The EMBO Journal vol.9 no.9 pp.
2865-2875, 1990
The A- and B-type cyclin associated cdc2 kinases in
Xenopus turn on and off at different times in the
cell cycle
Jeremy Minshull1, Roy Goisteyn,
Caroline S.Hill and Tim Hunt
Department of Biochemistry, University of Cambridge, Tennis Court
Road, Cambridge CB2 IQW, UK
'Present address: Department of Physiology, School of Medicine,
University of California, San Francisco, USA
Communicated by T.Hunt
Cyclins play a key role in the induction of mitosis. In this
paper we report the isolation of a cyclin A cDNA clone
from Xenopus eggs. Its cognate mRNA encodes a protein
that shows characteristic accumulation and destruction
during mitotic cell cycles. The cyclin A polypeptide is
associated with a protein that cross-reacts with an anti-
body against the conserved 'PSTAIR' epitope of p34*2,
and the cyclin A -cdc2 complex exhibits protein kinase
activity that oscillates with the cell cycle. This kinase
activity rises more smoothly than that of the cyclin
B-cdc2 complexes and reaches a peak earlier in the cell
cycle; indeed, cyclin A is destroyed before nuclear
envelope breakdown. None of the cyclin-cdc2 complexes
show simple relationships between the concentration of
the cyclin moiety and the kinase activity. All three cyclin
associated kinases (A, Bi and B2) phosphorylate identical
sites on histones with the consensus XSPXK/R, although
they show significant differences in their substrate
preferences. We discuss possible models for the different
roles of the A- and B-type cyclins in the control of cell
division.
Key words: cyclins/cdc2 kinases/mitosis/Xenopus
Introduction
Entry into mitosis and resumption of meiosis involve the
action of a protein kinase encoded by the product of the
cdc2+ gene in fission yeast or its homologues in other
eukaryotes (Lee and Nurse, 1988; Nurse, 1990). The activity
of cdc2 kinase at the G2-M transition is regulated by
translational and post-translational mechanisms. The trans-
lational control appears to involve new synthesis of cyclin
subunits, which associate with p34cdc2 and are absolutely
required for the mitotic activity of the cdc2 kinase (Murray
and Kirschner, 1989b). The post-translational control prob-
ably includes dephosphorylation of tyrosine 15 on p34cdc2
by a protein phosphatase that is presumably activated at the
threshold of mitosis (Dunphy and Newport, 1989; Gautier
et al., 1989; Gould and Nurse, 1989; Labbd et al., 1989b;
Morla et al., 1989). At the end of mitosis the cyclins are
destroyed, which leads to rapid loss of cdc2 kinase activity
(Murray et al., 1989). Despite the clear evidence for their
role in turning cdc2 kinase on and off, however, it is not
clear how the cyclin subunits perform this function.
A further complexity is that there are two distinct types
of mitotic cyclin, cyclins A and B. Cyclin A was first
Oxford University Press
detected in clam embryos and was the first cyclin to be
cloned and sequenced (Swenson et al., 1986). Swenson et al.
also provided the first direct evidence that cyclin was
involved in the G2- M transition by showing that micro-
injection of clam cyclin A mRNA into stage VI Xenopus
oocytes promoted their maturation. Cyclin A was sub-
sequently identified in Drosophila by Lehner and O'Farrell
(1989) who found that it was encoded by an abundant
maternal mRNA in fly embryos. They found that new
transcription of this gene was essential for the division of
neuroblasts later in Drosophila development. In the absence
of cyclin A mRNA, the neuroblasts arrested in G2. Cyclin
B has been isolated from a wide variety of organisms that
include yeast, sea urchin, starfish, clam, frog, fly and man
(Booher and Beach, 1988; Pines and Hunt, 1987; Labbe
et al., 1989a; Westendorf et al., 1989; Minshull et al.,
1989a; Whitfield et al., 1989; Pines and Hunter, 1989). It
is encoded by the essential cdcl3 gene in fission yeast, and
probably exists in all eukaryotes. Like cyclin A, cyclin B
mRNA promotes frog oocyte maturation and the injection
of both types of mRNA is reportedly more effective than
either alone (Swenson et al., 1986; Pines and Hunt, 1987;
Westendorf et al., 1989).
Almost nothing is known about the significant functional
differences between A- and B-type cyclins. In clams, both
cyclins were found to be associated with p34cdc2, and anti-
bodies against either cyclin A or cyclin B immunoprecipitated
histone HI kinase activity (Draetta et al., 1989). There is
an important difference between cyclin A and B in un-
fertilized clam oocytes, however, since these cells contain
a considerable maternal endowment of cyclin B, but no cyclin
A protein. After fertilization, the oocytes do not require new
protein synthesis to complete first meiosis, which seems to
imply that cyclin A is not strictly required to enter M phase.
However, the second meiotic division, which in clams follows
-
15 min after first meiosis, shows disastrous abnormalities
in the absence of protein synthesis. The abnormalities may
stem from lack of cyclin A, since the maternal cyclin B
protein pool does not appear to be significantly depleted after
meiosis I in contrast to anaphase of meiosis II, when it is
degraded (Westendorf et al., 1989). In the early mitotic
divisions of clam eggs the two cyclins show slightly different
kinetics of destruction during mitotic division-cyclin A is
degraded before cyclin B-and these differences are empha-
sized when microtubule inhibitors are added (Westendorf
et al., 1989; Minshull et al., 1989b). In Drosophila, mRNAs
for the different cyclins accumulate in different tissues during
early development, cyclin B showing strong localization to
the presumptive germline (Whitfield et al., 1989). None of
these observations provide strong clues as to why there
should be two cyclins or what their respective roles are
during cell division.
One attractive idea is that cyclins act as 'targeting sub-
units' that guide cdc2 kinase to particular mitotic substrates.
The problem with this idea, however, is that the best
2865
J.Minshull et al.

* * ~~~~~M
R R S M A s N G H
characterized activity of p34cdc2 is as a histone HI kinase GAaTTCCGATCTGCGCTTAGTGCTTCTGGTTGTAGCAGTTGACCTAAATTTGAACAGACGATGCGGCGCAGTATGGCTTCCAATGGGCAC

(Lake 1973; Bradbury et al., 1974; Erikson and Maller, I L T A S VV AS S A F Q N P C L A K V E

ATCCTTACAGCATCCTCTGTGGTGGGAGCCAGCAGTGCATTCCAGAATCCATGCCTAGCCAAGGTAGAAGTCCAGCCTAACCTGCCTCAA
V Q P N L P Q

1989; Langan et al., 1989), and as already mentioned, R T V L G V I G D N E Q R R R S V S R G G V

AGAACTGTGTTGGGTGTTATCGGTGACAATGAACAGCGCAGAACa!AGTTAGTCGGGGTGGTGTCCCTGCAAAGAGTCTACCTGGAATT
P A K S L P G I

Draetta et al. (1989) found that cyclin A-cdc2 and cyclin E N V L A F P G E I L S A N P A P V A P K P S F T V Y V D E

B-cdc2 complexes from clams both showed HI kinase GAAAATGTTCTTGCTTTCCCTGGAAAAATCCTGTCTGCAAACCCAGCGCCTGTTGCTCCAAAGCCAAGTTTTACAGTCTATGTGGATGAG

activity. It seems unlikely, however, that the only role of P T E T Y S V E I D C P S L G D E D S N I V K

CCAACAGAAACCTATTCAGTTGAAATAGATTGTCCCAGTTTGGGTGATGAGGATTCAAATATTGTCAAGCAGAACATTCACCTGCTCCTA
D N I H L L

p34Cdc2 kinase in vivo is to phosphorylate histone H1. If D I S E A S P M V V D T S T P E D D S V

GATATCAGTGAAGCTTCTCCGATGGTGGTTGACACGTCACCCCAGACAAGCCCAGAGGATGACTCTGTAACAGACCCTGATGCTGTAGCT
T D P D A V A

p34Cdc2 is a 'master regulator' which causes entry into V S E Y I H E I H Q Y L R E A E L K H R P K A Y Y M R K SP

M phase, it more likely does so by activating secondary GTGTCTGAATATATACATGAAATTCACCAGTACCTTCGAGAGGCTGAGTTGAAACACAGACCAAAAGCATATTACATGCGTAAGCAGCCA
D I T S A M R T I L V D W L V E V G E E Y K L H T E T L Y L
regulators such as pp6osrc (Morgan et al., 1989; Shenoy GACATTACTTCAGCAATGCGCACGATACTTGTGGACTGGCTAGTAGAAGTTGGTGAGGAATACAAGCTGCACACGGAGACTCTTTATCTG

et al., 1989), rather than by phosphorylating histone HI. A M N Y L D R F L S C H S V L R G E

Q L V G T A A I L L A
L
GCTATGAATTATTTGGACCGCTTCCTATCATGCATGTCTGTACTCCGGGGAAAACTGCAGCTTGTAGGCACAGCAGCTATTTTATTGGCA
Moreover, there is some doubt whether the chromatin of S K Y E E I Y P P D V D E F V Y I T D D T Y S K K Q L L R M
TCAAAATAT AAGAAATCTACCCTCCTGATGTTGATGAATTTGTGTACATAACTGATGATACATACTCAAAGAAACAGCTTCTGCGCATG
Xenopus early embryos even contains histone HI (Dilworth E H V L L K V L A F D L T V P T V N Q F L L Q Y L Q R H A V
et al., 1987), and there is little direct evidence that chromo- GAGCACGTTCTGTTGAAGGTTCTGGCCTTTGACCTTACTGTACCTACAGTCAACCAATTCTTGCTACAGTATCTGCAGAGACATGCAGTC

some condensation is brought about simply by phosphoryla- S V K M E H L A M Y M A E L T L L E V E P F L E

AGTGTGAAGATGGAGCACCTTGCAATGTACATGGCAGAACTAACTCTGCTTGAAGTGGAGCCATTCTTGAAGTACGTTCCTTCACTTACA
Y V P S L T

tion of histone HI (Lennox and Cohen, 1988). A few other A A A A Y C L A N Y A L N E V F W P D T L E A

GCTGCTGCAGCCTATTGTCTTGCCAATTATGCACTCAACAAAGTGTTCTGGCCTGACACACTGGAAGCCTTTACTGGCTACGCACTGAGT
F T G Y A L S

proteins besides histone Hi and pp6osrc have been shown D I A P C L S D L H Q F C L G A P Y Q A Q Q A I R E K Y K T

to be phosphorylated by p34 cdcL kinase. These include
MAP2 (Erikson and Maller, 1989), SV40 large T antigen
(McVey et al., 1989), RNA polymerase II (Cisek and
Corden, 1989), the retinoblastoma gene product (Cooper and
Whyte, 1989; DeCaprio et al., 1989) and the nucleolar pro-
teins nucleolin and N038 (Peter et al., 1990). It remains to
be determined what other substrates of cdc2 kinase exist,
and how many of them change their activity in such a way as
to contribute to the cellular transformations that occur in
mitosis.
In this paper, we describe the isolation of a cyclin A clone
from Xenopus and show that it is associated with a protein
kinase that binds to pI3SuCl and reacts with an anti-PSTAIR
antibody, presumably p34 d.2* We compare the timing of
activation of cyclin A and B associated histone kinases in
the cell cycle and show that both types of enzyme phos-
phorylate several serine residues in the context SPX(R/K)
in the same positions in sea urchin sperm histones HI and
H2B. There are endogenous (non-histone) substrates for both
types of enzyme in frog eggs, some of which are preferential
substrates for A- or B-type kinases. We discuss the relation-
ships between the two types of cyclin and their possible roles
in cell cycle control in the light of these findings.
Results
Isolation and sequencing of an A-type Xenopus cyclin
The first two cyclin cDNA clones we isolated from Xenopus
oocyte cDNA libraries encoded B-type cyclins (Minshull
et al., 1989a). Since A-type cyclins have been found in
clams, flies, humans and sea urchins (Swenson et al., 1986;
Lehner and O'Farrell, 1989; Wang et al., 1990; Pines and
Hunter, 1990; M.Winkler, personal communication) we
expected Xenopus to possess cyclin A. Moreover, a candidate
polypeptide was detected in a mitotic cell-free system labelled
with [35S]methionine (C.Ford, personal communication).
To isolate clones encoding A-type cyclin(s), XgtIO cDNA
libraries from frog ovary were screened with a redundant
oligonucleotide encoding the conserved amino acid sequence
KYEE[M/I]YPP[E/D] in clam cyclin A and sea urchin cyclin
B, with the nucleotide sequence underlined in Figure 1 (see
Materials and methods). Positive plaques were isolated and
counter-screened with a mixed frog cyclin B 1 /B2 probe to
identify the B-type cyclin clones (see Minshull et al., 1989a).
Most of the oligonucleotide-positive clones annealed strongly
with the cyclin B-specific probes, but four out of 64 plaques
GACATAGCACCTTGCCTTAGTGATTTGCACCAATTCTGTCTTGGTGCCCCTTATCAGGCTCAGCAAGCAATAAGGGAAAAGTACAAGACC
L N
T X Y M Q V S L L E M P S I L P *
ACCAAGTATATGCAAGTGTCTCTTCTGGAGATGCCGTCAATACTTCCCCTCAACTGAAGCCTTCCAGAGTGGACGCACACACAGCACTTA
CCTCGTBaZDAACAGTGTTGGTCTTTTTATGAAGACACTGCAGGCCAAGTGGCCAATGGAGCTATTTTATTTATTGACCTTCATACCAAG
ACTCCTGTGCTTTTATAATGTACTTTTTATTCTGTGTAAACTATAGGACCTTATTTATAACAAAGCCTCAGATTGGACACTAGTTGCTGA
CTGTGGGATTTAGTCTATGGACATCAATCATGTCTAAAAGTCACTTAGTTGGGATGTACTACTACAAATCAGAACTCTATTGGTAGTGCA
CTGGTTAACAGACATCAGTATTCTATTAGACTTGGACAATOCTGACTGTCTCTCCTACAGTCGAGCTTCTCCAGGAAATTAAGTGTTTTT
TTGTTAAACATCGTCGACATTGAACTGCTTCATTTTCCCAGGTTCTTAACTTGTGATGGTGTTAAGTGTTTTTAATAAACTGACTTTACT
CAAAAAGGAATTC
Fig. 1. Nucleotide and derived amino acid sequences of X.laevis
cyclin Al. The nucleotide sequence of XLcycAl was determined by
dideoxynucleotide sequencing of randomiy selected M13 subclones as
described by Minshull et al. (1989a). The sequence begins and ends
with an EcoRI linker. The 3' end of the clone contains a
polyadenylation signal AATAAA 17 bases from the end of the clone.
The in-frame termination codons in the 5' untranslated region and at
the end of the coding region are indicated by asterisks. The position of
the redundant oligonucleotide used to screen the library is indicated by
underlining. The Sau3A sites (GATC) surrounding the portion of the
clone used to produce antigen in E.coli are indicated in bold and
underlined.
gave a relatively weak signal with B-probes. DNA was
prepared from these clones, and the sizes of the inserts
checked by gel electrophoresis. The largest insert (- 1900
bp) was subcloned into the EcoRI site of pGEM1. Figure 1
shows the sequence of this clone (XlcycAI). Xenopus cyclin
Al contains 419 amino acids with a predicted molecular
weight of 46 773, though like other cyclins it migrates
anomalously on SDS -polyacrylamide gels with an apparent
molecular weight of -60 000.
Figure 2 compares the derived amino acid sequence of
frog cyclin Al with A-type cyclins from clam, fly and
human, which clearly identifies frog cyclin Al as an A-type
cyclin. From residue 160 onwards the frog sequence is very
similar to that of the other species' cyclin A-there are 100
completely conserved amino acids, and substitutions are for
the most part highly conservative. Apart from the motif
RXXLGVI starting at residue 41, conserved in the clam but
appearing as KXXLAVI in the fly, the N-termini show
essentially no sequence conservation. This island of N-
terminal conservation has its counterpart in the B-type
cyclins, where it occurs in a similar place as RXXLG
(D/E/N)I. Only 40 of the completely conserved residues in
cyclin A are found in the same places in the B-type cyclins,
and it is striking that in the corresponding region, the A-
type cyclins show considerably higher sequence identity with
each other than do the B-type cyclins. One other A-type clone
was also sequenced, but it was truncated at the 5' end,
lacking -80 amino acids from the N-terminus. It showed
few and only conservative changes from the amino acid

2866

MKMSQPFALHHDGENQMQRRGKMNTRSNGLSGQKRAALGVITNQVNQQ Clam A
HASFQIHQDMSNKENPGIKIPAGVKNTKQPLAVIGGKAEKN Fly A
MLGNSAPGPATREAGSALLALQQTALQEDQENINPEKkAPVQQPRTRAkLAVLKSGNPRGL
MRRSMASNGHILTASSVVGASSAFQNPCLAKVEVQPNLPQRTVLGVIGDNEQRR
R
L VI
LG I N
VRIQPSRAAKPKSSEFNIQDENAFTKKNAKTFGQQPSQFSVFVDPTPAAPVQKAPTSHVT Clam A
ALAPRANFAVLNGNNNVPRPAGKVQVFRDVRNLNVDENVEYGAKSNVVPVVEQFKTFSVY Fly A
AQQQRPKTRRVAPLKDLPVNDEHVTVPPWKANSKQPAFTIHVDEAEKEAQKKPAESQKIE Human A
RSVSRGGVPAKSLPGIENVLAFPGKILSANPAPVAPKPSFTVYVDEPTETYSVEIDCPSL Frog A
EDTQVAPSGKSLASLVDKENHDVKFGAGGKELVDYDLDSTP
MSVTDVQSPMSVDRSILGVIQSSDISVGTETGVSPTGRVKELPPRNDRQRFLEVVQYQMD Fly A
REDALAFNSAISLPGPRKPLVPLDYPMDGSFESPHTMDMSIVLEDEKPVSVNEVPDYHED Human A
GDEDSN-------IVKQNIHLLLDISEASPMVVDTSPQTSPEDDSVTDPDAVAVSEYIHE Frog A
V Y
D D P SEY
IYNYLRQAEMKNRAKPGYMKRQTDITTSMRCILVDWLVEVSEEYKLHRETLFLGVNYIDR Clam A
ILEYFRESEKKHRPKPRYMRRQKDISHNMRSILIDWLVEVSEEYKL TETLYLSVFYLDR Fly A
IHTYLREMEVKCKPKVGYMKKQPDITNSMRAILVDWLVEVGEEYKLQNETLHLAVNYIDR Human A
IHQYLREAELKHRPKAYYMRKQPDITSAMRTILVDWLVEVGEEYKLHTETLYLAMNYLDR Frog A
I Y R ZR
IY Y LB
K TM Q DI
y
MR IL DWILVEV EEYKL KIL L
E MR IL DW Q F
Y DR A consensus
LLQET
FLSQMAVVRSKLQLVGTAAMYIAAKYEEIYPPEVGEFVFLTDDSYTKAQVLRMEQVILKI
FLSSMSVLRGKLQLVGTAAHLLASKFEEIYPPEVAEFVYITDDTYTKKQVLRMEHLVLKV
FLSCMSVLRGKLQLVGTAAILLASKYEEIYPPDVDEFVYITDDTYSKKQLLRMEHVLLKV
FLS V R KLQLVG A A KIZ P? V Et TDD Y Q LRI
V LQLVGVT A KYZMP DV D TT I M
LTFDVAVPTTNWF-CEDFLKSCDADDKLKSLTMFLTELTLIDMDAYLKYVPSITAAAALC Clam A
LSFDLCTPTAYVFINTY-ALLCDMPEKLKYHTLYISELSLMEGETYLQYLPSLHSSASVA Fly A
LTFDLAAPTVNQFLTQYFLHQQPANCKVESLAMFLGELSLIDADPYLKYLPSVIAGAAFH Human A
LAFDLTVPTVNQFLLQY-LQRHAVSVKMEHLAMYMAELTLLEVEPFLKYVPSLTAAAAYC Frog A
L FD PT J K Ef L Y PS
L GRPL LHrLRR SK HT AKYIMEL 18 IAA
LARYSLGMEPWPQNLVKKTGYEIGHFVDCLKDLHKTSLGAESHQQQAVQEKYKQDKYHQV Clam A
LARHHILGMEMWYPRIEEITTYKLEDLKPVVLHLCHTHKTAELN-TQAMREKYNRDTYKKV Fly A
LALYTVTGQSWPESLIRKTGYTLESLKPCLMDLHQTYLKAPQHAQQSIREKYKNSKYHGV Human A
LANYALNKVFWPDTLEAFTGYALSDIAPCLSDLHQFCLGAPYQAQQAIREKYKTTKYMQV Frog A
LA
L L
1
11
T Y
Y E
L A QA EXT
V K
Y V A consensus
B consensus
SDFSKNPVPHNLALLAL Clam A
AMMESVENSKDDFDQLCEAYNCKQKEDEHQQPDINTKSNVNLFYKF Fly
SLLNPPETPNL
A
Human A
SLLEMPSILPLN Frog A
B consensus
S
Fig. 2. Comparison of frog cyclin Al with other published cyclin A
sequences. The amino acid sequences of A-type cyclins from the clam
Spisula solidissima (Swenson et al., 1986), the fly Drosophila
melanogaster (Lehner and O'Farrell, 1989) and human (Wang et al.,
1990) were manually aligned with that of frog cyclin Al. Cyclin A
specific consensus sequences are indicated and compared with those
for B-type cyclins. Bold letters in the consensus indicate conservation
between A- and B-type cyclins.
sequence shown in Figure 1, and overall resembled cyclin
Al much more closely than do frog cyclins BI and B2.
The level of cyclin Al mRNA in oocytes and early
embryos was -4 x I07 molecules per oocyte as measured
by RNase protection mapping. This is similar to that of cyclin
Bi and B2 mRNAs (H.Kobayashi and J.Minshull, unpub-
lished results).
A and B type cyclins are associated with p340tdc2
Purified MPF from Xenopus eggs contains B-type cyclins
associated with p34 OC2 (Gautier et al., 1990). To test
whether the A-type cyclins were also associated with
p34CdC2, mitotic extracts of activated Xenopus eggs (Murray
and Kirschner, 1989a) were immunoprecipitated with affinity
purified anti-cyclin antibodies, Western blotted and probed
with an anti-PSTAIR antibody. Figure 3A shows that
immunoprecipitates with all three cyclins contained an
immunoreactive band of -34 kd (lanes 1-3), which co-
migrated with a strongly reactive band present in unfraction-
ated frog cytoplasm (lane 4). As a control, we used an
antibody against a cdc2 related polypeptide (J.Paris and
M.Philippe, unpublished), which did not detect any cyclin
associated material (lanes 5-7). Thus a 34 kd PSTAIR-
containing polypeptide, presumably p34cC2, was associated
with each cyclin. In addition to the 34 kd band common to
all the anti-cyclin immunoprecipitates, cyclin Bl and B2 were
Cyclin A and cdc2 kinase
associated with a slightly more slowly migrating form of
Human A
Frog A p34Cdc2. This probably represents a phosphorylated form of
A consensus
B consensus
p34cdc2, but these data do not exclude the possibility that the
bands associated with each cyclin may in fact be different
polypeptides. Further work is required to settle this important
question.
We also performed the converse experiment, to see what
newly synthesized polypeptides were associated with p34',2
Fly A
Clam A in mitotic cyclin extracts. Extracts of activated Xenopus eggs
prepared according to Murray and Kirschner (1989a) were
A consensus labelled with [35S]methionine for 45 min and then incubated
B consensus
with p1 3sucl -Sepharose, the selective affinity matrix for
p34cdc2 (Brizuela et al., 1987; Dunphy et al., 1988; Labbe-
et al., 1989a). The bound proteins were analysed on SDS
I DR B consensus polyacrylamide gels. Figure 3B, lane 13, shows that the
Clam A
pI3Sucl affinity matrix selectively retained four bands from
Fly A
Human A
the complex mixture of labelled proteins seen in lane 9. The
LK
Frog A
A consensus
uppermost band corresponded to cyclin A, as shown by its
L B consensus identical mobility with immunoprecipitated cyclin A (lane
12), its periodic destruction in each cell cycle (see below,
Figure SB) and by the presence of an immunoreactive band
A A consensus in the same position when a Western blot of the pI3sucL-
B consensus
bound material was probed with anti-cyclin A antiserum (data
not shown). The lower bands corresponded to the B-type
cyclins. Essentially no other proteins were retained by the
pl3SUC" matrix. The absence of any newly synthesized
p34CdC2 is noteworthy. In the experiment shown in Figure
3B, the yield of cyclin A appeared to be lower than that of
cyclins B 1 and B2. To determine the fraction of each cyclin
present in complexes with p34cdc2, an extract was labelled
with [35S]methionine for 45 min and immunoprecipitations
performed with anti-cyclin antibodies before and after
passage over a column of p13Sucl-Sepharose. Over 80%
of the immunoprecipitable B-type cyclins bound to the
column, whereas 71 % of the A-type cyclin was bound.
Figure 3B also shows that the anti-cyclin antibodies were
highly specific for their cognate antigens. Immunoprecipit-
ates using anti BI (lane 10), anti B2 (lane 11), anti Al (lane
12) did not contain detectable amounts of the other labelled
cyclins, which means there was no evidence for the forma-
tion of mixed B1/B2 or A/B heterodimers.
Bacterially expressed cyclin B 1 and B2 are substrates for
purified cdc2 kinase (Gautier et al., 1990). The binding of
cyclins to pl3sucl and their association with polypeptides of
34 kd containing the conserved PSTAIR sequence led us to
conclude that cyclins were tightly associated with p34cdc2
and hence, that anti-cyclin immunoprecipitates should display
protein kinase activity. To test this, well-washed anti-cyclin
immunoprecipitates or pl3sucl beads were incubated with
[_y-32P]ATP in kinase buffer without any additional sub-
strates and analysed on a polyacrylamide gel. Figure 3C
shows a small number of autophosphorylated bands, whose
mobilities corresponded to the [35S]methionine-labelled
cyclins seen in Figure 3A (compare lanes 10 and 15, 11 and
16, 12 and 17, and 13 and 14). Labelled bands of the same
mobility were found in anti-cyclin immunoprecipitates from
[_y-32P]ATP-labelled Xenopus eggs extract (data not shown).
The kinase activity of cyclin - cdc2 complexes
fluctuates during the cell cycle
The best characterized substrate for cdc2 kinase is histone
HI. Figure 4 shows an experiment in which a mitotic extract
was labelled with [35S]methionine and duplicate samples
2867
J.Minshull et al.

T BI B2 A p13 p13 Bl B2 A

97.0 97.0

66.5 66 5 -
41 B2 81 - A f32 B5 T .:

5746-1 6",
57.6 55.6
55.6
50-0 43. I
43 1

35.7
35.7 -
0

2~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
-.

6w
a 8<

2o1
so
.....
20 1 -

9 10 11 12 13 14 15 16 17

A) Western bJlotsn of anti-cyclirn (B) Immunoprecipitates (C) Autophosphorylation ot

immunoprecipitates, probed from 35S-labeled extract immunoprecipitated kinases
with anti-PSTAIR antibody

Fig. 3. Xenopus cyclins are associated with p34cdc2 and the complexes undergo autophosphorylation. Panel A. A cell-free extract of Xenopus eggs
was prepared according to Murray and Kirschner (1989a) and incubated for 45 min. Samples were immunoprecipitated with affinity purified anti-
cyclin antibodies as described in Materials and methods. The samples were analysed by electrophoresis on a 15% SDS-polyacrylamide gel,
transferred to nitrocellulose and blotted with an anti-PSTAIR antibody (lanes 1-4), which was detected by alkaline phosphatase coupled second
antibody. Lane 1, anti-cyclin A immunoprecipitate; lane 2, anti-B2 immunoprecipitate; lane 3, anti-Bi immunoprecipitate; lane 4, total mitotic egg
cytoplasm (0.3 y1 of extract). The major bands seen at 25 kd and 50 kd represent residual light and heavy chains respectively of the anti-cyclin
antibodies. Lanes 5-8 show a duplicate blot of the same samples probed with a polyclonal antibody raised against p32Egl, a cdc2 related polypeptide
identified in Xenopus eggs by J.Paris and M.Philippe. Panel B. Immunoprecipitates or pl3suc -Sepharose precipitates were prepared as described in
panel A, except that the extract was labelled for 45 min with [35S]methionine. The immunoprecipitates were boiled in SDS sample buffer containing
2-mercaptoethanol and analysed on a 15% polyacrylamide gel. Lane 9, total labelled extract; lane 10, anti B1 immunoprecipitate; lane 11, anti B2
immunoprecipitate; lane 12, anti-cyclin A immunoprecipitate; lane 13, p13"uc1 -Sepharose bound material. Panel C. A sample of Xenopus egg
extract was prepared and immunoprecipitated as for panel A. The precipitates were autophosphorylated by the addition of kinase buffer containing 50
AM [y_-32P]ATP, 1.6 mCi/ml, and analysed on a 15% polyacrylamide gel. Lane 14, pl3SUCl; lane 15, anti-BI; lane 16, anti-B2; lane 17, anti-Al.
The positions of mol. wt markers are indicated in kd.

/s f /§D /G /tCh CR /@ /t /H
Ct ,NN0°N
OC tim e (m inutes)
66.5 --

57.6
55.6 - k_;g""il|"|_
. _ S.W,,,Lo* =_]cycins B1 & B2 'S
,, _ _
-3

43 1 -

- Histone Hl i 32P
4mgIm. ON VW

Fig. 4. Fluctuation of histone HI kinase activity during the cell cycle. A cell-free extract of Xenopus eggs, prepared according to Murray and
Kirschner (1989a), was incubated at 23°C in the presence of [35S]methionine. Samples were taken into liquid nitrogen at 5 min intervals for analysis
of protein synthesis and histone HI kinase (see Materials and methods). The times from the beginning of the incubation are shown. Positions of mol.
wt markers on the 15% SDS-polyacrylamide gel are indicated in kd.

were taken for analysis of protein synthesis and histone
kinase activity. As shown previously by others, the activity
of cdc2 kinase towards this substrate fluctuates dramatically
with the cell cycle, showing maximal activity during mitosis
(Meijer and Pondaven, 1988; Murray and Kirschner 1989a;
Moreno et al., 1989). Two cycles of B-type cyclin accumu-
lation and destruction were apparent in this experiment,
which correlated with two peaks of histone HI kinase
activity. As is often the case, it was difficult to detect the
synthesis and destruction of cyclin A in the total pattem of

2868

methionine-labelled polypeptides. This is largely because
cyclin A is present at only 1/10th the concentration of the
-
sum of cyclin B in early frog embryos.
To compare the individual activities of cyclin A-cdc2 and
cyclin B-cdc2 complexes during the cell cycle, the ex-
periment shown in Figure 4 was repeated, taking samples
in sextuplicate for analysis of total protein synthesis, for
cytological examination of the state of the added nuclei, for
affinity absorption on p1 3suc1 - Sepharose, and for immuno-
precipitation with each of the three cyclin antibodies. The
cycle time was longer in this experiment. Cyclin A was
degraded between 70 and 80 min and the B-type cyclins were
degraded between 80 and 90 min. Nuclear envelopes were
still intact at 70 min, broke down at 80 min and had reformed
by 100 min.
Figure 5B shows the analysis of samples that were affinity
absorbed onto p1 3s" - Sepharose. The washed beads were
incubated with mixed sea urchin sperm histones and
['y-32P]ATP and analysed by SDS -PAGE. The synthesis
and destruction of 35S-labelled cyclins (particularly of cyclin
A) is much clearer in this panel than in Figure 5A. The
cyclins were bound by the p13suc' beads and hence associ-
ated with p34CdC2 from the earliest times onwards. The
histone HI kinase activity of the beads peaked strongly at
70 and 80 min, corresponding to the onset of nuclear
envelope breakdown, and then fell rapidly. The kinase
activity associated with the beads did not parallel the level
of cyclins; indeed, at the time of the peak of activity at 80
min, all of the cyclin A and a considerable fraction of the
B-type cyclins had already been destroyed. The level of
p34cdc2 in all these samples was essentially constant as
measured by Western blotting (data not shown). This
suggests that some of the cdc2 kinase molecules may retain
kinase activity for a short time after the destruction of the
cyclins.
A surprising feature of these data was that the kinase(s)
bound to p13suc' beads displayed strong activity towards sea
urchin sperm histone H2B, and the cell cycle kinetics of the
H2B kinase activity were not the same as those of the HI
kinase. Considerable H2B kinase activity was present early
in the cell cycle, and this increased steadily towards the peak
at M phase instead of showing the sharp peak of activity
of the H1 kinase. Moreover, even after destruction of the
cyclins, residual H2B kinase activity was associated with the
pl3sucI beads. As will be shown below, residual H2B kinase
activity is found associated with cyclin A immunoprecipitates.
Many proteins besides p34Cdc2 and cyclins bind to pl3Sucl
beads, however, and we cannot exclude the possibility of
the presence of an H2B kinase that shows little or no cell
cycle related variation in activity. No such activity was bound
by control antibodies or blank Sepharose.
Each cyclin - cdc2 kinase complex shows
characteristic kinetics of activation
Figure 5C shows the histone kinase activity of the individual
anti-cyclin immunoprecipitates. They each phosphorylated
sea urchin sperm histones HI and H2B, showing character-
istic cell cycle related fluctuations in activity. The kinase
activity of the immune complexes was lost when the cyclins
were destroyed; in the absence of cyclins, p34cdc2 should
not bind to the antibodies or the protein A beads. The kinetics
of activation of the three different cyclin associated kinases
showed marked differences. Cyclin A immunoprecipitates
Cyclin A and cdc2 kinase
showed a gradual rise in histone kinase activity almost in
parallel with the amount of cyclin present. The cyclin B1
immunoprecipitates showed a more abrupt peak of mitotic
activation, and B2 immunoprecipitates had an extremely
sharp peak of kinase activity with essentially no detectable
activity before the onset of mitosis (see the curves in Figure
5D). The activation of the B2 kinase complex correlated with
the disappearance of a faster migrating form of cyclin B2
and the appearance of a slower electrophoretic variant that
appears to be due to its phosphorylation (Gautier et al., 1990;
R.Golsteyn and T.Hunt, unpublished results), similar to that
seen for sea urchin cyclin B (Meijer et al., 1989).
Figure 5D shows the quantitation of these data. Cyclin
A immunoprecipitates showed a peak of HI kinase activity
10 min earlier than that of the B immunoprecipitates. The
cyclin A associated kinase activity fell to a low level by the
time of nuclear envelope breakdown, whereas the kinase
activity of cyclin B immunoprecipitates (and of the pl3sucl
beads) were still high at this point. This was a reproducible
finding; the A-type cyclins were always destroyed as the
B-type cyclin associated kinase activity rose. These results
suggest that the A- and B-type cyclin kinase complexes have
different roles. In the Discussion we explore what kind of
'crosstalk' may occur between the different cyclin-cdc2
complexes.
Kinases associated with cyclins A and B
phosphorylate histones at different rates
As Figure 5 showed, sea urchin sperm histone H2B was an
unexpectedly good substrate for the cyclin-cdc2 protein
kinases; indeed, in the case of cyclin A-cdc2 complexes,
histone H2B was a much better substrate than histone HI.
To examine the substrate specificities of the individual
kinases in more detail, samples were taken from an extract
in mitosis and immunoprecipitated with affinity purified
antibodies. These immunoprecipitates were tested for their
ability to phosphorylate increasing concentrations of a
mixture of sea urchin sperm histones HI and H2B. Figure
6 shows that the kinases immunoprecipitated by anti-cyclin
A antibodies phosphorylated histone H2B more strongly than
histone HI at all histone concentrations tested. At low histone
concentrations, cyclin B associated kinases phosphorylated
histone HI at about twice the rate of histone H2B, but as
the concentration rose above 3 ItM, the phosphorylation of
histone HI declined and histone H2B became a relatively
better substrate. The Lineweaver-Burke plots of these data
were non-linear and showed clear evidence for high substrate
inhibition in all cases, which was most marked for histone
HI as a substrate for the cyclin A -cdc2 kinase activity. At
the low concentrations of histone HI used in Figure 5 (- I
PM), however, cyclin A immunoprecipitates were as efficient
at phosphorylating histone HI as cyclin B immunopre-
cipitates.
The reason why sea urchin sperm histone H2B is a
substrate for cdc2 kinase was suggested by its sequence (von
Holt et al., 1984); unlike the core histones from most
species, it contains cdc2 kinase consensus motifs, X[S/T]
PX[K/R] (Shenoy et al., 1989). When we tested the activity
of the cyclin - cdc2 complexes on rat liver and chicken
erythrocyte histones, we found that histone HI and histone
H5 were good substrates for both A- and B-type cyclin
immunoprecipitates, whereas essentially no activity was
detected against the core histones of these species. In rat and
2869
J.Minshull at al.

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Fig. 5. Immunoprecipitation of cell cycle dependent histone kinase activities with anti-cyclin antibodies. A cell-free extract of Xenopus eggs, prepared
according to Murray and Kirschner (1989a), was incubated at 23°C in the presence of sperm nuclei and [35S]methionine. Samples were placed in
liquid nitrogen at 10 min intervals for subsequent immunoprecipitation, or into fix for cytological examination. Panel A. Total labelled extract
analysed by electrophoresis on a 15% polyacrylamide gel. Panels B and C. Samples (10 pl) were immunoprecipitated with affinity purified
antibodies or p135UC1 -Sepharose as described in Materials and methods. The beads used to bind the antibodies were suspended in 10 ,ul of
phosphorylation mix containing 50 AM [-y-32P]ATP and mixed sea urchin histones (140 pg/mi total histones; 1 pM histone HI, 1.75 pM histone
H2B) in Hi kinase buffer, and incubated at 30°C for 10 min. SDS sample buffer (20 pl) was added, samples were boiled for 90 s and 10 pl loaded
onto a 17.5% polyacrylamide gel. Positions of mol. wt markers are indicated in kd. Panel D. A lighter exposure of the gel shown in panel C was
analysed on a scanning densitometer, and the histone HI kinase activity of each immunoprecipitate was plotted against the time that the sample was
taken.

2870

_._IMD
Fig. 6. Substrate specificities of kinases associated with cyclins Bi, B2
and Al. Mitotic cytoplasm from activated eggs was immuno-
precipitated with affinity purified antibodies. The p34cdc2 -cyclin
precipitates were assayed for their ability to phosphorylate an
equimolar mixture of purified histones HI and H2B from the sea
urchin Echinus esculentus, as described for Figure 5. Phosphorylated
histones were analysed by electrophoresis on a 17.5% SDS-
polyacrylamide gel. Concentrations indicated are for each histone.
chicken, only histones HI and H5 contain the XSPX[K/R]
motif.
To confirm that the cyclin associated kinases could be
ascribed to cdc2 activity, and not some other enzyme, a
mitotic extract was passed over a p13sucl column before
immunoprecipitation of the cyclin associated kinases. The
flow-through from the pl3sucl column lost >98% of anti-
cyclin immunoprecipitable kinase activities. Thus both A-
and B-type Xenopus cyclins are associated with kinase
activity that is bound by pl3sucl, which presumably re-
presents p34cdc2 or a very closely related kinase.
We were concerned that the substrate preferences shown
in Figure 6 might be an artifact produced by the anti-cyclin
antibodies interfering with kinase activity. To test this, an
alternative method was used to generate specific cyclin
associated kinases: a frog egg extract was depleted of endo-
genous mRNA by treatment with pancreatic RNase followed
by RNasin, as described by Murray and Kirschner (1989a).
Each cyclin mRNA was then separately translated in the
extract for 170 min. Such extracts passed through one round
of mitosis and cyclin destruction, and into a second round
of mitosis (they rarely if ever performed a further round of
cyclin destruction within 4 h). Samples were taken from
these extracts, absorbed onto pl3sucl beads, and tested for
their kinase activity as before. The only mRNA present was
added by us, and any endogenous cyclin proteins that might
have been synthesized before nuclease treatment should have
been destroyed during the first in vitro mitosis, so we could
be reasonably confident that these preparations contained
only the desired cyclin -cdc2 complexes. The pl3sucl
Sepharose kinases prepared from these extracts had identical
patterns of substrate preferences as those prepared by
immunoprecipitation (data not shown), suggesting that the
substrate preferences shown by the cyclin A and B immuno-
precipitates were a true reflection of their intrinsic activity
and not an artifact caused by the antibodies.
A and B kinases phosphorylate the same sites on
histones Hi and H2B
We next tested whether the differences in substrate pre-
ferences shown in Figure 6 reflected different affinities for
the same site(s), or whether there were specific cyclin
A- and B- cdc2 kinase target sites on the various histones.
To distinguish between these possibilities, sea urchin sperm
histones H I and H2B were used as substrates for the kinase
activity of anti-cyclin immunoprecipitates. Incubations of up
Cyclin A and cdc2 kinase
.-* w
e
*** *
;
B .
Fig. 7. Phosphopeptide analysis of histones HI and H2B
phosphorylated by cyclin associated kinases. Panel A. Histones HI or
H2B were phosphorylated by kinase immunoprecipitated from mitotic
Xenopus extract with either anti-BI or anti-Al antibodies.
Phosphorylated histones were TCA precipitated and digested with
trypsin as described in Materials and methods. The digestion products
were loaded onto TLC plates, electrophoresed at 1000 V for 17 min in
pH 6.5 buffer, then chromatographed in butanol/acetic
acid/water/pyridine for 2 h. The large negatively charged spot is
probably free phosphate. Panel B. A peptide corresponding to the
N-terminal 24 amino acids of sea urchin histone H2B2 was
phosphorylated by cyclin-cdc2 kinases and the phosphorylation sites
determined as described in Materials and methods. Phosphorylated
serines are indicated by asterisks, and non-phosphorylated serines by
vertical lines.
to 3 h were used in order to achieve maximum phosphoryl-
ation of the sites. The phosphorylated proteins were digested
with trypsin and fingerprinted. Phosphoamino acid analysis
(Cooper et al., 1983) showed only phosphoserine in all cases
(data not shown). We were quite surprised to find that
phosphorylation of histones by either A- or B-cyclin
associated kinases produced identical phosphopeptide maps
(Figure 7A). The phosphorylation sites on histone H2B (see
Figure 7A) were determined by sequence analysis of the
eluted peptides. The faster moving spot was an equimolar
mixture of KA[S-phosphate]PK and KG[S-phosphate]PR,
whereas the slower moving spot corresponded to the N-
terminal peptide, PK[S-phosphate]PSK (see Figure 7B). No
evidence for phosphorylation of serine 8 was found in this
- analysis, but when a synthetic peptide corresponding to the
N-terminus of sea urchin histone H2B2 was used as a
substrate for the kinases, sequence analysis of the phos-
phorylated peptide showed that serines 3, 8, and 13 were
all phosphorylated. In this case, the analysis did not reach
serine 18. All these serine residues have proline residues
as their C-terminal neighbours, whereas serines 5 and 7,
which do not have C-terminal neighbouring prolines, were
not detectably phosphorylated (see Figure 7B). In the case
of histone HI, the predominant peptide is probably a mix-
ture of the peptides [K/R][S-phosphate]P[K/R], which occurs
eight times in this protein. Apparently the phosphorylated
serine inhibits trypsin cleavage at the adjacent basic residue.
These results suggest that the sequence SP forms an
essential part of the recognition site for cdc2 kinase, whereas
the nature of the flanking residues is less critical. The
simplest way to account for the different rates of phosphoryl-
2871
J.Minshull et al.
ation of the various histones shown by the A- and B-type
cyclin-cdc2 complexes is to suppose that the different cyclin
subunits confer different affinities for particular targets on
the kinase subunit; but once the kinase meets its substrate,
it shows strong local target site preferences. These results
and the Western blot shown in Figure 3A imply, although
they do not rigorously prove, that the different cyclins are
associated with the same cdc2 subunit. Had the phosphoryl-
ation sites been different, we would have suspected
otherwise.
Xenopus eggs contain substrates for cyclin A- and
B-associated protein kinases
The foregoing results indicated that A- and B-type cyclins
were associated with protein kinase activities that differed
in their apparent affinities for different histones, although
they phosphorylated the same sites on these histones. We
suspected that this might reflect a difference in the normal
cellular substrates for A- and B-type cyclin associated
kinases. To see if frog eggs contained endogenous proteins
which could be phosphorylated by cyclin- p34cdc2 com-
plexes we prepared 'substrates' as follows: first, an inter-
phase Xenopus egg extract was depleted of endogenous
p34cdc2 by absorption over a pl3`1 column. Next, the
extract was fractionated by gel filtration on an AcA34
column. Individual column fractions were tested for cdc2
kinase substrates by incubation with [Fy-32P]ATP in the
presence or absence of anti-cyclin immunoprecipitates
prepared from a mitotic extract. This fractionation procedure
simplified the pattern of endogenous phosphorylation, and
proved a reliable way to generate substrates for the individual
cyclin-p3402 kinases. The phosphorylated polypeptides in
these fractions need not necessarily be direct substrates for
the added cyclin -cdc2 kinases. Some of them could be
substrates for kinases that were present in the fraction, which
were themselves activated by cyclin-cdc2 kinase. Never-
theless, the appearance of new phosphorylated proteins upon
addition to the cyclin -cdc2 kinases shows that the column
fraction contains a substrate of the added kinase, whether
the actual bands are direct or indirect targets.
Figure 8 shows results from two column fractions con-
taining polypeptides that were substrates for cyclin-cdc2
kinases. These polypeptides fell into several categories as
indicated by the arrows. Some polypeptides (ppl09, pplO2,
pp32) were equally well phosphorylated by anti-cyclin A and
anti-cyclin B immunoprecipitates. We suspect that ppl09
corresponds to the 110 kd mitotic phosphoprotein previously
described by Karsenti et al. (1987). Other bands were
phosphorylated preferentially by the B-type kinase complexes
(pp65) or by the A-type complexes (pp74), but not by both.
Thus, there are proteins in Xenopus eggs which are differ-
entially phosphorylated by the kinases associated with A-
and B-type cyclins, but we are more struck by the similarities
in target specificity at this level of discrimination than the
differences. None of these endogenous target proteins are
yet identified, and as discussed above, we cannot be absolutely
certain that they are direct substrates for the cyclin-cdc2
kinases.
Discussion
We have been studying the behaviour of cyclin A during
the Xenopus cell cycle. We describe the isolation and
2872
Isss:
Gil..
Fig. 8. Frog egg proteins phosphorylated by p34cdc2_cyclin kinases.
A Xenopus egg extract was depleted of p34cdc2 and cyclins as
described in Materials and methods. The extract was gel filtered on an
AcA34 column, and fractions numbers 21 and 43 (corresponding to
native mol. wts of - 470 kd and 25 kd respectively) were used as
substrates for phosphorylation by cyclin-cdc2 kinases. The substrate
was made 50 ,uM in [,y-32P]ATP, 10 Id aliquots were transferred to
tubes containing cyclin immunoprecipitates and incubated for 15 min at
30°C. Samples were processed as decribed for Figure 5, and analysed
on a 15% SDS-polyacrylamide gel. Positions of mol. wt markers are
indicated in kd. Lanes 1-3, no added substrate, immunoprecipitates
with: lane 1, B1; lane 2, B2; lane 3, A1. Lanes 4-7, fraction no. 21;
lanes 8-11, fraction no. 43. Immunoprecipitates used as kinases were:
lanes 5 and 9, B1; lanes 6 and 10, B2; lanes 7 and 11, Al. Lanes 4
and 8 contained no immunoprecipitated kinases and show the
autophosphorylation of the two fractions.
sequence of a Xenopus oocyte A-type cyclin cDNA. Frog
cyclin A mRNA is about as abundant as that of each of the
B-type cyclins, but cyclin A makes up only 10% of the -
total cyclin synthesis, which makes the synthesis and
destruction of the polypeptide difficult to detect in cycling
cell-free extracts labelled with [35S]methionine. The major-
ity of cyclin A is found in a complex with a p34cd2 subunit.
This complex shows protein kinase activity which rises more
steadily and earlier in the cell cycle than that of the cyclin
B -cdc2 kinase; indeed, the kinase activity of the cyclin
A - cdc2 complexes peaks before nuclear envelope break-
down occurs, and cyclin A is destroyed earlier in the cell
cycle than cyclin B. The cyclin A immunoprecipitates lose
HI kinase activity at this time. It is not possible to say if
the cdc2 kinase subunits that lose their cyclin A subunit
immediately lose their kinase activity. The pl3suc -Sephar-
ose beads lose kinase activity shortly after the destruction
of the cyclins, however, so inactivation of cdc2 kinase must
follow the loss of cyclin quite rapidly.
The cyclin A -cdc2 and cyclin B -cdc2 kinases show
different preferences for sea urchin sperm histones HI and
H2B as substrates, although the same sites on these proteins
are phosphorylated by both enzymes. This suggests that the
catalytic subunits of the A- and B-type kinases are similar,
and may be identical. We also show that endogenous (non-
histone) substrates for the cdc2 kinase complexes can be
found in Xenopus eggs. At least some of these substrates
are preferentially phosphorylated either by the A-cyclin or
by the B-cyclin complexes, consistent with idea that cyclins
can act as targeting subunits of cdc2 kinase, although con-
siderable overlap in substrate preferences appear to exist.
It is uncommon for protein kinases to have targeting
subunits. The regulatory subunit of cyclin AMP-dependent
protein kinase acts as an inhibitor of the enzyme, and cAMP
activates kinase activity by promoting the dissociation of the

rcydrn A targe(
protons
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OdC2
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orfation
A: A 'parallel' model for the control of ntosis: two
or three kinases are required for M-phase
-yclin cydi r7
LA Aphc6pnatase A
cdc IT L free system, which suggests either that cyclin A is super-
B-phosphatase
(3rl
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ormalion
B: A 'seral' (cascade) model of cdc2 acftvaton.
Fig. 9. Possible models of cyclin A and B collaboration in controlling
entry into M phase. Two possible ways are shown in which cyclin
A-cdc2 and cyclin B-cdc2 complexes may act together to cause M
phase. In A, the two cyclin-cdc2 complexes phosphorylate different
substrates. Differences in kinetics of activation of these kinases may
reflect the requirement for the phosphorylated target proteins at
different times in M phase. In model B, association of cyclin A with
cdc2 makes cdc2 a substrate for a phosphatase which activates it. The
activated cyclin A-cdc2 kinase then phosphorylates cyclin B-cdc2
phosphatase and activates cyclin B-cdc2 complexes. These in turn
may activate a phosphatase which dephosphorylates and activates free
cdc2. All these reactions are presumed to be reversed by a cdc2 kinase
(not shown). Destruction of cyclin turns off the activation reaction,
which allows the rephosphorylation and inactivation of cdc2 kinase.
subunits (Builder et al., 1981). A similar kind of mechanism
appears to operate in the control of protein kinase C, which
contains a pseudo-substrate sequence in its regulatory domain
(House and Kemp, 1987). It is perhaps easier to imagine
how regulatory subunits mask kinase activity than to see how
they might direct the enzyme to specific target proteins,
particularly since there is evidence to suggest that cdc2 kinase
may have activity, against histone HI at least, in the absence
of cyclin subunits (Labbe et al., 1989b; Hutchinson et al.,
1989); but histone HI kinase activity may represent a kind
of default substrate preference, and the 'real' targets in the
cell may be poor substrates unless cyclins are present to guide
the cdc2 subunit. This idea is presented in Figure 9A, which
suggests a model in which there are distinct cyclin A and
cyclin B cdc2 kinase targets with complementary roles in
-
the cell cycle. Another kind of way in which cyclin subunits
might act is to influence the subcellular distribution of their
associated kinase subunits. Pines and Hunter (1990) have
presented evidence that human cyclin A shows predomin-
antly nuclear localization in HeLa cells, whereas cyclin B
is found in the cytoplasm until nuclear envelope breakdown
occurs.
Cyclin A and cdc2 kinase
The evidence for the existence of distinct substrates for
the A- and B-type cyclin-cdc2 kinase complexes suggests
that the two cyclins collaborate in inducing mitosis.
Drosophila cyclin A mutants are unable to perform the cell
divisions required for cell division in neuroblasts, which
arrest in G2 (Lehner and O'Farrell, 1989), and in the
fission yeast Saccharomyces pombe, elimination of the cdc13
(cyclin B) gene also causes G2 arrest (Booher and Beach,
1988). Molecular and biochemical evidence from other
organisms is as yet less clear-cut. In starfish and Xenopus,
cyclin B is necessary for activation of cdc2 kinase, MPF
and entry into M phase (Picard et al., 1989; Minshull et al.,
1989a; Murray et al., 1989). Murray and Kirschner (1989a)
found that translation of a sea urchin cyclin B mRNA alone
could support multiple rounds of mitosis in the Xenopus cell-
fluous, or that the sea urchin cyclin can to some extent mimic
the properties of frog cyclin A, or that cyclin A performs
a subtle role that is inadequately monitored in the cell-free
system.
Implicit in the foregoing discussion is the idea that the
cyclins are required for kinase activity of p34cdc2 because
they are integral subunits of the kinase. An alternative view
is that cyclins are not primarily kinase subunits at all, but
exist mainly to permit the activation of cdc2 kinase by post-
translational modifications. According to this model, cyclins
do not act solely as targeting subunits of the kinase, but also
function as targeting subunits for putative p34cdc2 kinases
and phosphatase(s). In this scheme, illustrated in Figure 9B,
association of cyclin with p34lcdc2 would allow dephos-
phorylation and activation of the cdc2 subunit. A circuit of
the kind suggested by the classical evidence for positive
feedback control of MPF (Wu and Gerhart, 1980; Masui
and Clarke, 1979) could be provided by the two cyclin-cdc2
kinases (Figure 9B). Cyclin A would combine with a
molecule of p34cdc2, thereby making it a target for a
phosphatase. This active cyclin A -cdc2 kinase would then
phosphorylate and activate the phosphatase that recognizes
cyclin B -cdc2 complexes, which would thereby be activated
and might in turn promote the activation of p34cdc2 that is
not associated with cyclin subunits. As long as cyclins are
associated with cdc2, the mitotic phosphatase(s) would stay
switched on, but once the cyclins had been destroyed, the
cdc2 subunit could no longer be maintained in its dephos-
phorylated state. Activation of the cdc2 phosphatase by
phosphorylation is consistent with the observation that
inhibitors of type 1 and 2A phosphatases result in activation
(and therefore dephosphorylation) of cdc2 kinase (Picard
et al., 1989; Felix et al., 1990; Hunt, 1989).
Whichever of these models is correct, we strongly suspect
that there is important collaboration between these closely
related yet clearly different cyclins. The kinetics of activation
of the A- and B-type kinase complexes in the cell-free mitotic
extract is consistent with the idea that cyclin A may be
involved in the activation of cyclin B - cdc2 kinase in some
sort of kinase cascade, or performs some essential function
that sets up the correct conditions for the B-type cyclins to
act. We are presently attempting to test these ideas directly.
Materials and methods
Isolation and sequencing of cDNA clones
Cyclin A clones were isolated from frog oocyte poly (A)' RNA cDNA
libraries in XgtlO provided by John Shuttleworth and Doug Melton. The
2873
J.Minshull et al.
libraries were screened with an oligonucleotide with the sequence
5'-TCTGGRGGRTAYATCTCYTCRTATTT-3' (corresponding to the
amino acid sequence KYEEIYPPD) in parallel with a mixed probe made
from full-length Xenopus cyclin Bi and B2 clones. Screening and sequencing
procedures were performed as described by Minshull et al. (1989a).
Raising and purifying antibodies
Polyclonal anti-cyclin antibodies were raised in rabbits by injection of gel
purified proteins made in Escherichia coli as described in Gautier et al.
(1990). Anti-Bi antibodies were raised against protein expressed by the
cyclin BI-pWEV construct (Gautier et al., 1990). Anti-cyclin B2 antibodies
were raised against the complete cyclin B2 protein (Minshull et al., 1989a),
which was expressed from a subclone of the coding region of XlcycB2 in
pET3b in the strain BL21 (DE3) (Studier and Moffatt, 1986). Anti-cyclin
A antibodies were raised against a fusion protein generated by subcloning
the largest Sau3A fragment of XLcycAl (indicated by bold underlining of
the sequence GATC in Figure 1) into the BamHI site of pET3a (Studier
and Moffatt, 1986). This protein lacked the N-terminal 55 amino acids of
cyclin Al, and the authentic frog sequence SVSRGG... follows the vector
encoded sequence MASMTGGQQMGRG as its N-terminus (see Figure 1).
Antibodies were affinity purified as described by Harlow and Lane (1988).
Affinity columns were prepared from the bacterially expressed antigens
solubilized in SDS and coupled to cyanogen bromide-activated Sepharose
according to the manufacturer's instructions.
Preparation of Xenopus egg extracts
Cell-free extracts of Xenopus eggs were prepared according to Murray and
Kirschner (1989a). Mitotic egg cytoplasm as a source of cyclin associated
kinases was made by incubating an extract at 23°C until it entered mitosis.
Samples (50 yd) were frozen in liquid nitrogen and stored at -80°C until use.
Preparation of p136I1 - Sepharose
pl3suc -Sepharose was prepared by a modification of the procedure
described by Brizuela et al. (1987). pI3suc1 was prepared from E.coli strain
BL21(DE3) transformed with a plasmid containing the sucl+ gene under
the control of the T7 gene 10 promoter. The bacteria were grown to an
A6W of 0.5, and then induced with 0.5 mM IPTG for 3- 10 h. The bacteria
were spun down and stored frozen at -80°C. They were thawed in lysis
buffer (20 mM HEPES, 2 mM EDTA, 1 mM DTT and 1 mM PMSF,
pH 7.5). Lysozyme was added to 2 mg/ml, incubated on ice for 15 min,
and the lysate sonicated until no longer viscous. Insoluble material was
removed by centrifugation for 20 min at 10 000 g, the supernatant dialysed
against lysis buffer and loaded onto a DEAE column. The column was eluted
with a gradient of NaCl in lysis buffer: pl3SUCl eluted between 30 and 60
mM NaCl. p13sucl containing fractions were pooled and concentrated by
adding ammonium sulphate to 65% saturation. The pellet was taken up in
a small volume of coupling buffer (500 mM NaCl, 100 mM sodium
carbonate buffer, pH 8.3) and fractionated on a gel filtration column
equilibrated with coupling buffer. pI3sucl eluted as a low molecular weight
protein (10-20 kd), and was coupled to CNBr-activated Sepharose 4B
(Pharmacia) at 2 mg pl3SuC1 per ml of beads, following the manufacturer's
instructions.
p 138UC1 affinity absorption of Xenopus extracts
Xenopus egg extracts were affmity absorbed onto pI3SUCl -Sepharose using
a procedure adapted from Dunphy et al. (1988) and Meijer et al. (1989).
The extract was diluted at least 5-fold in HI kinase buffer (80 mM sodium-
(3-glycerophosphate, 20 mM EGTA, 15 mM MgCl2, 1 mM DTT), then
spun in a microfuge at 14 000 r.p.m. for 2 min. The supernatant was trans-
ferred to a tube containing p13suc1 -Sepharose (20 al of packed beads per
10 11 of extract) equilibrated with H1 kinase buffer and rotated end over
end for 30 min at room temperature. The beads were washed three times
in bead buffer (50 mM Tris-Cl pH 7.4, 5 mM NaF, 250 mM NaCI, 5
mM EDTA, 5 mM EGTA, 0.1% Nonidet P-40, 1 ag/ml leupeptin, 2 pg/mn
aprotinin, 10 jg/ml soybean trypsin inhibitor, 100 pg/mn benzamidine) with
a change of tube at the second wash, then twice in H I kinase buffer. The
beads were then either resuspended in SDS sample buffer or used
immediately for HI kinase activity determination.
Immunoprecipitation of Xenopus cyclins
Cyclins were immunoprecipitated from Xenopus egg extracts using affinity
purified antibodies. Extracts were diluted at least 5-fold in HI kinase buffer
and incubated with antibody for 1 h on ice. The tubes were then spun in
a microfuge at 14 000 r.p.m. for 2 min. The supernatant was transferred
to a tube containing protein A-Sepharose equilibrated with Hl kinase buffer,
and rotated end over end for 30 min at room temperature. The beads were
washed and used as described for pl3suc affinity absorption.
2874
Analysis of immunoprecipitates by Western blotting
To detect the presence of p34cdc2 in anti-cyclin immunoprecipitates, the
immune precipitates bound to protein A-Sepharose were suspended in SDS
sample buffer containing 100 mM N-ethylmaleimide in place of
2-mercaptoethanol and analysed on SDS-polyacrylamide gels. The 'NEM
sample buffer' maintains the first antibodies' disulphide bonding so that they
run on the subsequent SDS-polyacrylamide gel with a molecular weight
> 100 kd and do not interfere with subsequent antibody detection (both the
anti-cyclin and anti-p34cdc2 antibodies were raised in rabbits). Proteins were
transferred to nitrocellulose, a marker strip stained with Ponceau S so that
the portion of the gel containing proteins > 65 kd could be removed. The
low molecular weight region of the blot was blocked with TBST (10 mM
Tris-Cl, pH 8.0, 150 rmM NaCl, 0.05% Tween-20) with 4% w/v dried
skimmed milk and subsequently transferred to TBST containing 2% milk
and the primary (anti-PSTAIR) antibody at 4°C overnight. The antibodies
were located with alkaline phosphatase conjugated goat anti-rabbit IgG from
Bio-Rad.
Preparation of Xenopus egg protein kinase substrates
An interphase extract was prepared from Xenopus eggs which had been
activated in the presence of cycloheximide. This extract was depleted of
endogenous p34cdc2 by diluting 2-fold in HI kinase buffer and passing it
down a p13SUC. column. Next, the extract was fractionated by gel filtration
on an AcA34 column equilibrated with HI kinase buffer. Fractions were
phosphorylated by incubation with [-y-32]ATP (50 /AM, 1.6 mCi/ml) in the
presence or absence of immunoprecipitated kinases prepared from a mitotic
extract.
Phosphorylation of proteins by cyclin associated kinases
[-y-32P]ATP (1 mM ATP and 32 mCi/ml 32P04 at the activity date) was
prepared from carrier-free phosphoric acid by the method of Glynn and
Chappell (1964).
The protein kinases were usually bound either to pI3SuCI -Sepharose,
or to protein A -Sepharose via one of the anti-cyclin antibodies. A packed
volume of 5- 10 ul beads was mixed with 10 tl of a reaction mix containing
the substrate protein (normally mixed histones at a final concentration of
0.25 mg/mi) and ATP (50 jsM and 0.3 mCi/ml) in HI kinase buffer. The
bead suspension was incubated for 10 min at 30°C, then the reaction
terminated by the addition of 20 Al SDS sample buffer (Anderson et al.,
1973) containing 25 mM EDTA. Crude extract was assayed for kinase
activity as described by Cicerelli et al. (1988).
Purification of histone substrates
Histones were prepared from nuclei of sperm of the sea urchin Echinus
esculentus (Thomas et al., 1986). The nuclei were lysed in 0.2 mM Na2
EDTA, extracted with 0.2 M H2SO4 on ice for 45 min, and then neutral-
ized with 1 M triethanolamine. The solution was dialysed at 4°C against
several changes of 0.1 % w/v ammonium bicarbonate and lyophilized. We
assume that 1 mg/mi mixed histones had an absorbance at 230 nm of 3.3
(Camerini-Otero etal., 1976). Histones were individually purified by reverse-
phase HPLC using a C4 Hi-Pore column (BioRad) as described by Hill
and Thomas (1990). The peptide corresponding to the first 24 amino acids
of sperm histone H2B2 from Echinus esculentus, PKSPSKSSPRKGSP-
RKASPKRGGK, was synthesized by the Peptide Synthesis Facility at the
Institute of Animal Physiology and Genetics, Babraham.
Phosphopeptide analysis of histones
To analyse the peptides in histones that were phosphorylated by various
kinases, 25 jig of histones [either purified HI or H2B2, 1 mg/mi solutions
of which have A230 of 1.85 and 4.2 respectively (Camerini-Otero et al.,
1976; Thomas and Butler, 1977)] were incubated with 2 mM ATP and
immunoprecipitated kinase from 25 pl of mitotic Xenopus extract in a reaction
volume of 100 ,d, at 30°C for 3 h. Phosphorylated histones were removed
from the bead-bound kinase, TCA precipitated and redissolved in 40g1I of
0.13 mM ammonium bicarbonate containing 0.5 itg of trypsin. This was
incubated at 37°C for 4 h, lyophilized, redissolved in 3yd of 5 mM acetic
acid and spotted onto POLYGRAM® SIL G TLC plates. Peptides were
mapped by electrophoresis in the first dimension for 17 min at 1kV in
pH 6.5 buffer (0.5% v/v acetic acid, 10% v/v pyridine in water), and
developed in a second dimension of butanol/acetic acid/water/pyridine
15:3:12:10 v/v. Phosphorylated peptides were located by autoradiography
and eluted from the chromatograms with 0.1 M HCI. Their sequence was
determined on an Applied Biosystems gas phase sequencer. Phosphoserine
was identified by the formation of an adductbetween its,8-elimination product
(dehydroalanine) and dithiothreitol as previously described in Hill et al.
(1990). We are most grateful to Dr Len Packman for performing these
analyses.

The synthetic peptide was phosphorylated as described for full-length
histones. Phosphorylated and non-phosphorylated forms were separated by
reverse-phase HPLC using a C-18 Spherisorb 3 ODS II column.
Phosphoserines were identified as described above.
Analysis of labelled proteins on SDS - polyacrylamide gels
Labelled proteins were analysed by electrophoresis on SDS-polyacrylamide
gels made according to Anderson et al. (1973). Proteins labelled with
[35S]methionine (Amersham SJ1515) were detected by autoradiography on
Amersham 'Hyperfilm (3-Max'. Proteins labelled with 32p04 were detected
by fluorography on Fuji RX film. Molecular weight markers were made
as described by Evans et al. (1983).
Acknowledgements
We thank Chris Ford for showing us the first direct evidence that cyclin
A existed in frogs, to Andrew Murray for sharing his protocol for making
'cycling extracts' and to Len Packman for performing sequence analyses.
Doug Melton and John Shuttleworth generously provided cDNA libraries.
We also thank Jean Gautier, Eric Karsenti, Jim Maller, Paul Nurse, Michel
Philippe and Jian Kuang for much helpful advice and encouragement. This
work was supported by the CRC, the MRC and the Wellcome Trust, which
also supported oligonucleotide and peptide synthesis. R.G. holds a Fellowship
from the Commissioners of the Exhibition of 1851, and C.S.H. holds the
Gateway Research Fellowship at New Hall, Cambridge.
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Received on May 17, 1990
Note added in proof
The sequence reported here has been submitted to the EMBL database with
the accession number X53745, and the title X.laevis cyclin A cDNA.
2875