fer of amino acids from aminoacyl- METHIONINE tRNA’s, After 10 minutes more, the cells were lysed by suspension in 5 and 0.0015M MgCl. containing 15 mg of purified bentonite per milliliter (8) and by repeated freezing (in a mixture of dry ice and acetone) and thaw- ing. The lysate was extracted with a OD AT 260mp volumes of 0.01M tris, 0.0144 KCl, 0 EMBRYO SERINE half volume of carbon tetrachloride, and the upper phase was shaken for 15 minutes with an equal volume of twice-distilled, buffer-saturated phenol. The aqueousphase was extracted again with phenol, and traces of phenol were removed with cold, peroxide-free ether. The solution was then adjusted to 0.144 with sodium acetate buffer (44/, pH 6), and then 2 volumes of cold ethanol were added. After several hours at —20°C, the precipitated RNA was collected by centrifugation, washed with cold ethanol, and dried. The yields of tRNA from the above-mentioned volumes of packed cells were 1.3 optical density (O.D.) units (260 my) and 0.85 O.D. units for embryonic and adult cells, respectively. The MAK column (1 by 3 cm) was similar to that of Kano-Sueoka ef al. (2); it contained 1 g of kieselguhr and 0.25 ml of 1-percent methylated albumin, because only small quantities of material were available for analysis. The second change was the use of a shallower gradient of 0.20 to 0.65M NaCl (250 ml in total volume). The freon column used was a modification of the method of Weiss et al. (6). Radioactive aminoacyl-tRNA’s, to- gether with 5 mg of carrier unlabeled tRNA’s (9), were applied to a column FRACTION NO, Fig. 1. Elution profiles of aminoacyl-tRNA’s of blood cells from embryonic and adult chickens on MAK columns. Dotted lines, optical density at 260 mu; solid lines, radioactivity. Amino acids labeled with “C were used in the preparation of all aminoacyl-tRNA’s. exception of the methionyl-tRNA, which gave a clear alteration in the methionyl-tRNA adult and the embryonic methionyltRNA’s gave two resolvable peaks, the proportions of the two peaks differed. The ratio of the peaks was 1.3 for the embryonic cells and 2.7 for the adult cells. These results were similar to those found with the MAK column. In addition, the two peaks for the em- change during development in the pro- third peak was observed, but its significance is doubtful. elution profiles, there were no signifi- cant differences in the profiles between the aminoacyl-tRNA’s from the two sources. Under the present chromatographic conditions, both the adult- and the embryonic-cell showed two major peaks in MAK chromatography. However, a dramatic portion of methionyl-tRNA in the two major peaks was observed. The ratio of the areas under the peaks which was 1.1 in the embryonic cells shifted to 3.8 in the adult cells. Figure 2 shows the elution patterns of the aminoacyl-tRNA’s on the freon column. The better resolution of this chromatographic system revealed the presence of multiple peaks for several of the amino acids tested leucine, and methionine). No significant difference was observed between the elution patterns of the embryonic and adult cells of the amino acids minute intervals. Optical density was measured at 260 my» on alternate fractions. A sample (1 ml) of each fraction was then mixed with 10 ml of Bray’s solution (J0), and the radioactivity was measured in an Ansitron liquid-scintillation coun- However, the elution profiles of the ter. Aminoacyl-tRNA’s isolated from erythrocytes of 4-day-old chick em- bryos and from reticulocytes of adult chickens were compared first by chro- matography on the MAK column and then on the freon column. The specific tRNA’s examined cine, methionine, Typical results of graphs are shown 8 DECEMBER 1967 were: arginine, leuserine, and tyrosine. the MAK chromatoin Fig. 1. With the The chromatograph of leucyl-tRNA from avian reticulocytes differed strikingly from that from E. coli B on both (arginine, (0.5 by 250 cm). Elution was effected with a concave NaCl gradient contain- ing 0.01M sodium acetate and 0.01M MgCl, (pH 4.5) at room temperature. Fractions (2 ml) were collected at 12- bryonic cells were further apart; a small arginine, leucine, serine, and tyrosine. methionyl-tRNA’s from cells of the two different developmental stages showed a striking alteration. Although both the 0.30 }- ~ = 2 wo N or a = o & 030 4200 Fig. 2 (right). Elution profiles of aminoacyltRNA’s on freon columns. Avian amino- C1C pe The following samples were prepared with “C-labeled amino acids: arg (A), arg (E), leu (E), met (A), ser (E), and tyr (A), ozs; SER tar acyl-tRNA’s were prepared as described. whereas leu (A), met (E), ser (A}, and tyr (E) were acylated with *H-labeled amino acids. Escherichia coli leucyl-tRNA was prepared as described by Kano-Sueocka and Sueoka (2). Dotted lines, optical density at 260 mu: solid lines, radioactivity. (In the double-labeling experiments, “C is shown by a solid line, and *H is shown by a dashed line.) o 1 Ra [i 15 be i ooste | ; a 100 opsTYRIAE) i i i 4 PN EA, [SERED 25 —7200 cls —ico | —s0 200 , (~ 100 200 > |ee 200. eg —j!00 ‘ f aosi J (|i Sf © as iS La00 |; ' | —— 4 | | oes: ‘: 200 ¢ 100 eh en,{50 | 100 200 FRACTION NO. 1331