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Downloaded from symposium.cshlp.org on May 12, 2016 - Published by Cold Spring Harbor Laboratory Press Control of the Cell Cycle in Early Embryos J. RUDERMAN,* F. LUCA,* E. SHIBUYA,* K. GAVIN,* T. BOULTON,* AND M. COBBt *Department of Anatomy and Cell Biology, Harvard Medical School, Boston, Massachusetts 02115; tDepartment of Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas 75235 At the end of oogenesis, oocytes exit from the cell cycle and arrest at the G2/M border of meiosis I. In clams, fertilization provides the extracellular signal that breaks this cell cycle arrest and initiates a series of rapid cell division cycles. In this paper, we consider two aspects of this process: the rapid, one-time activation of a member of the ERK/MAP kinase family that appears to be a key player in breaking cell cycle arrest, and the repetitive role of cyclin accumulation and destruction in driving cycles of cdc2/28 activation and inactivation. Oocytes of marine invertebrates provide excellent material for identifying some of the molecules involved in regulating the cell cycle. The full-grown oocytes contain large stockpiles of virtually all the enzymes and structural proteins needed to sustain the rapid, postfertilization cleavage division cycles, yet they remain arrested. Their responses to fertilization are abrupt, synchronous and, most important, dramatically exaggerated. This makes it easier to spot important regulatory components, ones that are often barely detectable in tissue culture cells. Indeed, the cyclins were first seen in clam embryos, where their synthesis is turned on strongly within minutes of fertilization (Rosenthal et al. 1980). The discovery that their levels oscillated across the cell cycle (Evans et al. 1983) focused attention on potential cell cycle roles for these proteins. Early work established that the rise and fall in cyclin levels in the embryonic cell cycles are due to continuous synthesis followed by a very brief interval of selective proteolysis (Swenson et al. 1986; Standart et al. 1987; Minshull et al. 1989a; Hunt et al. 1992). The levels of cyclin A and cyclin B show offset kinetics of cycling, with cyclin-A levels dropping in mid-mitosis and cyclin-B levels falling several minutes later, just at the metaphase/anaphase transition (Westendorf et al. 1989). It is now well established that, during the abbreviated cell cycles of early embryos, it is the rise in cyclin that drives cells from interphase into mitosis. The first direct evidence for this role came from the demonstration that the introduction of cyclin A into frog oocytes, which are arrested at the G2/M border of meiosis, caused those cells to enter M phase and resume the meiotic cell cycle (Swenson et al. 1986). B-type cyclin also had the same effect (Westendorf et al. 1989). Further support for an M-phase-promoting role came from the demonstration that the depletion of cyclin mRNA (via antisense constructs) prevented cellfree extracts from entering mitosis (Minshull et al. 1989a,b). The development of an mRNA-dependent cell-free system from frog embryos allowed Murray and Kirschner (1989) to establish definitively that the synthesis of a single protein, cyclin B, is sufficient to drive multiple cycles of certain cell cycle events, including nuclear envelope breakdown and reformation, chromosome condensation and decondensation, and the rise and fall in histone H1 kinase activity. Finally, genetic studies in yeast revealed a gene, cdc13 +, required for entry into mitosis whose sequence placed it firmly in the cyclin-B family (Booher and Beach 1988; Goebl and Byers 1988; Solomon et al. 1988). Cyclins exert their effect by binding to and activating the protein kinase cdc2/28. Originally identified in genetic studies of yeast (Hartwell et al. 1974; Nurse and Bissett 1981), cdc2/28 encodes a 34-kD protein kinase (Reed et al. 1985) that is required at least twice in each cell cycle, once in G 1 for commitment to the cell division cycle and again in G 2 for entry into mitosis (for review, see Ghiara et al. 1991; Surana et al. 1991; see also other papers in this volume). The M-phase role of cdc2/28 is best understood: It is the catalytic subunit of a multiprotein complex called MPF (for maturation- or M-phase-promoting factor). MPF was discovered as an activity in metaphase-arrested frog eggs that, when transferred by microinjection into interphase-arrested oocytes, would drive those cells into M phase (Ecker and Smith 1971; Masui and Markert 1971). MPF activity was subsequently found to cycle during each meiotic and mitotic cell cycle; new protein synthesis was required in each cell cycle to generate the rise in MPF activity and entry into mitosis (Wasserman and Smith 1978; Wagenaar 1983; Gerhart et al. 1984). Partially purified preparations of MPF from frog and starfish eggs, which displayed a high kinase activity with strong preference for the substrate histone H1 (Lohka et al. 1988), were discovered to contain a 34-kD protein kinase representing the homolog of cdc2/28 (Arion et al. 1988; Dunphy et al. 1988; Gautier et al. 1988; Labb6 et al. 1988). The connection with cyclins was fueled by the observation that monomeric cdc2/28 by itself is inactive, whereas the active form associates with other proteins (Reed et al. 1985; Draetta and Beach 1988). The nature of the cdc2/28-associated proteins was first identified by Draetta et al. (1989), who found that newly synthesized cyclin in clam embryos binds to and activates cdc2/28, forming independent complexes of cyclin A-cdc2/28 Cold Spring Harbor Symposia on Quantitative Biology, VolumeLVI.9 1991 Cold Spring Harbor LaboratoryPress 0-87969-061-5/91$3.00 495 Downloaded from symposium.cshlp.org on May 12, 2016 - Published by Cold Spring Harbor Laboratory Press 496 RUDERMAN ET AL. and cyclin B-cdc2/28, each of which displays high H1 kinase activity. This discovery led to the proposal that the cyclins act as positive regulatory subunits for cdc2/ 28, the catalytic subunit of MPF, and that the periodic rise and fall of MPF activity is due to the periodic accumulation and destruction of the cyclins across each cell cycle (Draetta et al. 1989). This view has now been amply confirmed (see, e.g., Labb6 et al. 1989; Murray and Kirschner 1989; Pines and Hunter 1989; Gautier et al. 1990; Solomon et al. 1990). It is thought that cyclin A and cyclin B, as well as the more recently discovered cyclin types, confer some sort of specificity on the timing of cdc2 activation, its substrate specificity, or its localization. Uncovering these differences has been hampered by the fact that cyclins A and B can substitute for each other in virtually all assays tried so far. For example, both can induce meiotic maturation (Westendorf et al. 1989), phosphorylate the same sites on histone H1 (Minshull et al. 1990), and induce DNA synthesis in G~ lysates (D'Urso et al. 1990). However, a good case can be made for the idea that cyclin B targets cdc2/28 for its functions in mitosis: Cyclin B is high in M-phase cells (A is low or absent), it is present in partially purified MPF (A is not), and loss-of-function cyclin-B mutants in yeast arrest just before they enter M phase (Booher and Beach 1988; Minshull et al. 1989b; Westendorf et ai. 1989; Kobayashi et al. 1991). The role of cyclin A is more mysterious. The fact that it peaks slightly ahead of cyclin B (Pines and Hunter 1989; Westendorf et al. 1989) and enters the nucleus well in advance of cyclin B (Pines and Hunter, this volume) suggests that cyclin A plays a role earlier in the cell cycle, with initiation of S phase as an obvious candidate. The results of antisense experiments further support this view (C. Brechot, pers. comm.). However, the role of cyclin A may be more complicated, as suggested by the finding that, in Drosophila, a cyclin-A mutation arrests in G2, not in G I (Lehner and O'Farrell 1989). Up to now, most molecular work has concentrated on establishing how the cyclin binding activates cdc2/28 and promotes entry into M phase (Draetta et al. 1989; Murray et al. 1989; Solomon et al. 1990; Kumagai and Dunphy 1991; Parker et al. 1991). Less effort has gone into studying how cells exit from mitosis and enter into the next interphase. The importance of programmed cyclin destruction in driving this cell cycle transition is suggested by two kinds of observations. First, when mitotic exit is prevented by colchicine-mediated dispersion of microtubules, cyclin A drops on schedule, but cyclin B remains high (Hunt et al. 1992). Second, synthesis of a truncated, stable version of cyclin B in a cell-free system keeps cdc2/28 hyperactivated and nuclei arrested in M phase (Murray et al. 1989; Glotzer et al. 1991). To learn more about the regulatory processes that control transitions from one cell cycle stage to another, we have used a cell-free system (Luca and Ruderman 1989) that reproduces many cell cycle changes occurring in intact cells. These include the activation of ERK/MAP kinase within minutes of fer- tilization, the subsequent activation of cdc2/28, and the programmed destruction of the cyclins at the end of each cell cycle. METHODS The materials and methods used in these experiments have been described previously in the following papers: Swenson et al. (1986), Shibuya and Masui (1989), Draetta et al. (1989), Luca and Ruderman (1989), and Boulton et al. (1991). RESULTS AND DISCUSSION Fertilization Leads to the Rapid Tyrosine Phosphorylation of pp42 ERI</MApki.... and Its Activation Clam oocytes are arrested at the G2/M border of meiosis I. Like other oocytes, they contain a maternal stockpile of cyclin B and cdc2/28 (Arion et al. 1988; Draetta et al. 1989; Dunphy and Newport 1989; Westendorf et al. 1989) associated as an inactive complex that can be isolated using p13 ~uc~beads (Fig. 1). Curiously, in clam oocytes, only a very small portion of the inactive cdc2/28 is phosphorylated on tyrosine. Entry into M phase is accompanied by shifts in the mobility of cdc2/28 and changes in its pattern of tyrosine phosphorylation (Fig. 1). With the aim of following the kinetics of this switch more carefully, we fertilized oocytes, took 4-minute time points and followed tyrosine dephosphorylation of cdc2/28 by blotting total cell proteins with an antiphosphotyrosine antibody. As shown in Figure 2, its dephosphorylation occurred gradually rather than showing an abrupt switch preceding entry into first meiotic M phase (GVBD, or germinal vesicle breakdown) at 11 minutes after fertilization. In contrast to this leisurely decline in cdc2/28 phosphotyrosine levels, a strong phosphotyrosine signal appeared on a 42-kD protein at 3-4 minutes, stayed high for the duration of this experiment (20 min), and disappeared at anaphase of meiosis I (about 30 rain), never reappearing during subsequent meiotic or mitotic divisions (not shown). The speed of this response and the fact that many cell-surface receptors contain tyrosine kinase activities or can activate tyrosine kinases (for review, see Boulton et al. 1991; Ferrell et al. 199t) suggested that this might be a very early step in the signal transduction pathway turned on by the binding of sperm to its receptor. The size of this protein and its pattern of phosphorylation suggested that it might be a member of the ERK (extracellular signal regulated kinase) family identified in mammalian tissue culture cells (Cooper et al. 1984; Boulton et al. 1990) and a homolog of the 42-kD MAP kinase (mitogen-activated protein kinase) activated during frog oocyte maturation (Ferrell et al. 1991). In support of this idea, antisera raised against rat ERK-2 recognized a 42-kD protein in Downloaded from symposium.cshlp.org on May 12, 2016 - Published by Cold Spring Harbor Laboratory Press CELL CYCLE CONTROL IN EMBRYOS Figure 1. Oocytes contain a preformed cyclin B-cdc2/28 complex that is modified after fertilization, pl3SUCl-bound material from lysates of unfertilized oocytes (GV) or 15-min postfertilization embryos (GVBD) was electrophoresed on an SDS-polyacrylamide gel, blotted to nitrocellulose, and reacted with a mix of cyclin B and cdc2 antibodies (left) or antiphosphotyrosine antibodies, which were generously provided by Brian Drucker and Tom Roberts (Harvard Medical School, Boston) (middle). The positions of cyclin B and cdc2/28 are indicated. Three different cdc2 bands (1, 2, and 3) can be distinguished by slight differences in their electrophoretic mobilities and reactivity with antiphosphotyrosine antibodies, pl3SUCl-bound material from embryos (GVBD) and oocytes homogenized at the prefertilization pH (6.8) or postfertilization pH (7.2) was assayed for histone H1 kinase activity (right). Immunoblot anti-cyclin B anti-cdc2 Cyclin B ~ GVBD 4 8 T12 I I I I GV GVBD i ~ ~ 16 20 I I p4z c d c 2 r- Figure 2. Oocyte activation is accompanied by the tyrosine phosphorylation of a 42-kD protein. Oocytes were activated by the addition of additional 40 mM KCI to the seawater, a treatment that mimics fertilization. Aliquots were removed at 4-min intervals, electophoresed on an SDS-polyacrylamide gel, and blotted with antiphosphotyrosine antibodies. anti-p-tyr H1K Activity GV 6.8 GV G V B D i i GV G V B D 7.2 I ~ cdc2 [" *'"~ ~ clam oocytes that became tyrosine phosphorylated after fertilization (not shown). These and other experiments have now established that this clam pp42 which becomes phosphorylated on tyrosine within 3-4 minutes of fertilization is a member of the E R K family. Recent work from other laboratories has highlighted the potential importance of ERKs in early signal transduction pathways (for review, see Boulton et al. 1991). In particular, Anderson et al. (1990) argued that, because the activity of these kinases requires phosphorylation on both serine/threonine and tyrosine residues, they are excellent candidates for coordinating input signals and activating downstream targets. Experiments from our laboratory confirm this view. The addition of molybdate to oocytes at any time between 0 and 3 minutes after fertilization blocks the tyrosine phosphorylation of clam pp42 ERK (which would normally appear about 4 min postfertilization) and arrests cells in inter- 0 497 I I ~ 9_/1 --2 phase; addition of molybdate just 1 minute later, at 4 minutes, allows the appearance of phosphotyrosine pp42 ERK and subsequent entry into M phase (not shown). This result indicates that the activation of cdc2/28 kinase activity is a late event and strongly suggests that it depends on the prior activation of pp42 ERK. It should be noted that during meiotic maturation of frog oocytes, Ferrell et al. (1991) and Gotoh et al. (1991a) have described a related pp42 MAPki that is activated after cdc2/28 activation. Unlike the clam pp42 ERK, which undergoes a one-time activation during release from cell cycle arrest, that enzyme is activated during each cell cycle. It probably represents a different member of the E R K / M A P kinase family that has a true mitotic function (Gotoh et al. 1991b). The development of cell-free systems that can go through one or more rounds of cell cycle events in vitro and the availability of active, purified recombinant cell cycle proteins have greatly facilitated investigations of the molecular mechanisms leading to cycles of cdc2/28 activation and repression during mitosis (Lohka and Maller 1985; Newport and Spann 1987; Luca and Ruderman 1989; Murray and Kirschner 1989; Solomon et al. 1990). In hopes of learning more about the molecular mechanisms of oocyte activation, we developed a cell-free system from quiescent oocytes that reproduces several of the events occurring after fertilization, including activation of pp42 ERK and cdc2/28. Oocyte lysates, which contain inactive cyclin B cdc2/28 complexes, do not develop active cdc2/28 when maintained at the prefertilization pH of 6.8. When the cytoplasmic pH was raised, cdc2/28 kinase activity appeared (as assayed by activity of p13 suc~bound material toward histone H1), suggesting that postfertilization rise in pH has a role in the activation of the preexisting complexes (Fig. 3) (see also Westendorf et al. 1989). Lysates were also activated by the addition of purified recombinant cyclin A or cyclin B protein (not shown), reproducing in vitro the original observations that the addition of cyclin to intact, G2-arrested .... Downloaded from symposium.cshlp.org on May 12, 2016 - Published by Cold Spring Harbor Laboratory Press 498 RUDERMAN ET AL. p H 6.8 I I '0! 60' ! pH8.0 I0 I 10' 20' 30' 60' 211 60 I ! m Figure 3. In vitro activation of the stored pre-MPF complex by elevated pH. Oocytes were lysed in buffer held at the prefertilization pH of 6.8, and a 12,000g supernatant was prepared. Aliquots of the lysate were mixed with an equal volume of pH 6.8 or pH 8.0 buffer, and histone H1 kinase activity was assayed at 0 and 60 min later. oocytes (Swenson et al. 1986) induces entry into M phase. We also used these lysates to look for activators of pp42 ERK. Purified recombinant rat ERK-2 protein by itself has very low kinase activity toward the substrate myelin basic protein and remains inactive when added to the prefertilization oocyte lysate. In contrast, rat ERK-2 protein added to lysates made from oocytes taken at 15 minutes after fertilization became active (not shown), indicating that the activators are present and maintained in an active state in lysates from postfertilization cells. This assay system provides an excellent opportunity to identify and isolate activators of pp42 ERK,including its own tyrosine kinase, and to work backward through the signal transduction pathway. Control of Programmed Cyclin Destruction in a Cell-free System To ask what controls the periodic accumulation and destruction of the cyclins across the cells, we developed a cell-free system that reproduces several aspects of cyclin destruction in vitro (Luca and Ruderman 1989). The onset of cyclin destruction and the appropriately staggered disappearance of cyclins A and B are correctly regulated (Fig. 4). Just as in the intact embryo, lysates made from early interphase require further protein synthesis to reach the cyclin destruction point, whereas lysates made from cells past the mitotic commitment point do not. By combining lysates from different cell cycle stages, we found that the timing of cyclin destruction is set by the cell cycle stage of the cytoplasm and not the cell cycle stage of the substrate cyclins themselves (Luca and Ruderman 1989). One feature of cyclin destruction that was reproduced too well in vitro, however, was the brief and transient window of cyclin destruction, considerably less than 5 minutes, making it hard to prepare an extract containing the destruction machinery in its active form. Thus, biochemical analysis of the proteolytic pathway and its regulation remained difficult. Murray et al. (1989) provided an insight into this problem when, in the course of adding cloned cyclin mRNAs into their frog embryo cell-free system, they ~'~j.:g~ 9 9 RRe Figure 4. Cyclin destruction in vitro. Embryos were labeled with [35S]methionine during the first mitotic cell cycle, and a lysate was prepared from early M-phase cells and incubated at 18~ After the start of the incubation in vitro (t = 0 min), samples were taken at the indicated times and analyzed by gel electrophoresis followed by autoradiography. The positions of cyclin A, cyclin B, and ribonucleotide reductase (RR) are indicated. The dashes on the right denote the positions of molecular-weight markers, from top to bottom, 116, 94, 56, and 40 kD. (Reprinted, with copyright permission of the Rockefeller University Press, from Luca and Ruderman 1989.) found that a sea urchin B-type cyclin missing the first amino-terminal 90 amino acids remained stable, whereas the full-length version was destroyed on schedule. This truncated, stable cyclin also had the very interesting property of permanently activating the cyclin destruction machinery toward full-length cyclin B. The importance of the amino-terminal region was further confirmed by the observation that fusion of that region to a marker protein led to the temporally regulated destruction of the fusion protein. In this amino-terminal region, Glotzer et al. (1991) identified a short stretch of amino acids (RxxLxxlxN) conserved in all B-type cyclins. When the arginine was changed to cysteine by site-directed mutagenesis, the resulting cyclin proved resistant to stage-specific mutagenesis. Related regions noted among the A-type cyclins seemed likely to be involved in their specific proteolysis but had not been tested. Here we have directly tested the importance of the amino-terminal region of cyclin A, which contains the candidate A-type motif RAALGVITN. Clam cyclin proteins lacking amino-terminal regions were produced by using convenient restriction sites to remove regions encoding the first 60 amino acids of cyclin A and the first 97 amino acids of cyclin B. The truncated proteins, termed AA60 and BA97, were synthesized in Escherichia coli using the pET5 expression Downloaded from symposium.cshlp.org on May 12, 2016 - Published by Cold Spring Harbor Laboratory Press CELL CYCLE CONTROL IN EMBRYOS a CaB 499 H1 Kinase Assay b 0' M ~A ~B 5' 10' 15' 30' 60' 2tl 4h ;. + Buffer ,., 9 116 .... 96 ,-- H1 68~ +A~60 56 ~ H1 +Baa7 31 21 -'- Figure 5. Bacterially expressed clam cyclin AA60 and BA97 can each induce H1 kinase activity when added to interphase lysates lacking endogenous cyclins. (a) Purified cyclin AA60 (AA) and BA97 (AB) produced in E. coli were electrophoresed on a 15% polyacrylamide gel and stained with Coomassie brilliant blue (CBB).(b) A 150,000g supernatant lacking endogenous cyclins was prepared from two-cell embryos arrested in interphase. Portions of the lysate were incubated with buffer, cydin AA60 or cyclin BA97, and incubated at 18~ At the indicated times, aliquots were taken and assayed for H1 kinase activity. Autoradiograms of the 32p-labeled histone H1 from each reaction are shown. vector (Studier et al. 1990), gel purified, and renatured by step-wise dialysis (F. Luca et al. in prep.). The truncated cyclins were active as M-phase inducers in several different types of assays. For example, both were capable of inducing G2-arrested frog oocytes to resume meiosis (not shown). When added to emetinearrested interphase lysates, which contain inactive cdc2/28 and lack endogenous cyclins, both AA60 and BA97 induced histone H1 kinase activity, a marker for activated cdc2/28 (Fig. 5). Further experiments established that the Acyclins induced this activity by binding to and activating cdc2 (not shown). Just as in cyclin B (not shown), removal of the amino terminus from cyclin A converted it into a stable cyclin but did not inhibit destruction of endogenous, fulllength cyclins (Fig. 6). Not unexpectedly, the presence of stable AA60 in lysates kept cdc2/28 kinase activity high for hours (not shown). To compare the effects of Anti-cyclin A Anti-cyclin B + Buffer w cyc A- 30' 45' 60' 90' 2h 3h 5h 0' 3 0 ' 4 5 ' 6 0 ' 9 0 ' 2 h 3h 5h --- .cyc B .X . 9 o ' , 3 o ' 45' ~ go' : ~ ~ sh m* 9 r 30' ~ - ; A " ~:-,-- - .,-: ~ 8o' ~ ~ ,-. :' , , - , ! , y ] - 7 o. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , :~ -cycB x ? i Figure 6. Cyclin AA60 is stable and does not inhibit the destruction of endogenous full-length cyclin A or B. 150,000g supernatants from embryos taken at mid-late interphase were incubated with buffer (top) or cyclin AA60 (bottom). At the indicated times after starting the incubation at 18~ aliquots were taken, electrophoresed on a 15% polyacrylamidegel, blotted to nitrocellulose, and probed with polyclonal serum raised against cyclin A (left) or cyclin B (right). X indicates a non-cyclin protein that cross-reacts with this particular cyclin-B antiserum. Downloaded from symposium.cshlp.org on May 12, 2016 - Published by Cold Spring Harbor Laboratory Press 500 RUDERMAN ET AL. AA60 and BA97 in living cells, each was injected into one cell of a two-cell frog embryo. Both AA60 and BA97 were potent inhibitors of cell division: 100% of the recipients in each set arrested without undergoing any further cleavage, whereas all the buffer-injected controls continued to divide normally for hours. All of the Acyclin-blocked cells contained highly condensed chromosomes reminiscent of metaphase arrest (not shown). of the other (not shown). Thus, regardless of any functional differences between the full-length A- and Btype cyclins when present at normal levels in the intact cell, the presence of high levels of either truncated cyclin in lysates had identical effects on the persistence of cyclin destruction in vitro. Both AA60 and BA97 Lead to Persistent Activation of the Cyclin Destruction Machinery by Preventing Cyclin Destruction from Being Turned Off To ask whether AA60 or BA97 could turn on cyclin destruction in an interphase-arrested lysate, an Mphase lysate was incubated with emetine for 4 hours at 18~ to take it well past the cyclin destruction point. Radioactively labeled proteins were then added and found to be stable over the next 4 hours, indicating that the lysate had indeed turned off the cyclin destruction system. When AA60 was added, it bound to and activated cdc2 kinase to high levels, but the lysate failed to activate cyciin destruction. In striking contrast, when BA97 was added, it was able to both activate cdc2 kinase and proceed to activate the cyclin destruction In normally dividing embryos, cyclin destruction is a brief and transient process, lasting for less than 5 minutes (Hunt et al. 1992). In contrast, lysates containing urchin AB displayed constitutive cyclin destruction (Glotzer et al. 1991). The presence of AA60 led to the same appearance of constitutive cyclin destruction activity that acted on both A- and B-type cyclins, indicating that one cyclin type is able to influence destruction a Cyclin-BA97-activated cdc2 Turns on Cyclin Destruction but Cyclin-AA60-activated cdc2 Does Not + Buffer + AA60 o' 5' 10' 15' 30' 60' 2h 5h 0' 5' 10' 15' 30' 60' 2h 5h 116 9668- A - ------------- 5640- b 0' 5' 10' 15' 30' 60' 2h 5h O' 5' 10' 15' 30' 60' 2h 5h 116-, 96-' 68- -A- 56 40 - ," ~ ~ ~'~'~'-'- Figure 7. Truncated cyclin A does not turn on or advance the onset of cyclin destruction. Mid-interphase lysates containing [35S]methionine-labeled cyclin A reticulocyte translation product were mixed with buffer or AA60 on ice and then incubated at 18~ Samples were taken at the indicated times and analyzed by SDS gel electrophoresis followed by autoradiography. 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Cold Spring Harb Symp Quant Biol 1991 56: 495-502 Access the most recent version at doi:10.1101/SQB.1991.056.01.056 References This article cites 52 articles, 12 of which can be accessed free at: http://symposium.cshlp.org/content/56/495.refs.html Article cited in: http://symposium.cshlp.org/content/56/495#related-urls Email alerting service Receive free email alerts when new articles cite this article sign up in the box at the top right corner of the article or click here To subscribe to Cold Spring Harbor Symposia on Quantitative Biology go to: http://symposium.cshlp.org/subscriptions Copyright © 1991 Cold Spring Harbor Laboratory Press