Transfer ribonucleic acid from Scenedesmus obliquus D3.

Scenedesmus obliquus is a green alga whose cells have a well-defined nucleus, and contain a chloroplast. Even when grown in the dark these cells retain their chloroplast. Because of the toughness of the cell wall, extraction of tRNA requires either vigorous homogenization of the cells before phenol extraction or prolonged phenol extraction at 60°C. Determination of the amount of tRNA and its purity, estimated by measurement of the methionine-accepting tRNA per g of cells and per E260 unit of isolated nucleic acid respectively, showed that the preferred method is by phenol extraction at 60°C. This method, however, is only acceptable when cells, harvested in the midexponential phase of growth, are used, since for older cells a toughening of the cell wall apparently prevents extraction of nucleic acid by phenol treatment. Determination of the base composition by the method described by Katz & Comb (1963) shows that tRNA from S. obliquus contains a considerably higher proportion of UMP and a somewhat lower proportion of CMP than other tRNA species, e.g. that from Escherichia coli (Zubay, 1962), yeast or rabbit liver (Cantoni et al., 1962). Optimal conditions for charging the tRNA with methionine by using a crude aminoacyl-tRNA synthetase isolated from S. obliquus requires 4m-Mg2+ and pH7.8. With a crude preparation of E. coli transformylase and [3H]N10-formyltetrahydrofolate approx. 20% of this methionyl-tRNA is formylated, whereas with a homologous enzyme preparation only 5 % formylation is observed. The species specificity of the tRNA and synthetase preparations from S. obliquus was studied by comparing the extent of [14C]methionyl-tRNA formation in homologous and heterologous systems. Whereas the synthetase from rat liver is able to charge the S. obliquus tRNA to an even greater extent than the homologous enzyme, synthetases from E. coli and Anacystis nidulans give a considerably lower charging level. The S. obliquus synthetase is able to charge methionine into tRNA from rat liver, wheat germ and E. coli to a degree only slightly lower than that obtained by using their homologous synt hetases . Conditions have been described (Marcu et al., 1973) whereby an unmodified uridine residue at position 23 from the 3'-end of the tRNA can be methylated specifically. This residue is normally the ribothymidine of the common sequence -G-T-Y-C-. By this procedure it is found that S. obliquus tRNA is methylated to about one-half of the extent to which rat liver and wheat-germ tRNA are methylated. Only a very low amount of E. coli tRNA is methylated, which agrees with the findings of Marcu et al. (1973). When tRNA, fractionated by BD-cellulose chromatography (Gillam et al., 1967), was examined by this method at least four separated species of s. obliquus tRNA were detected in which the uridine at this position is unmodified.