The effect of cycloheximide on Euglena gracilis phenylalanyl-tRNA synthetases.

The effect of cycloheximide on the chloroplastic, cytoplasmic and mitochondrial phenylalanyl-transfer RNA synthetases of Euglena gracilis was studied by growing both logarithmic and stationary phase cultures in the presence of the antibiotic. Enzyme activity was measured relative to untreated control cultures. At very low concentrations of cycloheximide (1 microgram/ml), all three log phase enzymes showed an increase in activity of 40-50%. At slightly higher concentrations (2.5 microgram/ml), the phenylalanyl-tRNA synthetase activities were comparable to those of the control cultures. At a cycloheximide concentration of 5 microgram/ml the enzyme activities from stationary phase cultures showed only very slight decreases (5-20%). The cytoplasmic and mitochondrial enzymes behaved similarly in log phase cultures at this concentration. However, the chloroplastic phenylalanyl-tRNA synthetase from log phase cultures treated with 5 microgram/ml cycloheximide showed a marked decrease in activity (70%). A further increase in antibiotic concentration to 10 microgram/ml resulted in significant losses of activity of all three enzymes, from both growth stages. The implications of the data with regard to identification of the site(s) of chloroplast enzyme synthesis are discussed.

A consideration of Euglena gracilis W3BUL as a cytoplasmic control for the wild-type phenylalanyl-tRNA synthetase system.

The studies described indicate that the UV bleached mutant, Euglena gracilis W3BUL does not serve as a suitable cytoplasmic control for the phenylalanyl-tRNA synthetase system. Chromatography of wild-type E. gracilis on Sephadex G100 revealed three peaks of activity identified as the chloroplastic, cytoplasmic and mitochondrial enzymes. The chloroplastic activity was greater in log than in stationary phase cells and was the only activity recovered from purified chloroplasts. Cell-free extracts of the achloroplstic mutant, E. gracilis W3BUL, contained wild-type levels of the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases. However, no chloroplastic synthetase was detected in the mutant extracts. Anomalies in the aminoacylation behavior of the W3BUL system were observed which suggest the possibility of a mutation affecting non-chloroplastic tRNAs in this UV-induced mutant. These anomalies significantly reduce the ability of the E. gracilis W3BUL mutant to serve as a cytoplasmic control in the phenylalanyl-tRNA synthetase system.

A reinvestigation of the sites of transcription and translation of Euglena chloroplastic phenylalanyl-tRNA synthetase.

An attempt was made to determine the sites of chloroplast phenylalanyl-tRNA synthetase transcription and translation. Inhibitors of bacterial RNA and protein synthesis were added to logarithmic and stationary phase cultures of Euglena gracilis wild-type B. Logarithmic phase cultures were sensitive to both types of inhibitors. In stationary phase cultures plastid synthetase was reduced by RNA but not by protein synthesis inhibitors. The effect of the antibiotics on the mitochondrial enzyme was also noted. Several possible explanations of these resuults are discussed.