ISOLATION OF POLYSOMES AND POLY(A)‐CONTAINING RNA FROM CHLORELLA FUSCA VAR. VACUOLATA

Abstract

SUMMARYRibonuclease activity in 120000 g supernatant extracts of Chlorella fusca var. vacuolata showed maximal activity at pH 5 and minimal activity at about pH 7·5 in the range 5 to 8·6. Dithiothreitol, high salt concentration and 100 mM tris significantly inhibited ribonuclease activity although the extent of inhibition was not uniform at all pH values. Heparin, poly‐vinylsulphate and sodium dodecylsulphate were largely ineffective as ribonuclease inhibitors under the same conditions.Polysomes were isolated using the method of Jackson and Larkins (1976) without the addition of heparin but including 10 mM dithiothreitol. Sucrose density gradient centrifugation analysis revealed profiles of polysomes consisting mainly of SOS ribosomes and containing a high proportion of large polysomes when cells were treated with 2·5μgml‐l cycloheximide before harvesting. The yield of polysomal material was about 50 A250 units per 1010C. fusca cells. Total RNA extracted from C. fusca was analyzed by acrylamide gel electrophoresis in 98 % formalide. Cells broken by liquid nitrogen grinding gave RNA preparations which were much less degraded than when the French pressure cell was used for cell disruption. Of three methods tested for isolation and deproteinization of RNA, phenol‐cresol deproteinization of extracts in trisbuffered triisopropylnaphthalene sulphonate/p‐amino salicylate gave the highest yield of RNA and was consistent with the conditions established for minimizing ribonuclease activity. Re‐extraction of the phenol phase was necessary to obtain a high yield particularly of poly(A)‐containing RNA. C. fusca cells growing exponentially in the light gave a greater proportion of poly(A)‐containing RNA than light‐limited cells.Poly(A)‐containing RNA was separated from total RNA and from polysomes on a column of oligo(dT)‐cellulose. This fraction, isolated from total RNA consisted mostly of molecules between 2 × 106 and 0·5 × 106 daltons, but a significant proportion of smaller molecules was present when polysomes were first isolated as the starting material. Poly(A)‐tracts obtained by oligo(dT)‐cellulose chromatography of 14C‐labelled poly(A)‐containing RNA were up to about 450 nucleotides in length with tracts 300 nucleotides long apparently most abundant. When the data were corrected for the increase in radioactivity with tract length and the logarithmic distribution of tract sizes on polyacrylamide gels, smaller tracts were shown to be predominant.