RNA from wild-type Euglena, aplastidic mutant cells, and purified chloroplasts was chromatographed on oligo(dT).cellulose at 4°. The poly(A)containing and poly(Alacking fractions from each were then tested in a cell-free protein-synthesizing system from wheat germ for their abilities to specifically stimulate synthesis of large subunit ribulosebisphosphate carboxylase [EC 4.1 (7). These studies formed the basis for the present work in which poly(A)-containing and poly(A)-lacking RNA fractions were isolated from purified Euglena chloroplasts, and from wild-type and mutant cells. These RNAs were then tested for their specific abilities to act as templates in the syntheses of the large subunit of the carboxylase (LS-carboxylase), using a cell-free proteinsynthesizing system from wheat germ. METHODSCell Growth and Chloroplast Preparation. Wild-type Euglena gracilis var. bacillaris was cultured phototrophically and harvested as described (6). The aplastidic mutants, WsBUL and WjoBSmL (courtesy of J. A. Schiff) were grown heterotrophically (5). Chloroplasts were prepared from freshly harvested phototrophic cells according to the procedure of Eisenstadt and Brawerman (8) except that cells were broken in a cooled Yeda press (9) under argon at 1500 lbs./ inch2 (10.4 MPa) and a flow rate of 6-8 ml/min. RNA Extraction. Packed chloroplasts, isolated from 40 g of packed cells, were lysed with 20-30 ml of 2% sodium triisopropylnaphthalene sulfonate, 1% NaCl, 2.5 mM MgCl2, and 0.05 M Tris-HCI, pH 7.6. RNA was extracted with phenol and chloroform as described by Penman (10) and precipitated with 0.2 M NaCl and 2.5 volumes of cold ethanol. All preparations were treated with 10 ,ug/ml of DNase I (RNase-free) for 15 min at 00 (11). The phenol-chloroform and alcohol precipitation steps were then repeated. The above procedure was also used for extraction of RNA from whole cells. All extractions were performed at 220.
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