoots of seedlings (Oh43) subjected to a 1 hour temperature shift (heat shock; hs), which upon translation in a cell-free system yielded a heat shock specific polypeptide of 18 kD. We have obtained further evidence that this polypeptide belongs to the 18 kD heat shock polvpeptide class (hsp class) described previously (Baszczynski et al., Can. J. Biochem. 60:569, 1982; Baszczynski, MGCNL 56:111, 1982).
Polyclonal antibodies were raised against the 18 kD hsp class using the method outlined by Baszczynski (MGCNL 58:135, 1984). These antibodies were used to recognize 18 kD heat shock polypeptides among translation products of RNA from different sources.
Translation products (25 ul) were treated with 0.5% (1.25 ul) of ribonuclease A (1 ug/ul) and 0.5% (1.25 ul) of ribonuclease T1 (1 U/ul), for 15 mins. at 30 C. Antiserum (50 uL) and immunoprecipitation buffer (12.5 uL; 10 mM NaPO4, pH 7.5, 150 mM NaCl, 10% NaDOC, 10% triton X-100) were added. After 20 mins. at room temperature 100 uL of a preparation of Staphylococcus aureus cells was added and the mixture was incubated for a further 20 mins. at room temperature. The samples were spun for 3 mins. in a microfuge and the pellets were washed 3 times in wash buffer (10 mM NaPO4, pH 7.5, 150 mM NaCl, 1% NaDOC, 1% triton X-100). After the final rinse, the pellets were suspended in protein extraction buffer (200 uM Tris pH 7.5, 5% SDS, 7.5% 2-mercaptoethanol, 1 mM PMSF) and placed in a boiling water bath for 3 mins. The samples were spun in the microfuge for 3 mins. and the supernatants were subjected to SDS-PAGE and fluorography.
It was demonstrated first that these polyclonal antibodies recognize only heat shock specific polypeptides amongst translation products of total RNA from shoots of seedlings subjected to heat shock and that they do not recognize any polypeptides synthesized in the cell-free system by RNA isolated from shoots of control seedlings. The antibodies were then used in immunoprecipitation experiments with translation products of the poly (A+) RNA size fraction (630-850 nucleotides) isolated from shoots of control and heat shock seedlings. The heat shock specific 18 kD translation product was immunoprecipitated using these antibodies. This confirms our suggestion that this polypeptide belongs to the heat shock polypeptide class previously described and it confirms the presence of a mRNA coding for a heat shock polypeptide among the RNA molecules of this size class (630-850 nucleotides). [Note of correction: In last year's newsletter (Rees, MGCNL 59:76, 1985) there is an error in one of the buffer recipes: Transfer buffer consisted of 10 mM Tris pH 7.6 500 mM NaCl, not 500 mM Tris pH 7.6.]
Carol A. B. Rees and David B. Walden
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