/. Embryo! e.vp. Morph. Vol. 30, 3, pp. 647-659, 1973
Printed in Great Britain
647
Changes in protein during development
of Triturus embryos
I. Contents and syntheses of soluble or basic
protein of cell components
By HIROSHI IMOH1 AND TSUTOMU MINAMIDANI1
From the Department of Biology, Miyazaki University
SUMMARY
The present paper reports basic data on DNA content, protein content, and protein
synthesis in Trituruspyrrhogaster embryos during development from cleavage to the hatching
stage. Except for measurements of DNA and total protein contents, embryos were labeled
with sodium carbonate-14C for 10 h and fractionated into embryonic cell components, i.e.
cytoplasmic mass, yolk and pigment granules, and nuclei, in a discontinuous density gradient
of sucrose. The protein content and the radioactivity incorporated into protein were measured
in each fraction. Those fractions combining protein soluble in buffer at pH 8-3 and in 025
N-HCI were further studied with polyacrylamide gel electrophoresis.
In the newt embryo, four stages of active DNA increase were observed when cultured at
constant temperature; they were gastrula, neurula, late tail-bud, and before-hatching stages.
Total protein per embryo decreased from 3 to 2 mg during the development studied. The
content of cytoplasmic soluble protein per embryo was low and constant throughout development. Synthesis of the fraction was observed at the earliest stage of development studied
though the rate was not high and specific activity of the soluble protein increased during
development. Qualitative changes in the newly synthesized protein were observed. With the
yolk fraction, synthesis of protein, other than from probable contamination with the cytoplasmic fraction, was not detected and a detailed description was omitted.
Changes were observed at two stages of development in the synthesis of nuclear protein
soluble in buffer at pH 83, the first at gastrulation and the second at late tail-bud stage. The
change at gastrulation seemed to be the start of syntheses of the nuclear soluble proteins,
while quantitative enhancement rather than qualitative change was noticed at late tail-bud
stage. Most of the nuclear protein soluble in 0-25 N-HCI was histone. The histone content
increased in accordance with increase in the DNA content and the rate of DNA accumulation
was accompanied by proportionate incorporation of radioactivity into histone. Among
histone fractions, unique behaviour of the very lysine-rich histone was observed.
The availability of [14C]sodium carbonate in rough estimations of protein synthesis in
embryos and significance of the data obtained have been discussed.
INTRODUCTION
Syntheses of proteins are some of the most fundamental events in the development of embryos and various proteins with their unique functions are
synthesized and degraded. This program, which differs for each organism, is
determined genetically and is carried out through intervention of messenger
RNAs. Though there can be no doubt that studies on regulation mechanisms of
1
Author's address: Department of Biology, Miyazaki University, Miyazaki 880, Japan.
42
niii
30
648
H. IMOH AND T. MINAMIDANI
protein synthesis in developing embryos, including regulation mechanisms of
gene transcription, are of utmost importance and that the studies have solved
not a few problems in developmental biology (see Davidson, 1968), it would be
useful to know the gross program of protein synthesis; that is, when and what
kind of protein is to be synthesized during development of the embryo, to
understand development on a molecular basis.
Concerning protein synthesis in amphibian embryos, soluble and pellet
protein in Rana (Brown & Caston, 1962) and cytoplasmic and nuclear protein
in Rana (Ecker & Smith, 1971) have been reported among others. The present
report is an analysis of protein synthesis in Triturus embryos during development.
MATERIAL AND METHODS
Embryos of Triturus pyrrhogaster (BOIE) were used throughout the experiments. They were cultured at 21 ± 1°C and staged according to the tables of
Okada & Ichikawa (1947).
(a) Labeling of embryos andfractionation of embryonic cell components
Preparation of embryos for labeling and the detailed procedures of labeling
have been reported elsewhere (Cohen, 1954; Imoh, Sasaki, Kawakami &
Hayashi, 1972). Embryos in various developmental stages were labeled together
in the same 50 ml vial with 100 fid of Na214CO3 (2-0 mCi/ml, the Radiochemical
Centre, Amersham) for 10 h and, at the end of labeling, they were washed with
Holtfreter's solution and grouped according to their developmental stages. The
stages and numbers of embryos are shown in Table 1. The availability of the
precursor in the study of protein synthesis in embryos during development is
considered in the Discussion.
Labeled embryos were homogenized with 10 ml of 0-88 M sucrose in Tris-HCl
(pH 7-2) containing 3 mM-CaCl2 in a glass homogenizer provided with a Teflon
pestle (clearance 0-08 mm) at 600 rev/min. The homogenate was floated on
two layers of sucrose solution, 14 ml of 1-8 M on 16 ml of 2-1 M, in a centrifugation tube. Each sucrose solution was prepared with 10 HIM Tris-HCl (pH 7-2)
containing 3 mM-CaCl2. After centrifugation in a Spinco SW 25 rotor at 23000
rev/min for 90min, embryonic cell components were recovered from each
density boundary or the bottom; nuclei were obtained from the bottom, yolk
and pigment granules from the boundary between the 1-8 and the 2-1 M sucrose
layer, and most of the remaining cytoplasmic mass from the boundary between
the 0-88 and the 1-88 M layer. The nuclei were almost free from contamination
by cytoplasmic or yolk material, although the latter was a little contaminated
with nuclei because of a few intact cells (Imoh & Negami, 1972).
The DNA content of embryos during development was measured by the
diphenylamine method (Burton, 1956) with the sample prepared by the procedures of Schmidt & Thannhauser (1945) from homogenate of whole unlabeled
embryos or with the nuclei isolated and freed from histones (Imoh & Kawakami,
Changes in protein during development
649
Table 1. Stages and number of embryos labeled
Stages before label*
Stages after labelt
Number of embryos
9-10
72
61
62
62
58
50
44
24
25
22
20
15
40
5-8
11
12a
12b
12c
14-15
16-17
16-17
18-19
19
21
24-25
25-26
26-27
27-28
29-30
28
31
33
35
36
32
34
35
36
* Embryos were staged according to Okada & Ichikawa (1947).
t Stages at the end of labeling are shown and these stages are referred to in the text;
9-10 blastula, 12b or 12c gastrula, 16-17 neurula, 26-27 tail-bud, 29-30 late tail-bud, 32
balancer developing stage, 34 the second foreleg stage, and 36 the third foreleg stage.
1973). About 90% of DNA in the homogenate was recovered in the nuclei
fraction.
(b) Measurement of content and synthesis of protein
The protein content of whole embryos was determined by the method of
Lowry, Rosebrough, Farr & Randall (1951) with a sample prepared by the
procedures of Schmidt & Thannhauser (1945) from homogenate of whole
unlabeled embryos.
The isolated cytoplasmic material from the homogenate of labeled embryos
was diluted with Tris-HCl (pH 7-2) and then ethanol was added to the sample
to make a final concentration of 67 %. This was then centrifuged at 6000 rev/
min for 10 min. The resulting precipitate was extracted with Tris-HCl (pH 8-3)
for 2 h in the cold and centrifuged at 10000 rev/min for 10 min to remove
unextracted components. Perchloric acid (PCA) was added to an approximately
equal aliquot of the supernatant, so as to bring the final concentration of PCA to
0-5 N; this was then kept at 90 °C for 20 min. The protein content of the sample
was determined by the method of Lowry et al. (1951). The same volume of the
sample was processed by the same procedures and the protein precipitate
produced by the PCA treatment was caught on Millipore filter and dried.
Toluene-based scintilation fluid was added for counting in a liquid scintilation
counter (Packard TRI CARB). The remainder of the extract of cytoplasmic
protein was subjected to electrophoresis on polyacrylamide gel.
The yolk fraction was diluted about fivefold with the buffer (Tris-HCl, pH
7-2) to reduce the sucrose concentration and centrifuged at 10000 rev/min for
10 min. The precipitate was extracted with Tris-HCl (pH 8-3) for 2 h in the cold
42-2
650
H. IMOH AND T. MINAMIDANI
and centrifuged for 10 min at 10000 rev/min. The extract was analysed by the
same procedure as for the cytoplasmic soluble protein and the precipitate was
extracted with 0-25 N HCl for 2 h in the cold. The acid-soluble protein was
analysed as above, except that the conditions of electrophoresis differed.
The isolated nuclei were washed briefly with Tris-HCl (pH 7-2) and extracted
with Tris-HCl (pH 83) for 2 h in the cold. The supernatant was analysed as
cytoplasmic soluble protein and the precipitate was extracted with 0-25 N-HC1
for 2 h in the cold and was analysed by the same procedure as for yolk acid
soluble protein.
(c) Gel electrophoresis of soluble and acid soluble protein
The electrophoresis of the protein soluble in buffer at pH 8*3 was performed
on 7-5% polyacrylamide gel by the method of Williams & Reisfeld cited by
Nagai (1966). Electrophoresis was conducted with current of 4 mA per gel until
the tracking dye, bromphenol blue, had run 5-0 cm. At the termination of
electrophoresis the gel was stained with amidoblack 10B solution for several
hours and destained electrically. The gel was sliced into disks 1 mm thick and
these were liquefied by treatment with 35 % H 2 O 2 at 60 °C for 1 h. To the lysate
were added ethanol and the toluene-based scintilation fluid for counting in a
liquid scintilation counter. The results were reproducible.
For fractionation of the protein soluble in 0-25 N-HC1, 15 % polyacrylamide
gel electrophoresis was used according to the method of Shepherd & Gurley
(1966) with a glycine buffer (pH 4-0) instead of the valine buffer. After electrophoresis the gel was stained with amidoblack 10 B solution for 12 h and destained electrically. The optical density of the stained protein bands was
recorded with a densitometer (Fujiriken, FD-A4). The quantity of protein in a
band was calculated from the densitometer trace with reference curves which
had been made with known amounts of protein. Radioactivities in the protein
bands were determined in the same way as with the protein soluble in buffer at
pH 8-3. The characterization of the acid-soluble protein from nuclei has been
reported elsewhere (Imoh & Kawakami, 1973).
RESULTS
(a) Changes in contents of DNA and protein during development
The changes in DNA content per embryo during development are shown in
Fig. 1. There were four stages where the increase of DNA was greater than at
other stages when cultured at constant temperature; this was recognized by
calculating the rate of DNA accumulation (Fig. 1, dotted line). They were
gastrulation (st. 12), middle neurulation (st. 17), late tail-bud formation (sts.
29-30), and late foreleg (sts. 37-39) stages.
Fig. 2 shows changes in the content of total or fractions of protein per embryo
during development. Total protein was about 2 mg/embryo at the foreleg stage.
Because of ethanol soluble lipoprotein, which had been removed in the procedure
651
Changes in protein during development
20 -
6
8
Davs
10
4
12
i
10 12 17 25 29 32 34
Stage
Fig. 1
36
6
8
Days
i
i
i
10
i
1012 17 25 29 32 34
Stage
12
i
36
Fig. 2
Fig. 1. Content of DNA and rate of DNA increase per embryo. From groups of newt
embryos cultured at 21 ± 1 °C. An appropriate number of embryos at a given stage
selected, homogenized, and processed by the method of Schmidt & Thannhauser
(1945). The DNA content of the sample was determined by the diphenylamine
method (Burton, 1965). From the data, /tg DNA per embryo (O
O) and the
rate of DNA accumulation per embryo, i.e./tg DNA increase per 12 h per embryo
(
), were calculated.
Fig. 2. Changes in the contents of protein fractions during development. Protein
contents of whole embryo and of cell components isolated by the sucrose density
gradient were determined at various stages of development. Notation: total protein
in mg embryo (A
A) and cytoplasmic soluble protein (><••• x), nuclear
soluble protein ( #
# ) , and nuclear acid-soluble protein in fig per embryo
(O— -O).
of determination, the data^were lower than the true value by about 1 mg/
embryo. The content of cytoplasmic soluble protein was low and constant
throughout^development; soluble protein obtained from the yolk fraction,
which is not shown in the figure, was lower than cytoplasmic soluble protein.
Nuclear protein soluble in buffer at pH 8-3 was low before the tail-bud stage,
increased rapidly at tail-bud stage, and remained almost constant thereafter.
The nuclear protein soluble in 0-25 N-HC1, most of which was histone, increased
throughout development in a pattern quite similar with that of the DNA
content.
(b) Quantitative changes in the syntheses of protein fractions
Changes in the rate of radioisotope incorporation into protein fractions per
embryo and in the specific activities of protein fractions during development are
652
H. IMOH AND T. MINAMIDANI
1000 -
2
4
6
8
10 12 17 25 29 32 34
Stage
Fig. 3
10
12
36
6
8
Days
1012 17 2529 32 34
10
12
36
Stage
Fig. 4
Fig. 3. Radioactivities incorporated into protein fractions. Embryos were labelled
with 14CO2 for 10 h and homogenized. The cell components were isolated and the
protein fractions were extracted from them. Radioactivities in the protein fractions
were measured and calculated on a per embryo basis. Notation: cytoplasmic soluble
protein (x •• • x), nuclear soluble protein ( #
# ) , and nuclear acid-soluble
protein in cpm per embryo (O
O).
Fig. 4. Changes in the specific activities of protein fractions during development.
The specific activities of protein fractions were calculated from the data shown in Fig.
2 and Fig. 3. Notation is the same as in Fig. 3.
represented in Figs. 3 and 4, respectively. The rate of cytoplasmic soluble
protein synthesis per embryo gradually increased during development and the
rate of soluble protein synthesis from the yolk fraction showed the same pattern
as cytoplasmic soluble protein synthesis, though much lower and not shown in
the figure, suggesting that soluble protein from the yolk fraction was contamination with cytoplasmic soluble protein. Synthesis of HC1 soluble protein in the
yolk fraction was not detected. Nuclear soluble protein synthesis was not
detected at the blastula stage, became positive at gastrula stage, and was
enhanced at the late tail-bud stage. It may be noted that the rate of nuclear acidsoluble (basic) protein synthesis was greater, as was the rate of DNA accumulation, at gastrula, neurula and tail-bud stages than at other stages. The specific
activity of acid-soluble protein from nuclei was very high at the gastrula stage
and decreased thereafter, while that of cytoplasmic soluble protein increased
throughout development with a temporal decrease at the late neurula stage, and
that of nuclear soluble protein was roughly constant after the gastrula stage
(Fig. 4).
Changes in protein during development
i
i
i
i
i
i
653
i
)
ICtlV lty (cpm)
100 -
b
300
-
~((t)l
>
.
200 -
1
100 -
A
i
10 20 30 40
10 20 30 40
Slice number
10 20 30 40
10 20 30 40
Slice number
10 20 30 40
Fig. 5. Polyacrylamide gel separation of radioactivities incorporated into cytoplasmic
soluble protein. About 10 /ig of cytoplasmic soluble protein from labeled embryos
was subjected to electrophoresis in 7-5 % polyacrylamide gel. After electrophoresis,
gel was stained with amidoblack, destained electrically, and sliced into 1 mm disks.
The slices were liquefied and radioactivities in them were measured. The sample gel
was at the side of slice number zero, (a) Blastula, sts. 9-10; (b) gastrula, st. 12; (c)
neurula, sts. 16-17; (d) tail-bud stage, sts. 26-27; (e) balancer developing stage-c,
st. 32; (/) the second foreleg stage-a, 34; (g) the third foreleg stage-a, st. 36.
(c) Qualitative changes in the newly synthesized soluble protein fractions
In Fig. 5 are shown electrophoretic patterns of the radioactivity incorporated
into the cytoplasmic soluble protein at several stages of development. As about
10 //g of soluble protein was applied to each gel, the patterns were comparable
to each other. At the blastula stage protein synthesis occurred, but only small
amounts of newly synthesized protein were found in the protein applied to the
654
H. IMOH AND T. MINAMIDANI
1
100
1
1
1
i
i
i
i
i
-
i
i
r
so
1. 300
~. 40
-
(f)
£ 20
% 200
•+-<**
\ -"V
-
J SO
|
^
100
60
40
1 •
10 2(1 30 40
]() 20 30 40
Slice number
Fig. 6
20
2 3 4
1 2
3 4
l-lectrophoreiie mobiliiv (cm)
Fig. 7
Fig. 6. Polyacrylamide gel separation of radioactivities incorporated into nuclear
soluble protein. About 10 /tg of soluble protein from nuclei was fractionated. For
details, see text or the legend of Fig. 5. (a) Blastula sts. 9-10; (6) gastrula st. 12; (c)
neurula sts. 16-17; (d) tail-bud sts. 26-27.
Fig. 7. Polyacrylamide gel separation of acid soluble protein from nuclei. The acidsoluble protein was extracted with 0-25 N-HCI from nuclei isolated from labeled
embryos and fractionated by 15 % polyacrylamide gel electrophoresis. The gel was
stained with amidoblack for 12 h, destained electrically, and traced with a densitometer. The amount of sample applied to the gel was about 005 ml and protein
content varied among samples, (a) Blastula sts. 9-10; (b) gastrula st. 12; (c) neurula
sts. 16-17; (rf) tail-bud sts. 26-27.
gel. The radioactivity incorporated in the protein increased with development
and at the tail-bud stage one fraction showed extremely high radioactivity (Fig.
5d), though it gradually decreased through later development (Fig. 5e-g). The
nature of the fraction has not been examined. Fig. 6 shows electrophoretic
patterns of newly synthesized nuclear soluble protein. Radioactivity incorporated into the protein at the blastula stage was not significantly above background
level. Incorporation into several protein fractions was evident after the gastrula
stage and the pattern was essentially identical with that of tail bud embryos in
the embryo beyond the tail-bud stage.
(d) Changes in contents and syntheses offractions of nuclear acid-soluble protein
Figs. 7 and 8 show electrophoretic patterns of nuclear protein soluble in 0-25
N - H C I , from embryos at four stages. In Fig. 7 traces of density patterns of the
stained gels are shown and the peaks observable between electrophoretic
mobilities of 1-5-3-3 cm arehistonefractions: very lysine-rich (fl), arginine- and
Changes in protein during development
655
1000 -
1000 -
500 -
10 20 30 40
10 20 30 40
Slice number
Fig. 8. Radioactivity in the nuclear acid soluble protein separated by polyacrylamide
gel electrophoresis. The gel used for preparation of Fig. 7 was sliced and radioactivities in the slices were measured. For details, see the legend for Fig. 7.
I
80
-
60
-
1
40
1
1
1
-
A
A
A
_
1
A
a
A
A
A A
A
-
-
X
20
n
~
i'f'
•%
X
#
x •
*
~
\
1
1
1
1
1
6
8
10
12
Days
I I
1012 17 25 29 32 34
Stage
36
10 12 17 25 29 32 34
36
Stage
Fig. 9. Ratio of each histone fraction to total histone. The content and radioactivity
in each histone fraction was measured from Fig. 7 and Fig. 8 respectively, and the
total histone content or the total radioactivity was determined as the sum of them.
The ratio of each histone fraction to the total was calculated in percent, {a) The
ratio in content; (b) the ratio in radioactivity. O
O, The very lysine-rich fl;
x
x, arginine and alanine-rich f3: A
A, two slightly lysine-rich f2b + f2a2:
#
# , arginine and glycine rich f2al.
656
H. IMOH AND T. MINAMIDANI
alanine-rich (f3), two slightly lysine-rich (lysine- and serine-rich f2b and
alanine- and leucine-rich f2a2) as one continuous peak, and arginine- and
glycine-rich (f2al) in the order of increasing mobility. It should be noted that fl
at the blastula stage consisted of two peaks and one of them with lower mobility
increased at the gastrula stage. The peak between fl and f3 has not been
identified. It should also be noticed that the mutual ratio between the three
peaks (f3, f2b + f2a2, and f2al) did not change much, depending on stage,
while the ratio of f 1 to the three peaks changed during development, being low
at blastula and tail bud stages and high at the neurula stage. This is shown more
accurately in Fig. 9 (a), which shows changes in the ratio of content of each
histone fraction to the total histone content during development. Though the
experimental values were considerably scattered, the ratio of f2b + f2a2, f2al,
or f3 could be regarded as constant during development. On the other hand,
the ratio of f 1 positively elevated at the neurula stage. Almost the same statements could be made about radioactivities incorporated into newly synthesized
histone fractions (Fig. 8, Fig. 9 b).
DISCUSSION
It is probable that permeability of embryonic cells to the radioactive precursor, 14CO2, may change during development and, furthermore, the ratelimiting steps in fixation of CO2 to amino acids are unknown. However, as noted
by Brown & Caston (1962), it may be assumed that once an amino acid has been
formed, it will be incorporated into the class of proteins being most actively
synthesized at that particular time, in the statistical sense. Thus, by using 14CO2
as precursor, a correct answer may be obtained concerning what class of proteins is more actively synthesized than others in the embryo, at a given stage of
development. This statement does not exclude the possible increase of radioisotope incorporation into all classes of proteins during development. Examination of the data on histone synthesis, however, suggests that the incorporation
of 14CO2 into the various protein classes can be used for rough comparison of
protein syntheses at different stages of development. The histone is synthesized
without any major qualitative changes in composition after gastrulation
(Kischer & Hnilica, 1967; Hnilica & Johnson, 1970) with a definite ratio to
newly synthesized DNA (Imoh & Kawakami, 1973), though histone synthesis
before gastrulation and synthesis of f 1 fraction during development are controversial (Vorobyev, Gineitis & Vinogradova, 1969; Asao, 1969). Therefore, a
major part of histone synthesis after gastrulation seems to take place in relation
to DNA synthesis and, in the present experiment, the rate of 14CO2 incorporation
into nuclear basic protein was roughly parallel to the rate of DNA accumulation
(compare Figs. 1 and 3). Accordingly, the rate of 14CO2 incorporation into
histone may roughly represent the true rate of histone synthesis. As the mechanisms of histone synthesis are the same as those of the synthesis of any other
Changes in protein during development
657
protein (Borun, ScharfT & Robbins, 1967; Kedes, Gross, Cognetti & Hunter,
1969; Nemar & Lindsay, 1969), it may be assumed that an approximately
correct estimate of the true rate of synthesis of each protein during development
would be obtained from determination of the rate of 14CO2 incorporation.
Cytoplasmic soluble protein was extracted from the cytoplasmic mass precipitated by ethanol. It is possible that not all the soluble protein was recovered
after ethanol precipitation because of denaturation, though our preliminary
study with polyacrylamide gel electrophoresis suggested that the qualitative
change in the protein fractions before or after ethanol precipitation was small.
The content of this fraction per embryo was almost constant throughout
development despite a tremendous increase in cell number. The result resembled
that of Brown & Caston (1962) on Rana soluble protein. The specific activity of
the fraction increased during development without accumulation of protein
content, suggesting a high rate of turnover. Localization to cytoplasm, solubility
in pH 8-3 buffer, and high rate of turnover suggested that this fraction was
composed of cytoplasmic enzymes. The fraction consisted of many protein
species. Their synthesis was found at the earliest stage of development studied
and one of them was synthesized at quite a high rate at the tail-bud stage though
its nature was unknown.
The nature of the yolk fraction protein soluble in buffer at pH 8-3 was identical
with that of cytoplasmic soluble protein; and acid-soluble protein of yolk
showed no incorporation of radioactivity.
In relation to nuclear protein soluble in buffer at pH 8-3, two stages of
development require comment. The first is gastrulation. At the blastula stage,
incorporation of radioactivity into the fraction was not found and no peak
relating to newly synthesized protein was observed in the electrophoretic
pattern. As incorporation of radioactivity into histone was fairly high and peaks
of the histone fractions were apparent in the electrophoretic pattern at the same
stage, the failure to detect radioactivity in the nuclear soluble protein could
not be attributed to deficiency of labeled amino acid and it must be concluded that synthesis of nuclear soluble protein at the blastula stage was nonexistent or very low. On the other hand, at the gastrula stage, incorporation of
radioactivity in the fraction and the specific activity of the fraction were higher
and several fairly high peaks were observed in the electrophoretic pattern. The
change at gastrulation thus seemed to be the start of synthesis of the fraction,
which might have some relation to the beginning of gene transcription occurring
at gastrulation in amphibian embryos (see Davidson, 1968). The second stage
to be noted is the tail-bud stage when the content of the fraction per embryo or
the rate of synthesis per embryo increased greatly. Electrophoretic analysis of
the newly synthesized protein suggests that the change was a quantitative enhancement of synthesis rather than a qualitative switching-over. The enhancement could be related to a probable increase in differentiating cells; but this
remains to be studied.
658
H. IMOH AND T. MINAMIDANI
Most of the nuclear protein soluble in 0-25 N-HCl was histone and it could be
fractionated into five major subfractions and identified (Johns & Butler, 1962;
Johns, 1967; Imoh & Kawakami, 1973). As discussed above, in rough approximation, the histone seemed to increase in relation to DNA increase. There are
a few points to be noticed, however. The very-lysine-rich, fl, fraction at early
stages of development consisted of a few subfractions. The heterogeneity of verylysine-rich histone reported in adult tissues (Nelson & Yunis, 1969; Panyim,
Bilek & Chalkley, 1971) seemed to have its origin in early development, though
examination of embryonic tissues by electrophoretic analysis with higher resolution will be needed. The ratio of very-lysine-rich histone to total histone, with
both content and incorporated radioactivity, changed during development; low
before gastrulation, high at neurulation, low again at late tail-bud formation,
and high again before hatching, while the ratios of other histone fractions to the
total histone were almost constant. The percentage of fl cannot be independent
from those of other histone fractions and the change of fl during development
would be emphasized by taking a fraction, e.g. f2b + f2a2, as unity, though
experimental fluctuation in the fraction taken as standard, e.g. f2b + f2a2, cannot be excluded. The significance of the f 1 change during development, especially
at early stages, is now under study.
Looking over the results, it may be noted that the rate of nuclear protein
synthesis exceeded largely that of cytoplasmic soluble protein, especially during
early development, and it was only after the tail-bud stage that the specific
activity of cytoplasmic soluble protein became higher than those of nuclear
proteins. Though insoluble protein has not been studied in the present experiments, the larger synthesis of nuclear protein than cytoplasmic protein observed
in the early development of fishes and echinoderms (Krigsgaber, Kostomarova,
Terekhova & Burakova, 1971) or in early cleavage of Rana (Ecker & Smith,
1971) may have some relation to the present observations.
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Changes in protein during development
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IMOH,
{Received 28 March 1973, revised 6 June 1973)