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Figure 1. The Patterns of Protein Synthesis in Eggs before and after Activation
?S-methionrne was added at a final concentration of 30 &i/ml to 15 ml of a suspension of 20,000 unfertilized eggs per milliliter. After 5 min, portrons of this
suspension were fertilized, or made 10 pM in A23187 by addrtion of IO mM stock in drmethylsulfoxide. or made 10 mM in NH, Cl by addition of 1 M stock.
Samples were taken for analysrs at 10 mm intervals starting at 25 min (lanes a) until 115 min (lanes j) after addition of the activator. Samples (lanes k) were
taken at 127 min. The last two lanes labeled Uk and Fk are lighter exposures of the last unfedilrzed and fertilized lanes, respectively. The exposure of the
autoradiograph in the unfertilrzed panel was four times longer than the other three to compensate for the low level of incorporation before fertilization. Bands
X, Y, and Z are marked as examples of polypeptides whose synthesis IS reduced after fertrlization; and A, B. and C as the major nonhrstone proterns whose
synthesis is activated.
ing change is the appearance of the prominent new bands however, all the proteins are less strongly synthesized than
A, B, and C after fertilization or activation with A23187, in activations that involve release of intracellular calcium.
much as happens in Spisula (Rosenthal et al., 1980).
The Synthesis of Some Proteins Stops after
However, closer inspection reveals other interesting fea-
Fertilization
tures, of which the most unexpected is the behavior of
Several proteins, like the examples labeled X, Y, and Z on
protein A, which we shall call “cyclin” henceforth. It is the
Figure 1, are made in the unfertilized egg but are very hard
most strongly labeled protein at early times after fertiliza-
to detect after fertilization, though they continue to be
tion, but by 85 min after fertilization (lane g, fertilized) it
made in ammonia-activated eggs. These comparisons are
has almost disappeared. It gets stronger again in lanes h
most easily made by examining the last three lanes of
and i, only to decline again in lane k. These oscillations in
Figure 1, in which light exposures of the final lanes (k) from
the level of cyclin are extremely reproducible, as can be
unfertilized (U) and fertilized (F) eggs are matched with the
seen in Figures 2, 3, and 6, which show similar behavior
final ammonia-activated lane. The decline in synthesis in
in different batches of fertilized Arbacia eggs.
prefertilization bands is easier to see in Figure 7, which
shows a similar experiment in Lytechinus pictus.
Cyclin Does Not Oscillate after Parthenogenetic
Activation Cyclin Is Destroyed Periodically at a Particular
Although cyclin synthesis is strongly induced by A23187 Point in the Cell Cycle
or NH&I treatment of unfertilized eggs, under neither of Figure 2 correlates the oscillations in the level of cyclin with
these circumstances does its level oscillate in the same the cleavage cycle. The experiment was conducted in the
way as in fertilized eggs. This is probably because the same way as the previous one, except that additional
eggs do not divide, a point enlarged upon below. samples from the suspension of fertilized eggs were fixed
in 1% glutaraldehyde for later examination under the mi-
Ammonia Activation Does Not Turn On the croscope. The dashed line in Figure 2 denotes the cleav-
Synthesis of All the Proteins age index, measured as described in Experimental Pro-
As mentioned above, activation of eggs with weak bases cedures. The other two curves show the relative intensities
like NH4+ causes only about half the stimulation of protein of cyclin and protein B, as determined by densitometry of
synthesis that A231 87 or fertilization gives. Is this because the autoradiographs. Label accumulates more or less lin-
the synthesis of all the proteins is half turned on, or early in protein B, whereas cyclin (band A) falls precipi-
because half the proteins are fully turned on? Figure 1 tously at the onset of cleavage, only to rise and fall again
shows that protein B did not appear after ammonia acti- during the second cell cycle. Label in protein C also rose
vation, though it is one of the most strongly labeled in linearly, but the data have been omitted from this figure
fertilized and ionophore-activated eggs. For the most part, for clarity of presentation.
Maternal mRNA and Mrtosis
391
Continuous FulSes
abcdefgh mabcdefghi
0 1 2 hours
1
Figure 2. Correlatron of the Level of Cyclin with the Cell Divrsron Cycle
A suspension of eggs was fertrlrzed, and after 6 mm, %S-methionine was
added to a final concentration of 25 pCr/ml. Samples were taken for
analysrs on gels at 10 min intervals, starting at 16 min after fertilization. Figure 3. Comparison between Continuous and Pulse-Labeled Embryos
Samples were taken 20-30 set later into 1% glutaraldehyde I” calcium- A batch of eggs was fertiltzed and splrt into two. The frrst 2 ml (30,ooO
free anifrcral seawater for later mrcroscoprc examination; the cleavage Index eggs) had Y&h volume of labeled methronine added 5 min after the sperm,
IS shown thus: O----El. The autoradiograph shown as an Inset was scanned and samples for analysrs on gels were taken at 10 mm intervals thereafter.
to yield the data plotted thus: cyclin, U; protern B, A. .A. The other batch was allowed to develop, and every 10 mm a sample of 0.1
ml was added to a tube containing 1 pl of 35S-methronine. mrxed, and
Incubated for IO mm before adding 0.2 ml 25% TCA. The letters above
The Cyclical Fluctuation in Cyclin Are Due to each lane correspond to the equrvalent endpornts. (a) continuous label for
Periodic Destruction and Continuous Synthesis 25 mm, pulse from 15 to 25 mm; (b) 35 mm continuous, 25-35 mm pulse;
It seemed most likely that the oscillation in the level of (c) 45; (d) 55; (e) 65; (1) 75; (g) 85; (h) 95: (i) 105 min. m: markers. Cleavage
cyclin was achieved by continuous synthesis punctuated occurred between lanes e and f
123456
116'
68-,
55--
42-
0 30 60 90 min
Figure 4. Rates of Syntheses of Three Proteins during the Frrst 2 h of
Cleavage .”
The relatrve intensitres of cyclin, protein C. and the putatrve hrstone band
were determined from the autoradrograph shown rn the right panel of Figure Frgure 5. Patterns of Syntheses at Later Stages of Development, Showing
3 by densitometry. the Disappearance of Cyclrn after Addrng Emetine
This figure shows the contrnuatron of the experiment described in Figure 3;
lane 1 is rn fact a second sample of lane c of that figure, 45 min after
stage). After the first three divisions, the synchrony of cell fertrlizatron and 40 min with !%-methionrne. Lane 2 was sampled 3 hr 21
division is lost (Harvey, 1956) and continuous labeling did min after fertrlization, wrth 55 min exposure to label; lane 3 represents the
not show the oscillations in cyclin levels seen during the same batch of embryos 30 min later, having been exposed to lOA M
first three cleavage divisions (data not shown). However, emetine during that trme. The next sample was taken 4 hr 51 min after
fertlizatron, the last 37 mm of that trme with label present. Lane 5 and 6
it seemed likely that cyclin was still being periodically were sampled at IO hr 14 min and 10 hr 44 mtn. respectively, after 45 mm
destroyed. To detect this, we blocked protein synthesis and 75 mm exposures to %-methronine. The last 30 min of lane 6 incubation
with emetine, and took samples 30 min later for analysis had IO4 M emetine present.
on gels. Cyclin disappeared from embryos exposed to the
inhibitor, as comparison of lanes 2 and 3 shows. Cyclin is NH&I, suggesting that cell division is necessary for its
still synthesized at a high rate at 5 h (lane 4) more than disappearance. Figure 6 shows that when eggs are fertil-
halfway through cleavage, but is hard to detect at 10 h, ized, but cell division is blocked by cytochalasin D or
by which time the rate of cell division has slowed consid- colchicine, the pattern of disappearance of cyclin is altered,
erably and the embryo is at the midblastula transition so that instead of its intensity suddenly declining at 60-70
(Newport and Kirschner, 1982; Wilson, 1896; Hinegardner, min, as in the controls, it disappears much later and much
1967; O’Melia, 1983). Careful inspection of lanes 5 and 6 more slowly. The significance of this slow degradation is
is required to detect the synthesis of cyclin at this stage, unclear. Both drugs allowed the normal activation of protein
for the band is only faintly visible. However, it disappears synthesis. Taxol had exactly the same effect (data not
after exposure to emetine, showing that it is still being shown). It seems that the rapid disappearance of cyclin
made and cycling. In contrast, proteins 6 and C are still depends on some aspect of normal cleavage. Whether
being synthesized and appear quite strongly at this time. the converse is true-i.e., that disappearance of cyclin is
essential for cell division-we cannot say.
Cytochalasin, Colchicine, and Taxol All Slow Down
the Disappearance of Cyclin There Are Two Cyclins in Lytechinus pictus
We have already seen that cyclin does not cycle normally We have looked for cyclin-like proteins in two other marine
when protein synthesis has been activated by A23187 or organisms; Lytechinus pictus, a California sea urchin that
Maternal mRNA and MITOSIS
393
diverged from Arbacia punctulata about 30 million years destroyed. But there are also significant differences from
ago; and a mollusc, the clam Spisula solidissima, which is Arbacia. First, Lytechinus has not one but two prominent
phylogenetically very distant from the echinoderms. Figure cyclins, and they oscillate in different phases. Moreover,
7 shows an experiment with Lytechinus performed along the lower molecular weight of the two cyclins shows
the same lines as Figure 1. In general terms, the results oscillatory behavior after ammonia activation, something
resemble those from Arbacia, in that a striking change in we did not see in Arbacia.
the pattern of protein synthesis occurs after fertilization,
and that some of the newly made proteins are cyclically Proteins A and B in Spisula Are Cyclins
As in Lytechinus, two proteins in Spisula showed oscilla-
tions correlated with the cell cycle (Figure 8); they are the
Cytochalasin proteins referred to as A and B by Rosenthal et al. (1980).
These proteins are scarce in unfertilized oocytes,
and their synthesis increases very abruptly after fertiliza-
1234567 8 9 10 11 12
tion; virtually all of the mRNA for protein A becomes
associated with polysomes by first cleavage (E. T. R.,
unpublished observations).
Discussion
21
Cell
394
man and Smith, 1978). Furthermore, MPF requires protein mm longer to undergo frrst cleavage than unlabeled controls. In one
synthesis for its formation, and presumably is a protein experiment not shown here we tried to use 3H-lysine, which completely
blocked cleavage. Evrdently one should be careful of the label in these
(Gerhart, 1980). The parallels between the behavior of MPF experiments; we do not know whether the delay in divrsion caused by the
and cyclin are striking, but whether there is a direct corre- methronine was due to radratron damage (to which eggs are known to be
spondence between the physiological entity and the chem- highly susceptible) or to the acetate and mercaptoethanol rn the solutron.
ical one remains to be determined. A preliminary test suggested that neither of these unlabeled compounds
alone slowed division at the concentrations resulting from their presence in
Experimental Procedures the labeled solution as supplied, but that the combinatron might be dam-
aging.
Materials It should also be mentioned that the overall krnetics of incorporation of
Arbacra punctulata and Spisula solidissima were obtarned from the Depart- methionrne into TCA-precipitable material were not b-rear, owing to the fact
ment of Marine Resources of the Marine Biological Laboratory Woods that by 2 hr of incubatron well over 50% of the label had been Incorporated
Hole, and kept In runnrng seawater at ambient temperature (about 20°C), Into protein. This IS very like the situation reported by Ecker and Smith
whereas Lytechinus pictus were obtained from Pacific Bromarine, Inc, (1966) rn frogs’ eggs; such eggs rely on stored yolk proteins for amino
Venice, Californra, and kept in chilled seawater tanks at about 15°C. acrds, whose pools are very small and constantly replenished from the
Gametes were obtarned from Arbacia by 12 V AC electrical stimulation, stores. This means that the specrfic activity of the methionine pool declines
from Sprsula by dissectron, and from Lytechinus by injection of 0.5 M KCI. during the course of the exparrments. We do not think thus affects the
?S-methronine was the generous grft of Amersham Corp. It was their rnterpretatron of our results, but it should be borne in mind.
SJ204. whrch contains 2.5 mM K acetate and 14 mM 2-mercaptoethanol. It
was drluted with water to a concentrabon of 5 mCi/ml and stored as small Sample Preparation and Analysis
alrquots at -70°C. Samples of 50 ~1 were withdrawn from the incubatron vessels with a Gilson
Rat monoclonal antr-yeast tubulrn antibody YOL1/34 coupled to Sepha- Pipetman whose tips were cut back about 1 mm to enlarge the holes. The
rose 48 was the generous gift of John Kilmartrn (Kilmartin et al , 1982). samples were delivered into tubes containing 100 pl of 25% w/v TCA. The
Radioactrvely labeled proteins for molecular weight standards were precrpitates were harvested by centrifugation, washed twice wrth acetone,
prepared from a mixture, each at 1 mg/ml, of purified proterns dissolved In drred under vacuum, and dissolved rn 30 pl of SDS-gel sample buffer.
water: cytochrome c, RNAase A, soybean trypsrn inhibitor, carbonrc anhy- Acrylamrde gels 0.8 mm thrck were run according to the formulas of
drase, glyceraldehyde 3 phosphate dehydrogenase, creatrne krnase. actin, Anderson et al. (1973). Each lane 2-3 mm wade, was loaded with IO-15
glutamate dehydrogenase, bovine serum albumin, and beta galactosrdase. pl, correspondrng to about 100-200 embryos.
This mixture was incubated at 50°C with 0.16 M Tris-Cl. 5 mM dithioerythritol The gels were stained with 0.5% Coomassie blue R250 in 45% methanol,
(pH 8.8) for 2 mm and then precipitated with 12.5% w/v tricholoroacettc 45% water, 10% acetic acrd; destained In 20% methanol, 75% water, 5%
acrd. The precrpitate was taken up rn 1 M HEPES, 2 mM EDTA, 1.5 mM acetrc acrd; and dried onto Whatman 3MM paper. The dried gels were
“C-N-ethylmalermrde, specific activrty 23.7 mCi/mmol (New England Nu- autoradrographed wrth Fuj Rx film.
clear), pH 7.4. and Incubated at 20°C for 30 min. It was then diluted tn SDS The autoradrographs were scanned with a Transrdyne 2955 scanning
gel sample buffer to a concentratron of about 0.1 mg/ml for each compo- densrtometer connected via an Interactive Microware Inc. 12.brt analog
nent (the exact dilution was judged by running serial dilutions on a gel) and drgrtal converter to an Apple II microcomputer. Thus permitted Integration of
stored at -70°C rn small aliquots. A parallel sample was Incubated directly peaks by a procedure formally analogous to cutkng and werghrng.
wrth ‘C-N-ethylmalermide (i.e., wrthout the reductant and wrthout TCA
precipitatron) at pH 7.4. This gave good labeling of all proterns except for Acknowledgments
RNAase and soybean trypsin inhibitor, whrch do not have free-SH groups.
Carbonic anhydrase appears not to contain any reactive groups at all. The The experiments in this paper were done during the 1982 Physiology
labeled markers in the figures of thus paper contained a mixture of these Course at the Marine Biological Laboratory Thanks go to Joel Rosenbaum,
two preparatrons. Lawrence Hobble, Martha Rerd, Steve Barnard, Julre Sternberg, and espe-
crally the permanent staff of the MEL, who drd more than anyone to make
Preparation and Incubation of Eggs and Embryos this possible. We are grateful to John Gerhart John Kilmartrn, Roger
Eggs were washed by repeated settling through Millipore-filtered seawater. Sloboda. and David Begg for crucral discussions and practrcal advrce.
They were suspended at a final density of 15,000-20.000 eggs/ml, and This work was supported by NIH trarnrng grant GM-31 136-04 to the
incubated rn 20 ml glass scrntillation veals standtng in shallow dishes of Physiology Course. E. T R was supported by Natronal Scrence Foundation
water perched In Styrofoam boxes to provrde a constant temperature grant PCM 7923046 to Dr. Joan Rudenan.
environment at 20°C; ice was added occasionally rf necessary. The vrals The costs of pubkcatron of thus arkcle were defrayed rn pan by the
usually contarned 2 ml of egg or embryo suspension at the start, and were payment of page charges Thus artrcle must therefore be hereby marked
shaken gently before sampling. More than 90% of the fertilized eggs rarsed “advertrsement” rn accordance wrth 18 U.S.C Sectron 1734 solely to
fertrltzatron membranes and showed normal cleavage in all the experiments rndrcate this fact.
described in thus paper.
Received January 27, 1983; revrsed March 10, 1983
The Cleavage Index
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