Nucleotide Sequences in the Yeast Alanine Transfer Ribonucleic Acid

Abstract

The purification of the yeast alanine-, tyrosine-, and valinetransfer ribonucleic acids by countercurrent distribution has been described (l), and preliminary data have been reported on the oligonucleotide compositions of these RNAs (2, 3).The present paper summarizes results of attempts to account quantitatively for all of the fragments obtained by digestion of the alanine-RNA with pancreatic RNase and with Taka-Diastase RNase Tl. Results of the analyses of these two digests are consistent, one with the other, and indicate that the alanine-RNA is composed of 77 nucleotides, including nine unusual nucleotides. EXPERIMENTAL PROCEDUREIsolation of Alan&e-RNA-The alanine-RNA was isolated by countercurrent distribution essentially as described (1, 3).In recent isolations, the first distribution has been run for 700 transfers, with the upper phase feeding in continuously at tube 1 and running out of the apparatus at tube 200.The alanine-RNA was then reisolated from tubes 150 to 190, and the peak of activity was redistributed once for 1000 transfers (1, 3).The alanine acceptor activity of the RNA was approximately that previously described, but has been somewhat variable ( ~~30%).It is possible that there is some subtle structural change that is related t,o this variation in activity, but if so, it has not been detected in the analyses.The analyses described below suggest a purity of approximately 90%.The alanine-RNA, after isolation as a dry solid (1, 3), has been stored at -2O", although, as far as is known, the dry RNA is completely stable at room temperature.Digestion with Pancreatic RNo.se and Chromatography on DEAE-Sephadex-The alanine-RNA (7.7 mg) was dissolved in 3.0 ml of water, and the solution was mixed with 0.37 ml of 0.10 M sodium phosphate buffer (pH 7.0), 0.37 ml of a 1 mg per ml solution of crystalline pancreatic RNase, and 0.15 ml of chloroform.The mixture was incubated for 14 hours at 37", and then was diluted to 10 ml with water before chromatography.DEAE-Sephadex A-25, medium (fines removed by decantation in water), was washed with 0.5 N hydrochloric acid, water, 0.5 N sodium hydroxide, water, 1 M ammonium carbonate, and finally with water.A column (0.25 x 225 cm) (two pieces of tubing 4 mm in outside diameter joined together with a small piece of Tygon tubing) was packed by gravity with a suspension of the DEAE-Sephadex in water.