Download Translation of Cyclin mRNA Is Necessary for Extracts of Activated

Cell, Vol. 56, 947-956,
March 24, 1989, Copyright
0 1989 by Cell Press
Translation of Cyclin mRNA Is Necessary
for Extracts of Activated Xenopus Eggs
to Enter Mitosis
Jeremy Minshull,’ J. Julian Blow,t* and Tim Hunt’
Department of Biochemistry
University of Cambridge
Tennis Court Road
Cambridge CB2 1QW
England
tCRC Molecular Embryology Group
Department of Zoology
University of Cambridge
Downing Street
Cambridge CB2 1QW
England
l
The cyclins are a family of proteins encoded by maternal mRNA. Cyclin polypeptides accumulate during interphase and are destroyed during mitosis at about
the time of entry into anaphase. We show here that
Xenopus oocytes contain mRNAs encoding two cyclins that an? major translation products in a cell-free
extract from activated eggs. Cutting these mRNAs
with antisense oligonucleotides and endogenous RNAase H blocks entry into mitosis in a cell-free egg extract. The extracts can enter mitosis if either of the
cyclin mRNAs is left intact. We conclude that the synthesis of these cyclins is necessary for mitotic cell cycles in cleaving Xenopus embryos.
Cyclins are proteins that accumulate steadily during interphase and are destroyed at the end of mitosis (Evans et
al., 1983; Swenson et al., 1986; Standart et al., 1987).
They were first recognized in marine invertebrate eggs as
major translation products of maternal mRNA, but up to
now have not been described in vertebrates. However, almost all eukaryotic cells require protein synthesis to pass
from interphase into mitosis or meiosis (Hultin, 1961;
Kishimoto and Lieberman, 1964; Dettlaff, 1966; Wilt et al.,
1967; Wasserman and Masui, 1975; Wagenaar, 1983;
Gerhart et al., 1984; Picard et al., 1985). This protein synthesis requirement could easily be explained if there were
proteins that were required for entry into M phase and destroyed as a result of it. Since cyclins behave in this way,
we set out to look for them in Xenopus eggs, aiming to exploit the experimental advantages and the wealth of previous work on the cell cycle dynamics of maturationpromoting factor (MPF) in this system, as reviewed briefly
below.
MPF is generally agreed to be the cytoplasmic agent
responsible for the initiation of M phase (for review see
Masui and Shibuya, 1987). MPF was originally identified
t Present address: Microbiology Unit, Department of Biochemistry,
University of Oxford, South Parks Road, Oxford OX1 3QU, England.
in unfertilized amphibian eggs by Masui and Markert
(1971) and Smith and Ecker (1971) as an activity capable
of inducing amphibian oocytes to enter meiosis. It has
subsequently been identified in a wide variety of meiotic
and mitotic cells from yeast to man (Kishimoto and
Kanatani, 1976; Wasserman and Smith, 1978; Sunkara et
al., 1979; Kishimoto et al., 1982; Weintraub et al., 1982;
Gerhart et al., 1984; Meijer and Guerrier, 1984). MPF can
also drive GP-arrested cells into mitosis (Miake-Lye et al.,
1983; Newport and Kirschner, 1984; Kirschner et al.,
1985). Thus, MPF is an almost universal concomitant of
M phase, and is probably responsible for catalyzing the
G2-M transition in all eukaryotic cells.
MPF has been purified from Xenopus eggs by Lohka et
al. (1988) and from starfish oocytes by Labbe et al. (1988a)
and by Arion et al. (1988). All these preparations contain
a 32-34 kd subunit, which by two independent criteria corresponds to the product of the Schizosaccharomyces
pombe c&2+ gene (Dunphy et al., 1988; Gautier et al.,
1988; Labbe et al., 1988b; Arion et al., 1988). The c&2+
gene encodes a protein kinase that is essential for the
G2-M transition in fission yeast (Nurse et al., 1976;
Simanis and Nurse, 1986; Lee and Nurse, 1987).
Although microinjection of MPF is sufficient to initiate M
phase in G2-arrested cells, newly synthesized protein(s)
is normally required to activate MPF during the cell cycle
(Wasserman and Smith, 1978; Ford et al., 1983; Miake-Lye
et al., 1983; Gerhart et al., 1984; Ford, 1985). It is probably
not necessary to synthesize MPF itself, because this protein can be detected in interphase cells as an inactive form
known as pre-MPF (Cyert and Kirschner, 1988; Dunphy
and Newport, 1988). Moreover, the c&2+-homologous
subunit of MPF is already present in unfertilized starfish
oocytes and is stable during meiosis while MPF activity
levels rise and fall (Labbe et al., 1988a). It is thus probable
that protein synthesis supplies an MPF activating protein
that needs to be synthesized anew in each cell cycle.
The behavior of cyclins makes them attractive candidates for MPF activator proteins. Direct evidence for the
ability of cyclins to activate MPF has so far been limited
to experiments in which frog oocytes were injected with cyclin mRNAs and scored for entry into first meiosis. Thus,
synthetic mRNAs encoding clam and sea urchin cyclins
are both capable of inducing germinal vesicle breakdown
(Swenson et al., 1986; Pines and Hunt, 1987). Experiments of this type do not, however, distinguish between
genuine MPF activators and more distal components of
the maturation pathway. An alternative approach is to
study cell-free extracts that require protein synthesis to enter mitosis and permit more than one round of DNA replication (Lohka and Masui, 1983, 1984; Hutchison et al.,
1987; Blow and Laskey, 1988). If it could be shown that cyclin synthesis is necessary and sufficient to promote mitosis in vitro, this would provide strong support for the cyclin-MPF connection.
In this paper we report the isolation and sequence of
two homologous (but not identical) cyclins from Xenopus
Cell
946
Xenopus cyclin Bl
I
MSLRVTR
~TTCCCACTTAGTGAGACGTCTCTTTCAGACTGGGGGGCTGCAGTGTGACTTGTGGGTACAAATAGTAAAGCTW\CTGCAGGTTTGTCACCAGA
NMLANAENNVKTTLAGKRVVATKPGLRPRTALGDIGNKAE
AACATGCTGGC~ATGCAGAAAACAATGTGAAAACCACTTTGGCTTTGGCTGGA~GAGGGTTGTTGCTACCA~CCAGGGTT~GACCTCGTA~GCCTTGGGA~~TTGG~AC~GGCA~G
VKVPTKKELKPAVKAAKKAKPVDKLLEPLKVIEENVCPKP
GTGAAAGTGCCAACAAAAAAGGRATTARAGCCAGCCAGCAGT~~GCTGCC~G~GGC-CCTGTTGAC~ATTGTT~AGCCTCTT~GTGATA~GA~TGTTTGCCCTA~CCT
AQVEPSSPSPMETSGCLPDELCQAFSDVLI
HVKDVDADDD
GCTCAGGTTGAACCCAFCTCACCAAGCCCMTGGAAACATCTGGTTGCCTCCCTGATGAGCTCTGCCAGGCTTTCTCTGATGTCCTCATTCACGTTAAAGATGTTGATGCTGATGATGAT
GNPMLCSEYVKDIYAYLRSLEDAQAVRQNYLHGQEVTGNM
GGCAACCCRATGCTGTGCAGTGk4TATGTCAAGGACATTTATGCTTACCTGAG~GCCTTGAGGATGCA~AGCAGTCAGACA~ACTACCTTCATGGACAGGAAGTCACAGGCAACATG
RAILIDWLVQVQMKFRLLQETMFMTVGI
IDRFLQEHPVPK
CGTGCCATTTTGATTGACTGGCTGGTCCAGGTGC~TG~TTCCGTCTACTGCAGGAGACAATGTTCATGACTGTTGGCAT~TTGACCGCTTTCTGCAGGAACATCCAGTTCCC~A
NQLQLVGVTAMFLAAKYEEMYPPEIGDFTF"TDHTYTKAQ
AACCAGCTACAGCTTGTGGGGGTCACGGCTATGTTCCTTGCTGCT~ATA~~~~~~~~~~~~~,~~,~.~.~.~.~~~~~TGGAGACTTTACATTTGTAACTGATCACACATACACAAAGGCTCAA
IRDMEMKILRVLKFAIGRPLPLHFLRRASKIGEVTAEQHS
ATTCGGGACATGGAAATGAAGATACTTAGGGTGCTAAAGTTTGCAATTGGCCGACCCTTACCCCTGCACTTTCTTCGGAGAGCTTCTAAAATTGGAW\GGTAACTGCTGCTG~CAGCATAGT
LAKYLMELVMVDYDMVHFTPSQIAAASSCLSLKI
LNAGDW
TTAGCCAAATATTTGATGGACTTGTGATGGTGGATTATGATATGGTACATTTCACGCC?TCCC~TAGCAGCTGCTTCCTCCTGCTTGTCTCTC~AATCTT~ATGCAGGTGACTGG
TPTLHHYMAYS
EEDLVPVMQHMAKNII
KVNKGLTKHLTVK
ACCCCAACACTCCATCACTATATGGCTTACTCTGAAGAAGATCTAGTCCCTGTTATGCAGCATATGGCCAAGG
NKYASSKQMKI
STIPQLRSDVVVEMARPLM'
AACMGTATGCTAGCAGCAAACRAATGRAGATG~~TCAG~CGATTCCACAGCTGAGGTCAGATGTTGTTGTGGAAATGGCCCGCCCACTCATGTGAAGGACTACGTGGCATTCCAATTGTGTA
TTGTTGGCACCATGTGCTTCTGTAAATAGTCTAGTGTT
Xenopus cyclin 82
MATRRAAIPREADNILG
GAMRSK
GAATTCCGGCTAGATTTTATCGGTTGGTTTTA?~CGTTATTTTACCGGAGATGGCTACTCGTCGCGCTGCTATTCCCCGT~GC~AT~TATCCTTGGGGGTGC~TGCGATCCA~G
GNKVTVRGKPPAVKQSSNAVAKP
VQMNSRRAALGEI
SKMA
TTCMATGAATAGCAGACGAGCTGCTTTGGGA~GATTGGCAACA~GTGACTGTGCGAGG~AACCACCTGCAGTA~GCAGTCTTC~TGCTGTGGCA~GCCTTC~A~TGGCAG
ATKVANVKTKHVPVKPVVAEAAPKVPSPVPMDVSLKEEEL
CAACTAAAGTGGCAAATGTTAAGACTAAGCATGTACCTGTGAAACCAGTTGTAGCTGAAGCTGCCCCCAAGTGCCTTCCCCTGTGCCGATGGATGTGTCETTGAAAGAGGAAGAGCTGT
CQAFSDALTSVEDIDADDGGNPQLCSDYVMDIYNYLKQLE
GCCAGGCATTCTCCGATGCACTGACCAGTGTTGAAGACATTGATGCAGATGATGGTGGAAACCCTCAATG
VQQSVHPCYLEGKE
INERMRAILVDWLVQVHSRFQLLQET
TTCAACAGTCTGTACATCCTTGCTATCTTGAAGGAAAAGATTAATGAGCGTATGAGAGCTATCCTAGTTGACTGGCTTGTTCAAGTGCATTCTAGGTTTCAGCTTCTTCAGGAGACTT
LYMGVAIMDRFLQVQPVSRSKLQLVGVTSLLI
ASKYEEMY
TATACATGGGCGTTGCR9TCATGGATCGCTTCTTACAAGTTCAGCCAGTCTCCCGCAGTAAGCTTCAGTTGGTTGGTGTTACTTCCCTACTAATTGCTTCAAAATATATGAAGAGATGTACA
. . . . . *. . . . . . . . . . . . . . . . . . . . .
REMEMII
TPEVADFVY
ITDNAYTASQI
LRLLNFDLGRPLP
CT,~,C~.~.~~~~TGCAGACTTTGTTTATATCACCGATAAATGATTATCCTTCGACTCCTCAACTTTGACCTTGGACGGCCATTACCTC
LHFLRRASKSCSADAEQHT
LAKYLMELTLI
DYEMVHIKPS
TCCACTTCCTCAGACGGGCTTCAAAATCTTGCAGTGCTGATGCAGAGCAACATACCCTTGCAAAATATATTTGATGGAGCTTACACTCATAGACTATG~TGGTCCACATCAAGCCTTCAG
EIAAAALCLSQKI
LGQGTWGTTQHYYTGYTEGDLQLIMKH
AAATTGCAGCTGCTGCCCTCTGCCTATCTCAA~AATTCTTGGCCAGGGAACCTGGGGTACCACTCAGCACTATTACACAGGCTACACAG~GGTGACTTGCAACTGATCATGAAGCATA
MAKNI
TKVNQNLTKHVAVRNKYASSKLMKISTLPQLMAPL
TGGCTAAGAACATAACCAAAGTCAACCAGAATCTAACRAAGCATGTGGCTGTGAGGAACAAGTATGGCACCCTTCCTCAGCTTATGGCTCCTCTAA
ITELAASLS*
TCACAGAGCTTGCTGCAAGTCTCTCTTAGAACTGTTAAGTTAAGTGACCCTTTCAAAGAGAACCACTAATTGCACTTTTAACGTTGCTGCTGGCA~TGCTGCTGTTCCTGT~ATATGTTTGTA
TTTTTATTGACTCATTTGTWACTTTGAGTAATGCTTTTTTTATTT
APAAAAAAAACGGAATTC
Figure 1. Nucleotide
and Derived Amino Acid Sequences
of X. laevis Cyclins Bl and 82
The nucleotide sequences and derived amino acid sequences of the LgtiO Xenopus cyclin clones Xlcycl and XlcycP are shown. The sequences
begin with an EcoRl linker, and contain the polyadenylation signal AATAAA about 16 nucleotides before a poly(A) tract at the end of the clone. Termination codons are indicated by asterisks; the single TGA marked in the 5’ untranslated leader of cyclin 81 is in the same frame as the coding region.
Positions complementary to the cyclin Bl- and BP-specific antisense oligonucleotides are marked with solid underlining, and positions corresponding
to the consensus oligonucleotides (antisense #426, sense #120) are marked with dotted underlining. The redundant oligonucleotide used to screen
the original Ml3 libraries overlapped and included this region. Sequence data have been submitted to the GenBanWEMBL data library (accession
nos. JO3166 and JO3167).
oocyte cDNA libraries. We present evidence that translation of these mRNAs is necessary for entry into mitosis.
Results
Isolation and Sequence of Cyclin cDNA Clones
Cyclins were first detected because they are major translation products of maternal mRNA in clams and sea urchins (Evans et al., 1983). Their destruction during mitosis
was easily observed owing to the natural division synchrony of fertilized eggs. Similar kinds of experiments
in Xenopus did not immediately reveal periodically degraded proteins (data not shown). We therefore decided
to look for frog cyclin mRNA in a cDNA library made from
fertilized Xenopus egg poly(A)+ RNA: Sau3A cDNA fragments were inserted into an Ml3 phage vector (see Experimental Procedures). The library was screened in parallel with a full-length sea urchin B-type cyclin cDNA clone
(cyc4; Pines and Hunt, 1987) and aconsensus oligonucleotide with a sequence based on comparison between the
published clam and sea urchin sequences (see Experimental Procedures). Several clones were positive for
both probes. The clones fell into two classes, cyclin Bl
(represented by clone R3, with 209 nucleotides) and cyclin
B2 (clone A22, with 158 nucleotides). Their sequences
overlapped by 133 residues and showed 88%~ identity at
the nucleotide level. In the region of overlap, 27 out of 44
amino acids were identical in the open reading frame cor-
t&ins
and Mitosis in Xenopus
(4
Frog (X. lawis) cyclin 82
;;i
+
300
200
/
3
g
li
Clam (S. solidissima) cyclin A
Sea urchin (A. punclulala)
/
s
.3
e$
I
Eggs
/
100
i-7
'
I
0
100
a0
300
400
(N
MSQPFALHHDGENNGLQMQRRGKMNTRSNQVSGQKRAALGKNQQVRIQP
MALGTRNMNMNLHGESKHTFNNENVSARLG
MSLRVTRNMLANAENNVKTTLAGK
MATRRAAIPREADNILGGA
MTTRRLTRQHLLANTLGNNDENHPSNHIARAKSSLHSSENSLVNGKKATVSSTNVPKKRHALDDBSNFHNKEGVPLASKNTNVRHTTASVS
cdcl3+
COIlSi=?KlSUS
Clam A
Sea urchin
Froo Bl
Fro; B2
SRAATKKSSEFNIQDENAFSVFNAKTFGQQPSQFPTSVDPTPAAPVQKAQRVHVTDIPAALTTLCLEPLTEVPGSPDIIEEEDSMESPMILDLPPEYKPL
GKSIAVQKPAQRAALGNISNVVRTAQAGSKKVVKKDTRQKAMTKTKATSSLHAVVGLPVEDLPTE~STSPDVLDAMEVDQAIEAFSQQLIALQVEDIDK
RVVATKPGLRPRTALGDIGNKAEVKVPTKKELKPAVKAAKKAKPVDKLLEPLKVIEENVCP~AQVEPSSPSPMETSGCLPDELCQAFSDVLIHVKDVDA
MRSKVQMNSRRAALGEIGNKVTVRGKPPAVKQSSNAVAKPSKMAATKVANVKTKHVPVKPVVAEAAPKVPSPVPSPVPMDVSLKEEELCQAFSDALTSVEDIDA
TRRALEEKSIIPATDDEPASKKRRQPSVFNSSVPSLPQHLSTKSHSVSTHGVDAFHKDQATIPKKLKKDVDERVVSKDIPKLHRDSVESPESQDWDDLDA
dd
DREAVILTVPEYEEDIYNYLRQAEMKNRAKPGYMKRQ-TDITTSMRCILVDWLVEVSEEYKLHRETLFLGVNYIDRFLSKISVLRGKLQLVGAASMFLAA
DDGDNPQLCSEYAKDIYLYLRRLEVEMMVPANYLDRQETQITGRMRLILVQFIAS
DDDGNPMLCSEYVKDIYAYLRSLEDAQAVRQNYLHGQEV--TGNMRAILIDWLVQVQMKFRLLQETMFMTVGIIDRFLQEHPVPKNQLQLVGVTAMFLAA
DDGGNPQLCSDYVMDIYNYLKQLEVQQSVHPCYLEGKEINER--M~ILVDWLVQVHSRFQLLQETLYMGVAIMDRFLQVQPVSRSKLQLVGVTSLLIAS
EDWADPLMVSEYVVDIFEYLNELEIETMPSPTYMDROKELAWK-MRGILTDWLIEVHSRFRLLPETLFLAVNIIDRFLSLRVCSLMKLOLVGIAALFIAS
d
p
sY
DI
YL 1E
Y
MRILDWL
V
F Ll ET
V
DRFL
A
LoLVG
KYEEMYPPEIGDFTFVTDHTYTKA~IR~MEMKILRVLKFAIGRPLPLHFLRRASKIGEVTA~QHSLAKYLMELVMVDYDMV~-FTPSQIAAASSCLSLKI
KYEEMYTPEVADFVYITDNAYTASQIREMEMIILRLLNFDLGRPLPLHFLRRASKSCSADAEQHTLAKYLMELTLIDYEMVH-IKPSEIAAAALCLSQKI
KYEEVMCPSVQNFVYMADGGYDEEEILQAERYILRVLEFNLAYPNPMNFLRRISKADFYDIQTRTVAKYLVEIGLLDHKLLP-YPPSQQCAAAMYLAREM
KYEE
P
F
D
L L
P
Flrr
SK
PS
AA
L
akyL E
LGMEPWPQNLVKKTGYEIGHFVDCLKDLH-----KTSLGAESHQQQAVQEKYKQDKYHQVSDFSKNPVPHNLALLAL
LDPETHSSWCPKMTHYSMYSEDHLRPIVQKIVQILLRDDSASQKYSAVKTKYGSSKFMKISGIAQLDSSLLKQIAQGSNE*
LNAGDWTP-----------------------AKNIIKVNK*
LGQGTWGT-----------------------AKNITKVNQAPLITELAASLS*
LGRGPWNRNLVHYSGYEEYQLIS-------VVKKMINYLQYASKKFNKASLFVRDWYKKNSIPLGDDADEDYTFHKQKRIQHDNKDEEW
L
KYsK
kS
(W)
Figure 2. Comparisons
of Frog Cyclins Bl and 62 with Each Other and with Published
Cyclin Sequences
(A) The DIAGON program of Staden (1982) was used to compare the predicted amino acid sequence of Xenopus cyclin Bl (vertical axis) with the
sequences of Xenopus cyclin 82, cyclins from the sea urchin A. punctulata (Pines and Hunt, 1987) and the fission yeast S. pombe (Booher and
Beach, 1988) and cyclin A from the surf clam S. solidissima (Swenson et al., 1988). A score of 135 for a window of 11 residues was used.
(B) Manual alignment of the predicted amino acid sequences of all the published cyclins. Amino acids that show identity between all five sequences
are indicated as uppercase letters in the consensus sequence; lowercase letters show the cyclin B consensus.
responding to the cyclin sequence. The region of overlap
started with the conserved sequence DRFL and included
the central portion of the “cyclin box;’ KYEEMY-PE, as
would be expected from the choice of oligonucleotide
probe, which corresponded to this portion of the sequence. Both clones contained single inserts flanked by
Sau3A recognition sites.
These two Ml3 clones were used to screen full-length
Xenopus ovary cDNA libraries contained in LgtlO. Positive
clones were identified and sequenced as described in Experimental Procedures. Figure 1 shows the sequences of
two clones (Xlcycl and XlcycP) that contained the complete coding regions of two highly homologous cyclins.
The cyclin Bl clone lacks approximately 30 residues at the
5’ untranslated region, as estimated by primer extension
with mRNA; the length of the cyclin 82 leader sequence
is not yet known. Neither of the clones contains an upstream AUG, and cyclin Bl contains an upstream termination codon in frame with the AUG preceding the large
open reading frame. We cannot rigorously rule out the
possibility that translation of cyclin 82 mRNA starts at an
AUG upstream of the XlcycP sequence, although, as
shown below (Figure 4), the translation product of synthetic mRNA from this clone migrates very close to that
of authentic egg mRNA. Cyclin Bl polypeptide contains
398 amino acids and has a predicted molecular weight of
44,674. Subject to the the caveat mentioned above, cyclin
82 has 392 amino acids and a predicted molecular weight
of 43,625. The two sequences show great similarity in the
C-terminal 300 residues, but, as Figure 2 shows, they diverge considerably at the N terminus, with only small islands of conservation in the first 100 residues. In addition,
Cell
950
3
4
Oocytes
5
6
7
8
9101
-
2
3
4
5
6
7
8
91011
Embryos
Figure 3. Cyclin mRNA Levels during Xenopus Early Development
Oocytes were staged as described by Golden et al. (1980), and embryos according to Nieuwkoop and Faber (1987). RNA was extracted
from oocytes or embryos; the RNA equivalent of a single egg was hybridized with an RNA probe for cyclin Bi mRNA and analyzed by
RNAase protection.
comparison of the sequences of clones obtained from
different libraries (from different frog colonies) showed a
significant number of nucleotide substitutions. In every
case, however, the differences were at third-base positions and did not alter the predicted amino acid sequences.
Figure 2A compares the amino acid sequence of frog
cyclin Bl with that of frog cyclin 82 and the published sequences of cyclins from the sea urchin Arbacia punctulata
and the yeast S. pombe and of cyclin A from the surf clam
Spisula solidissima, by means of DIAGON plots (Staden,
1982). In every case the sequences begin to match well
only after about 100 residues from the N terminus of the
frog polypeptide. The match with clam cyclin A is less
good, although it is still impressive between residues 150
and 300. As we shall show elsewhere, the sequences can
be classified into A and B types, and the frog cyclins described here belong in the B class together with sea urchin cyclin and S. pombe cdc73+. The manually aligned
sequence listings in Figure 28 show that the B-type sequences contain 64 identities in the central 205 residues,
of which 51 are shared with the clam A sequence. There
are other short islands of homology in both ends of the
molecule, but their spacing is variable with respect to the
central conserved portion, and they are not present in all
the B-type cyclin molecules.
Cyclin Bl mFtNA Appears Early in Oogenesis and
Persists Well after the Midblastula Transition
Figure 3 shows the levels of cyclin Bl mRNA during oogenesis and early development of X. laevis. The mRNA is
present in stage 3 oocytes (Golden et al., 1980) and persists at least to stage 11 embryos, well after the midblastula transition (Nieuwkoop and Faber, 1967) though
the level declines slightly. We have not yet checked
whether cyclin 82 mRNA shows a similar pattern. There
are approximately 5 x 10’ copies of each cyclin mRNA in
unfertilized eggs as measured by RNAase protection
mapping compared with known amounts of synthetic cyclin transcript (data not shown).
Cyclln 81 and 82 An? Major Translation Products
of a Xenopus Egg Cell-Free System
The two Xenopus cyclin clones were subcloned into
pGEM vectors in order to make mRNA in vitro with T7 RNA
cyclin
cyclrn
82 ~
Bl i
ABCDEFGHIJ
Figure 4. Identification of Xenopus Cyclins in the Cell-Free Extract by
Antisense Ablation
An activated egg extract was incubated with sperm nuclei. Oligonucleotides were added at time zero, and [35S]methionine at 30 min. Samples (2 al) were taken into sample buffer (28 al) at 120 (lanes A, D, F,
I) and 150 min (lanes B, E, G, J). Lanes A and 8, no oligonucleotide;
lanes D and E, anti-cyclin 81 oligonucleotide (20 nglml); lanes F and
G, anti-cyclin 82 oligonucleotide (20 pglml); lanes I and J, both anticyclin oligonucleotides (each at 10 ug/ml). Synthetic mRNAs for cyclins Bl and 82 were translated in the rabbit reticulocyte lysate. The
labeled translation products (5000 cpm) were mixed with unlabeled
frog extract to give the same loading as the other lanes (approximately
0.35 al of egg extract per lane). Lane C, cyclin 1; lane l-f, cyclin 2. Molecular masses of protein standards are indicated in kd. Total inhibition of
protein synthesis in this experiment was 37% by anti-cyclin Bl, 14%
by anti-cyclin 82, and 18% by the mixture.
polymerase. The mRNA was translated in a reticulocyte lysate cell-free system, and the labeled translation products
were run on SDS-polyacrylamide
gels alongside [%Imethionine-labeled Xenopus egg extracts prepared according to Blow and Laskey (1988). Figure 4 shows that a
cluster of at least three prominent labeled bands in the
Xenopus extract appear to correspond to the reticulocyte
translation products (compare lane B with lanes C and Ii).
Collectively, the three polypeptides formed 8.7% of the
methionine incorporation in the Xenopus extract.
The identification of the polypeptides corresponding to
cyclin Bl and 82 in the frog extract was confirmed by hybrid arrest of translation, relying on endogenous RNAase
H to cut the RNA strand of DNA-RNA duplex molecules
(Donis-Keller, 1979; Minshull and Hunt, 1988; Dash et al.,
1987; Shuttleworth and Colman, 1987). Antisense oligonucleotides complementary to the underlined region8 in Figure 1 were designed to cut cyclin Bl or cyclin 82 mRNA
without affecting the other: the rate, extent, and specificity
of cutting were checked by RNAase protection mapping
(see Figure 8). The “arrested” lanes revealed that at least
two major bands were ablated by the anti-cyclin Bl oligonucleotide (Figure 6, lanes D and E), one of which
comigrated with the product of synthetic mRNA (lane C).
;;ylins
and Mitosis in Xenopus
Phase contrast
Eggs
Hoechst fluorescence
(B) Anti-cyclin Bl and B2
Figure 5. Nuclear Envelope Breakdown and Chromosome
tion in the Presence of Anti-Cyclin Oligonucleotides
Condensa-
Extracts treated with anticyclin oligonucleotides
(see Figure 4) were
examined under phase-contrast and fluorescence microscopy after 6
hr. (A) Anti-cyclin 81 oligonucleotide alone at 20 @g/ml. (Essentially
identical results were obtained with 20 @g/ml anti-cyclin 82 oligonucleotide, a control oligonucleotide at 20 ug/ml, and no oligonucleotide.)
(B) Anti-cyclin Bl and anticyclin 82 oligonucleotides, each at 10 uglml.
(C) Edeine at 50 uM. DNA fluorescence is due to 1 frgglml Hoechst
33256.
The anti-cyclin 82 oligonucleotide ablated one major
band, which corresponded to a minor product of the synthetic mRNA (compare lanes B, G, and H in Figure 6). We
suspect that the bands that run more slowly than the primary reticulocyte translation products represent posttranslational modifications of cyclin, as has previously
been observed with sea urchin and starfish cyclins (Pines
and Hunt, 1987; Standart et al., 1967).
Ablation of Cyclins Bl and B2 Blocks the Cell Cycle
in a GP-like State
Lysolecithin-extracted sperm nuclei were added to the
Xenopus egg extracts in order to monitor their ceil cycle
state. The nuclei rapidly formed nuclear envelopes and
performed one round of DNA synthesis (see Figures 5 and
7). After 2 or 3 hr the nuclear envelope broke down and
the chromosomes condensed into a compact mass, indicating that the extract had switched from interphase into
mitosis (Figure 5A; see Blow and Laskey, 1986,1988). Protein synthesis continued for more than 1 hr in these extracts and was strongly inhibited by edeine, showing that
reinitiation of translation occurred (data not shown). The
protein synthesis “window” for mitosis was determined by
addition of cycloheximide or edeine at various times and
scoring for nuclear envelope breakdown 4-6 hr later. Between 10 and 20 min of protein synthesis was required for
subsequent entry into mitosis. Figure 6 shows that the antisense oligonucleotides described above caused complete cutting of their complementary cyclin mRNAs within
5 min and could therefore be used to determine whether
cyclin synthesis accounted for the protein synthesis requirement for entry into mitosis.
Extracts were incubated with antisense oligonucleotides directed against cyclin Bl, cyclin 82, a mixture of
both, or a control “sense” oligonucleotide and were assayed for chromosome condensation and nuclear envelope breakdown as indicators of entry into mitosis. A tube
with no oligonucleotide served as an additional control.
Ablation of either cyclin Bl or cyclin 82 alone caused
some delay in mitosis but did not prevent it (Figure 5A).
When both cyclin mRNAs were destroyed, however, the
chromosomes did not fully condense, and the nuclear
envelope remained intact (Figure 58). This was very similar to the effect of the protein synthesis inhibitor edeine
(Figure 5C).
In other experiments, we used the consensus oligonucleotide indicated by the dotted underlines in Figure 1,
and its complement as a”sense”control. In every case the
antisense oligonucleotide inhibited mitosis while the
sense oligonucleotide did not, and the only change in the
pattern of protein synthesis was a diminution of the cyclin
bands similar to that shown in lanes I and J of Figure 4.
We therefore interpret the inhibitory effect of antisense oligonucleotides on mitosis as being due to their inhibition
of cyclin synthesis and not to inhibition of synthesis of
other proteins. This does not mean, however, that the synthesis of other proteins is unnecessary for mitosis in these
extracts.
Ablation of Cyclins Limits DNA Synthesis
to a Single Round
As a further check that preventing cyclin synthesis
blocked the cell cycle at the G2-M transition, we tested
whether DNA re-replication could occur in nuclei that had
been incubated in an extract lacking cyclin mRNAs. As
shown previously, nuclei that have undergone a single
round of replication in the type of extract described here
can only re-replicate their DNA when transferred to fresh
extract if they have passed through a mitosis-like state
(Blow and Laskey, 1988). Thus, the presence of cycloheximide in the first incubation permits a single round of DNA
synthesis but prevents re-replication (Harland and Laskey,
1980).
Figure 7 shows that cyclin mRNA scission has an effect
on DNA replication similar to that of cycloheximide. Cyclins Bl and 82 were cut with the consensus antisense oligonucleotide indicated by the dotted underlines in Figure
1. The first round of DNA synthesis proceeded normally
Cell
952
cyclin 82
probe
+.$@
Q ,9?+@
\o
(8 & 3
6
anti-B1
Q cd d
---
622 /-cyclin Bl
probe
/
anti-B2
anti-B1 + 82
I,
*
0
527
-
cyclin 82
-
cyclin Bl
-
cyclin 82
-
cyclin 82
^. ,
_ 3’rragment of
cut cyclin 82
-
Figure 6. Antisense Oligonucleotides Specifitally and Rapidly Cut Cyclin mRNAs to Completion
Samples (1 uf) were taken from the tubes described in.Fig&e 4 at 5, IO, 15, and 20 min (i.e.,
before addition of [35S]methiinine). The RNA
was extracted and hybridized with RNA probes
for cyclin Bl and 82 and cytoskeletal actin for
RNAase protection mapping. Hybrids were
digested with 90 U/ml RNAase Ti and analyzed by electrophoresis and fluorography. Positions of Hpall-cut pBR322 markers are indicated. Lane A, undigested probes; lane B,
protected cyclin Bl probe; lane C, protected cyclin 82 probe; lane D, extract without oligonucleotides at time zero; lanes E-H, 20 uglml
anti-cyclin 61 oligonucleotide; lanes I-L, 20
pglml anti-cyclin 82 oligonucleotide; lanes
M-P, 10 fig/ml of each anti-cyclin oligonucleotide. Samples were taken at 5, 10, 15, and 20
min in each series with antisense oligonucleotides.
cyclin 81
5’fragment of
-cut cyclin Bl
-
ABC
DEFGH
5’fragment of
cut cyclin 82
IJKLMNOP
in the nuclei incubated in this cyclin-depleted extract, but
the nuclei did not enter mitosis. When these nuclei were
transferred to a fresh extract, no second round of replication occurred as shown by the low incorporation of
3H-nucleotide and the absence of a significant “heavyheavy” peak of re-replicated DNA. Nuclei that underwent
their first round of replication in an extract containing the
sense version of the oligonucleotide
entered mitosis and
re-replicated their DNA when added to a fresh extract.
Since the control oligonucleotide did not prevent rereplication, the failure to re-replicate the DNA was not a
trivial consequence of addition of oligonucleotides. Thus,
ablation of the cyclin mRNAs described in this paper inhibited entry into mitosis, whether a consensus antisense
oligonucleotide or a mixture of specific anti-cyclin Bl and
anti-cyclin 82 oligonucleotides was used.
Discussion
The results presented in this paper show that Xenopus oocytes and eggs contain two B-type cyclin mRNAs, which
we propose to call cyclins Bl and B2. Their homology with
each other is about the same as with sea urchin cyclin,
clam cyclin B, and S. pombe cdc73+, whereas their
match with clam cyclin A is less good. We have recently
identified two more cyclin mRNAs in Xenopus ovary cDNA
libraries. The predicted polypeptides show better homol-
ogy with clam cyclin A than with cyclin B (unpublished
data). All the B-type cyclins known so far contain the
amino acid sequence FLRR-SK (see Figure 2), which is
not present in the A-type cyclins. RRXSX is a potential site
for CAMP-dependent protein kinase (Edelman et al.,
1987), which may be significant.
Targeted destruction of the Xenopus B-type cyclin messages by antisense oligonucleotides prevented a cell-free
DNA replicating system from entering mitosis. Moreover,
the nuclei that had been incubated in an extract specifically depleted of cyclin mRNA failed to re-replicate their
DNA when added to a fresh uninhibited extract, although
the antisense oligonucleotides did not inhibit the first S
phase. If either of the cyclin mRNAs was ablated by itself,
the extract was still able to pass from interphase to M
phase. We interpret this to mean that the two cyclins described here are functionally redundant and are each
alone capable of driving entry into mitosis. The gene
duplication probably occurred when Xenopus became
tetraploid (Kobel and Du Pasquier, 1988).
It is possible that the antisense oligonucleotides inhibit
mitosis by inhibiting synthesis of unidentified proteins with
fortuitous homology to the particular sequences we have
used. Since we used oligonucleotides complementary to
two different locations on each cyclin mRNA, this is not a
very strong possibility. It is equally unlikely that the inhibitory effect on mitosis was due to nonspecific inhibition of
C&lins
and Mitosis in Xenopus
Eggs
(A) control oligonucleotide
12
10 7
?
-*-
first (32P)
-z--
second (3H)
20
10
fraction
30
number
(B) anti-cyclin oligonucleobde
12
t
I
11
I(
11
0
lb
- *-
first (32P)
-
second ( 3H)
20
30
Figure 7. Nuclei Incubated in Extracts with Anti-Cyclin Oligonucleotides Cannot Perform a Second Round of DNA Replication
A two-stage incubation to assess the ability of nuclei to perform a second round of DNA replication was performed as described by Blow and
Laskey (1986). In the first incubation, sperm nuclei were added to egg
extracts containing BrdUTP, [a-s2P]dATF! and 15 uglml of either the
antisense oligonucleotide #426 or the control oligonucleotide #120. After 4 hr the nuclei were transferred to fresh extract containing BrdUTP
and $H]dATP and incubated for a further 4 hr. No oligonucleotides
were present in the second incubation. DNA was isolated and fractionated on CsCl density gradients. (A) Control oligonucleotide. (B) Anticyclin oligonucleotide. Filled symbols show labeling in the first incubation, and open symbols the labeling in the second incubation. The
peak of re-replicated DNA (fully BrdlJ substituted) is marked H/H
(heavy-heavy), the peak of once-replicated DNA is indicated by H/L
(heavy-light), and unreplicated DNA is marked L/L (light-light). Total inhibition of protein synthesis in the first incubation was 25% by the antisense oligonucleotide and 11% by the sense oligonucleotide.
protein synthesis. First, all experiments had a “sense” oligonucleotide control added at the same concentration as
the antisense oligonucleotide, and in no case did this prevent entry into mitosis. Second, extracts that entered mitosis sometimes had much higher levels of nonspecific protein synthesis inhibition than extracts prevented from
entering mitosis by loss of both cyclins (see Figure 4).
Third, oligonucleotides gave essentially no inhibition of
protein synthesis for the first 30 min of the incubation (data
not shown). By 20 min, however, the extract had fulfilled
its protein synthesis requirement for entry into mitosis.
A close connection between cyclins and MPF has recently emerged from the fusion of biochemical and genetic approaches (reviewed by Murray, 1988). MPF has
been purified over 3,000-fold (Lohka et al., 1988), and
shown to contain the Xenopus homolog of the S. pombe
cell division mutant cd&
gene (Gautier et al., 1988;
Dunphy et al., 1988). Genetic evidence suggests that the
gene product of cd& interacts with that of cdcW (Booher and Beach, 1987), which is clearly S. pombe cyclin as
judged by its homology with published cyclin sequences
(Goebl and Byers, 1988; Solomon et al., 1988). The null
phenotype of cdcW is failure to enter mitosis (Nurse et
al., 1976; Booher and Beach, 1988), exactly like the cellfree extract lacking cyclin B mRNA. These genetic experiments do not make it clear, however, whether cyclin is required to activate MPF or whether it acts downstream of
MPF and executes MPF functions.
Cyclin does not appear to contain a kinase consensus
sequence, but MPF is thought to exert its effects via protein phosphorylation (Maller et al., 1977; Dorbe et al.,
1983; Karsenti et al., 1987; Lohka et al., 1987; Ozon et al.,
1987); it therefore seems more logical to view cyclin as an
activator of MPF, although there could be functions of
MPF that do not require protein phosphorylation for their
execution. Moreover, the protein synthesis requirement
for activation of MPF is much easier to explain if cyclin is
an MPF activator. Cyclin may act by displacing an inhibitor
of MPF or by affecting the activity of a phosphatase, for
example. However, we note that despite the seemingly
absolute requirement for cyclin to allow MPF activation
or function, there is at present no evidence that cyclin is
what actually determines the timing of MPF activation. In
fission yeast, genetic evidence identifies several other
genes, such as weel+, niml+, and cdc25+, that appear to
regulate this function (Russell and Nurse, 1986, 1987a,
1987b; reviewed by Lee and Nurse, 1988), but their homologs have not yet been identified in higher organisms. It
may be more appropriate to view cyclin as a kind of molecular latch, a component that is capable of and necessary
for holding MPF “on,” but not as the only factor involved
in the activation process. This view is more compatible
with the puzzling gap between the end of the protein synthesis requirement window at lo-20 min and entry into mitosis at 2 to 3 hr in the kind of extracts described here. If
cyclin levels simply had to reach a critical threshald in order to activate MPF, why not enter mitosis at 20 min?
Something else has to happen, and we do not know what
it is.
Until recently, cyclins had been detected only in the
eggs and oocytes of marine invertebrates, and the results
presented in this paper still leave open the possibility that
cyclins are important only for the rapid cell division cycles
of early cleavage. This is probably not the case, however,
since the sequence homology between cyclins and the S.
pombe cdcW gene (Booher and Beach, 1988; Goebl
and Byers, 1986; Solomon et al., 1988) suggests that they
are required for the activation or action of MPF in normal
vegetative cell cycles. Furthermore, the occurrence of cyclins over this wide phylogenetic range suggests they occur in all eukaryotes.
Cell
954
Experlmental
Procedures
cDNA Libraries
A cDNA library from activated Xenopus egg mRNA was constructed
in M13mp6. Poly(A)+ RNA was prepared from electrically activated
eggs that had been frozen in liquid nitrogen and finely ground in a pestle and mortar. Extraction buffer (200 m M LiCI, 50 m M Tris-HCI [pH
61, 10 m M EDTA) was made 0.5% in SDS and heated until a single
phase was produced. It was mixed with the frozen egg powder and
shaken vigorously. A ratio of 20 ml of liquid for each gram of tissue was
used to reduce the problems of emulsion formation. After two more
phenol extractions followed by an ether extraction, RNA was precipitated by adding 0.25 vol of 10 M LiCI.
Poly(A)+ RNA was isolated by two cycles of chromatography
on
oligo(dT)-cellulose
(type III from Collaborative Research). First- and
second-strand cDNA was synthesized as described by Pines and Hunt
(1967) except that random hexanucleotides (final concentration 400
&ml)
were used as primers for the first strand. The cDNA was
digested with Sau3A and ligated into BamHI-cut M13mp6 RF. This was
used to transform Escherichia coli strain TGl by the method of Hanahan (1965).
Xenopus oocyte cDNA libraries in kg110 were the gifts of D. Melton
(Harvard University), A. Colman (University of Birmingham), and C.
Dingwall (University of Cambridge).
Screening cDNA Libraries
Filters were prepared according to Mason and Williams (1965). Duplicate filters from the Ml3 library were probed with a redundant oligonucleotide (#40, see below), chosen to cover the region of best match between clam cyclin A and sea urchin cyclin (Swenson et al., 1966; Pines
and Hunt, 1967) and with the sea urchin cDNA clone cyc4 (Pines and
Hunt, 1967). Ml3 clones that were positive with both probes were isolated and sequenced. Recombinants containing cyclin inserts were
identified by conceptual translation of the sequence and used to probe
duplicate filters from the 1, libraries.
The oligonucleotide
was labeled with [Y-~~P]ATP according to
Maxam and Gilbert (1960). Clone cyc4 was labeled by the method of
Feinberg and Vogelstein (1963). Filters were probed overnight in 5x
SCP (500 m M NaCI, 150 m M Na2HP04, 5 m M EDTA [pH to 6.6 with
HCI]), 2% (wVvol) skim milk, 0.5% SDS, 10 uglml boiled herring sperm
DNA; DNA probes were incubated at 65%, oligonucleotide probes at
42%. Oligonucleotide-probed
filters were washed three times in 4x
SSC (600 m M NaCI, 60 m M sodium citrate), 0.1% SDS at room temperature. Filters probed with cyc4 were washed twice in 4x SSC and once
in 3x SSC at 37%. Filters screened with primed-cut probes were
washed twice in 2x SSC and twice in 0.1x SSC at 60%. All washes
lasted 10 min.
DNA Sequencing
The “extended” dideoxynucleotide
sequencing method designed for
modified T7 DNA polymerase (Tabor and Richardson, 1967) was used
with the Klenow fragment of E. coli DNA polymerase as described in
BRL Focus (Summer 1967). Inserts from lgtl0 libraries were subcloned into a plasmid (usually one of the Gemini transcription vectors
pGEM-1 or pGEM-2) and sequenced by the “shotgun” sonication and
random-selection
approach of Bankier et al. (1967). The Ml3 subclones selected for sequencing were identified by screening with the
purified cyclin insert.
In Vitro l?anscription
and Translation
Synthetic mRNAs were transcribed from cDNA subcloned into pGEM
transcription vectors from Promega Biotec. Capped transcripts were
synthesized using SP6 or T7 RNA polymerases (SP6 according to the
protocol of Melton et al. [I9641 and Krieg and Melton [1987]; T7 as described by Pines and Hunt [19m). RNAs were translated in the mRNAdependent reticulocyte lysate system as described by Jackson and
Hunt (1963).
Analysis of Labeled Pmtelns on SDS-Polyacrylamlde
Gels
Analysis of protein products was performed on lo%-20%
gradient
SDS-polyacrylamide
gels after dilution with 5-10 vols of SDS gel sample buffer (Anderson et al., 1973). The reticulocyte lysate translation
reactions were terminated by addition of 5 m M EDTA and 50 pglml
RNAase for 10 min at 20% before addition of sample buffer.
RNAase Protection Mapping
RNAase protection was based on the protocol described by Krieg and
Melton (1967). Inserts from Ml3 shotgun clones were subcloned into
pGEM transcription vectors, and RNA probes were made with T7 RNA
polymerase. Reaction conditions were as described by Pines and Hunt
(1967) except that no cap analog was used; ATR GTR and CTP concentrations were each 1 mM, and the only UTP in the 5 ul reaction was
2.5 ul of label (PB10163 from Amersham International), giving a final
concentration of 12.5 pM UTR For probes longer than 250 bases, addition of unlabeled UTP at 375 uM increased full-length transcripts by
at least IO-fold. The labeled probes were gel purified (Krieg and Melton, 1967).
RNA for analysis was prepared from oocytes, embryos, and extracts
by freezing the sample (one to five embryos or up to 5 ul of extract)
on dry ice. The sample was thawed and homogenized by vigorous
pipetting in 125 ul of extraction buffer (300 m M NaCI, 50 m M Tris-HCI
[pH 7.51, 1 m M EDTA, 1% SDS). RNA was extracted with phenol-chloroform, ethanol precipitated, and stored at -60%.
RNA was hybridized with probes as described by Krieg and Melton
(1967). RNAase concentrations
were titrated for each probe. In
general, 35 U/ml of BRL RNAase Tl and 100 nglml Sigma RNAase A
were used. Digestion and RNA analysis were performed as described
by Krieg and Melton (1967).
Dligonucleotides
Oligonucleotides were made by Margaret Franklin or Colin Denston in
the Department of Biochemistry on a Biosearch Inc. Cyclone DNA
Synthesizer donated by the Wellcome Trust. The redundant oligonucleotide #40 originally used to screen the Ml3 library was B’TCTGG[A/G]GG[A/G]TA[TIC]ATCTC[T/C]TC[A/G]TATTT-3’
and, like the anticyclin 82 oligonucleotide 5’-GGACACATCCATCGGCAC-3’,
was purified by several ether extractions followed by ethanol precipitation. The
anti-cyclin Bl oligonucleotide
5’CCATTGGGCTTGGTGAGC3’
was
purified on an Applied Biosystems OPC column according to the
manufacuterer’s instructions. Oligonucleotide #426 had the sequence
5’.ACCTCTGG[A/G]GTGTACATCTCTTC-3’
and is complementary
to
both cyclin 81 and 82, as indicated by dotted underlining in Figure 1;
oligonucleotide #120 is the complement of #426.
Cell-Free Egg Extract
Cell-free DNA replication systems were prepared and analyzed according to the protocols described by Blow and Laskey (1966, 1966)
based on the method of Lohka and Masui (1963, 1964). For analysis
of newly synthesized proteins, [35S]methionine (Amersham SJ 1515)
was added to a final concentration of 2 mCi/ml.
Acknowledgments
We are grateful to Jon Pines, John Gerhart, Mike Wu, Andrew Murray,
Fred Wilt, Eric Rosenthal, and Alan Colman for their collaboration in
the early stages of these experiments, and for the facilities they
provided for making oocytes, eggs, and RNA. We thank John Shuttleworth, Colin Dingwall, and Doug Melton for samples of their cDNA
libraries, Tim Mohun for his actin clone, David Judge for help with sequencing software, Pat O’Farrell, Joan Ruderman, and Will Whitfield
for letting us see their clam and fly cyclin sequences, Ron Laskey for
laboratory facilities and encouragement, and Phil Garrett for technical
support. Oligonucleotide synthesis was made possible by a grant from
the Wellcome Trust. This work was supported by an SERC studentship
to J. M., and by the CRC and MRC.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 16 U.S.C. Section 1734
solely to indicate this fact.
Received November
2, 1966; revised December
15, 1966.
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