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.)
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