Download Overexpression of a truncated cyclin B gene arrests Dictyostelium

3105
Journal of Cell Science 107, 3105-3114 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Overexpression of a truncated cyclin B gene arrests Dictyostelium cell
division during mitosis
Qian Luo1 Christine Michaelis1 and Gerald Weeks1,2,*
1Department of Microbiology and Immunology, and 2Department of Medical Genetics, University of British Columbia, Vancouver,
BC V6T 1Z3, Canada
*Author for correspondence at address 1
SUMMARY
A cyclin gene has been isolated from Dictyostelium discoideum and the available evidence indicates that the gene
encodes a B type cyclin. The cyclin box region of the protein
encoded by the gene, clb1, has the highest degree of
sequence identity with the B-type cyclins of other species.
Levels of cyclin B mRNA and protein oscillate during the
cell cycle with maximum accumulation of mRNA occurring
prior to cell division and maximum levels of protein
occurring during cell division. Overexpression of a N-terminally truncated cyclin B protein lacking the destruction
box inhibits cell growth by arresting cell division during
mitosis. The gene is present as a single copy in the Dictyostelium genome and there is no evidence for any other
highly related cyclin B genes.
INTRODUCTION
addition, there is some evidence that cells that are in the late
part of the G2 phase also have a preference to become prestalk
cells (Maeda et al., 1989). A variety of low molecular mass
factors have also been shown to influence the formation of both
stalk and spore cells, and these molecules have been proposed
as morphogens responsible for generating and maintaining the
spatial pattern (for review, see Weeks and Gross, 1991). It is
therefore possible that the initial heterogeneity within the cell
population is established by the position of the cell within the
cell cycle, and that morphogenetic gradients then promote
continued differentiation and regulate the pattern. The
molecular mechanisms that are responsible for the influence of
the cell cycle on cell type determination are not known.
The control systems involved in the G2 to M phase transition are highly conserved in eukaryotic cells. Initiation of
mitosis requires a protein kinase complex, consisting a
catalytic subunit (cdc2 protein kinase) (Dunphy et al., 1988;
Gautier et al., 1988) and a regulatory subunit (cyclin B) (Labbé
et al., 1989; Lohka et al., 1988; Draetta et al., 1989; Meijer et
al., 1989; Gautier et al., 1990). Cyclin B accumulates during
G2, reaching a maximum at the G2/M boundary (Evans et al.,
1983; Standart et al., 1987). It is then destroyed during mitosis,
which results in the inactivation of the cdc2 protein kinase and
allows cells to leave M phase. Cells expressing a stable cyclin
B arrest during mitosis (Murray et al., 1989; Ghiara et al.,
1991; Luca et al., 1991).
In view of the presumptive importance of the G2 to M phase
transition in cell cycle control of D. discoideum, we attempted
to isolate the cyclin B gene and use it as a tool to study the
influence of the cell cycle on Dictyostelium development. By
creating a dominant negative mutation in cyclin B, it might be
possible to derive an understanding of whether the continua-
The cellular slime mold Dictyostelium discoideum is a simple
eukaryote, whose life cycle consists of two distinct phases.
During the vegetative phase, the haploid amoebae feed on
bacteria, increase in size and undergo mitotic cell division.
However, upon starvation, the amoebae enter the development
phase by forming a multicellular aggregate in response to
cAMP pulses. The aggregate elongates to form a migrating
slug comprising prestalk and prespore cells that are spatially
organized; the anterior 20% are prestalk cells and the posterior
80% are prespore cells. These cells eventually differentiate into
the stalk and the spore cells that make up the final fruiting body
(Loomis, 1982). Although there is a decrease in cell mass due
to endogenous respiration during the differentiation process,
continued cell division and DNA replication occur (ZadaHames and Ashworth, 1978; Durston and Vork, 1978). It is not
known if this continuation of the cell cycle is necessary for the
process of differentiation.
During the vegetative growth phase, the Dictyostelium cell
cycle is characterized by short S and M phases, the absence of
a G1 phase and a very long G2 phase (Weijer et al., 1984a), suggesting that the G2 to M transition might be the important site
of cell cycle control in this organism. There is considerable
evidence that initial cell type determination in Dictyostelium is
influenced by the position of the cell in the cell cycle at the time
of starvation (Weijer et al., 1984b; McDonald and Durston,
1984; Gomer and Firtel, 1987; Ohmori and Maeda, 1987;
Maeda et al., 1989; Zimmerman and Weijer, 1993). There is a
general agreement that cells that are in the S and early G2 phases
have a greater tendency to become prestalk cells, while cells in
the remainder of the G2 phase become prespore cells. In
Key words: cell cycle, cyclin, Dictyostelium
3106 Q. Luo, C. Michaelis and G. Weeks
tion of the cell cycle during development is essential and to
gain an insight into the molecular mechanisms involved in the
role of cell cycle in cell type determination. In this report we
present our initial studies: the isolation of a cyclin B gene from
Dictyostelium and the creation of a truncated cyclin B gene that
causes mitotic arrest.
MATERIALS AND METHODS
Growth of D. discoideum
Strain V12-M2 was grown in association with Enterobacter
aerogenes on rich nutrient plates for 48 hours at 22°C at which time
visible clearing of the bacterial lawn had occurred. Cells were
harvested in Bonner’s salt solution (Bonner, 1947) and separated from
residual bacteria by several centrifugations at 400 g for 5 minutes.
Strain Ax-2 was grown in HL-5 medium (Watts and Ashworth, 1970)
in shake culture (150 rpm) at 22°C. The HL-5 medium was supplemented with 1 mM folate, where indicated. Cells were harvested at
the indicated stages of growth by centrifugation at 400 g for 3 minutes
and washed twice in 20 mM potassium phosphate, pH 6.0.
Growth was synchronized by a modification of the stationary phase
release method (Yarger et al., 1974). Exponentially growing Ax-2
cells were diluted into fresh culture medium containing Oxoid
peptone (14.3 g), Oxoid yeast extract (7.15 g), Na2PO4 (0.507 g),
KH2PO4 (0.48 g), maltose (18.0 g per liter), pH 6.5 (Satre et al., 1986),
and allowed to grow for 3-4 days at 22°C (180 rpm), at which time
cells had reached a stationary phase density of 2×107 cells/ml. Cells
were incubated for an additional 12 hours, and then diluted into fresh
medium at a density of 1×106 cells/ml. The subsequent increase in
cell number over time was determined at hourly intervals.
Polymerase chain reaction (PCR)
The primer extension reaction was used to generate single-stranded
cDNA from poly(A)+ RNA extracted from vegetative V12-M2 cells.
Poly(A)+ RNA was isolated from total RNA by oligo(dT)-cellulose
chromatography (Aviv and Leder, 1972) as described by Maniatis et
al. (1982). The 50 µl of reaction mixture contained 2 µg of poly(A)+
RNA, 1 µg of 3′ PCR primer KL-4 (5′ ATC GAA TTC T(C)TC CAT
T(A)AA(G) A(G)TA T(C)TT A(G,T)GC 3′), 50 mM Tris-HCl (pH
8.4), 50 mM KCl, 8 mM MgCl2, 5 mM DTT, 0.8 mM of each of
dATP, dCTP, dGTP, dTTP and 34 units of AMV reverse transcriptase (Pharmacia). The reaction was incubated at 42°C for 2 hours.
Degenerate oligonucleotide primers with NotI and EcoRI adapter
sequences were synthesized in order to try to amplify a Dictyostelium
cyclin B gene. The 5′ primer KL-3 (5′ATC GCGGCCGC GC(T,C,A)
T(A)C(G)T(A) AAA(G) TAT(C) GAA(G) GAA(G) A(G)T-3′) was
derived from the sequence encoding a conserved motif (ASKYEE)
and the 3′ primer KL-4 was derived from the sequence encoding
another conserved motif AKYLME. Both motifs are within the cyclin
box sequence of other B-type cyclins. The PCR reaction contained 1
µg of primer KL-3, 125 ng of primer KL-4, 200 µM of each of dNTPs,
2 units of Taq polymerase in 1× reaction buffer (Promega) in a final
volume of 50 µl. The PCR program was designed as following; 3
cycles (95°C, 30 seconds; 37°C, 2 minutes; 72°C, 3.5 minutes), then
another 35 cycles (95°C, 30 seconds; 48°C, 1.5 minutes; 72°C, 3.5
minutes) using an Ericomp thermocycler. All the PCR products
ranging from 150 to 600 bp were precipitated with 95% ethanol and
2.5 M of ammonium acetate then digested with NotI and EcoRI for
subcloning into the Bluescript KS+ vector. Single-stranded plasmid
DNA was sequenced by the chain termination method (Sanger et al.,
1977) using the M13 universal primer.
Screening of cDNA and genomic DNA libraries
Three libraries were employed in the search for a full-length cyclin B
gene: a λZapII cDNA library constructed using mRNA from vegeta-
tive V12-M2 cells, a λgt11 cDNA library prepared from mRNA from
Ax-3 cells 4 hours after starvation and a genomic library containing
Dictyostelium Ax-2 nuclear DNA that was partially digested with
Sau3A to give an average size of 5 kb (Pears et al., 1985). Approximately 300,000 plaques of each cDNA library were screened
according to standard protocols (Maniatis et al., 1982).
DNA sequencing
PCR products were subcloned into Bluescript KS+ vector and cDNA
from the 4 hour λgt11 library was subcloned into pTZ19R. cDNA
from the 0 hour λZapII library was rescued according to the Stratagene in vivo excision protocol. A 2.5 kb insert from the genomic clone
was digested into three restriction fragments and subcloned into Bluescript KS+ and pTZ19R vectors, respectively. Single-stranded DNA
was isolated and sequenced by the chain termination method using
universal or reverse primers initially and then internal primers that
corresponded to already sequenced portions of the presumptive cyclin
B gene.
cDNA probes and hybridization procedures
The purified PCR product, a 1.5 kb HincII-EcoRI fragment and a 514
bp SnaBI-BglII fragment of the clb1-c2 cDNA clone were labeled by
the random oligonucleotide primer method (Feinberg and Vogelstein,
1983) using [α-32P]dCTP (ICN). Filters were prehybridized for 2-3
hours and then hybridized overnight at 42°C in a solution containing
50% formamide, 5× SSC, 5× Denhardt’s, 0.5% SDS and 250 µg/ml
of sheared and denatured salmon sperm DNA.
Southern blot analysis
Nuclei were isolated from vegetative cells of strain V12-M2 as
described previously (Cocucci and Sussman, 1970) and the genomic
DNA was extracted by standard protocols (Maniatis et al., 1982). A
5 µg sample of genomic DNA was subjected to restriction digestion
overnight, size fractionated on a 0.8% agarose gel and then transferred
to a nylon membrane. The filter was prehybridized and hybridized as
described above and washed twice for 20 minutes in 2× SSC, 0.1%
SDS at 42°C to provide low stringency conditions. After exposure to
X-ray film, the filter was re-washed twice for 15 minutes in 0.1× SSC,
0.1% SDS at 65°C, to provide high stringency conditions, and then
re-exposed to X-ray film.
Northern blot analysis
Total RNA was extracted from cells by the acid guanidinium thiocyanate method (Chomczynski and Sacchi, 1987). In order to reduce
the hybridization background caused by the presence of the high
copy number of the transgene in the transformants, the RNA preparations were resuspended in 500 µl of RES-1 buffer (0.5 M LiCl, 1
M urea, 1% SDS, 20 mM sodium citrate, 2.5 mM trans-1,2diaminocyclohexane-N,N,N′,N′-tetra-acetic, pH 6.8) and 4 µl of 2 M
acetic acid. The RNA was selectively precipitated by the addition of
an equal volume of 5 M LiCl/ethanol (3/2, v/v) and the mixture was
stored overnight at 4°C to allow complete precipitation. The supernatant was removed after centrifugation for 15 minutes in a
microfuge and the pellet was washed several times with 75% ethanol
to remove traces of LiCl (Birnboim, 1988). For northern blot
analysis, 20 µg of total RNA was denatured for 10 minutes at 70°C
in a solution containing 50% formamide, 17.5% formaldehyde, 20
mM MOPS, pH 7.0, 5 mM sodium acetate and 0.5 mM EDTA, pH
8.0. The RNA was size fractionated on a 1.25% agarose-formaldehyde gel, assessed for equal loading by ethidium bromide staining of
ribosomal bands, and then transferred to a nylon membrane. The
filter was prehybridized and hybridized as described above except
that 30 µg/ml of polyadenylic acid was included in the prehybridization and hybridization solution. The filter was then washed
twice for 15 minutes in 0.5× SSC, 0.1% SDS at 60°C and exposed
to X-ray film for an appropriate time.
Truncated cyclin B in Dictyostelium cell division 3107
pVEII vector construction
The pVEII vector containing the discoidin-Iγ promoter (Blusch et al.,
1992) was digested by KpnI, blunt-ended with T4 DNA polymerase,
dephosphorylated and then gel purified with Gene-clean (BIO Scientific). The clb1-c2 cDNA clone was digested with SnaBI-EcoRI to
yield a 1145 bp fragment that encodes a truncated cyclin B protein
that stretches from N106 to the C-terminal F436, and then continues
to the 3′ end of the poly(A)+ tail (Fig. 1). The fragment was end-filled
with DNA polymerase Klenow fragment, gel purified and then
inserted into the blunt-ended KpnI site of the pVEII vector. The amino
acid sequence of the predicted encoded product of this construct
includes a N-terminal methionine and six downstream amino acids
encoded from the polylinker of the vector and N106-F436 encoded
from the clb1-c2 cDNA. The correct junction between vector and
insert was confirmed by sequencing.
Transformation and selection of Dictyostelium
transformants
A 10 µg sample of the pVEII-truncated cyclin B vector DNA was
used to transform exponential phase Ax-2 cells by the CaPO4 DNA
precipitation method (Nellen et al., 1984) in bis-Tris HL-5 (Egelhoff
et al., 1989). Stable transformants were selected and grown clonally
in HL-5 medium containing 10 µg/ml of G418, 50 µg/ml of streptomycin, 50 µg/ml of ampicillin and 1 mM folate.
Preparation of anti-cyclin B antibody
A 633 bp BglII-EcoRI fragment of clb1-c2 cDNA, which encodes a
truncated product stretching from I277 to the C terminus (Fig. 1), was
cloned into a T7 expressing vector pRSET(A) at its BglII-EcoRI sites
(Invitrogene). The pRSET vector contains a histidine tag that allows
the His-cyclin B fusion protein to be affinity purified with Ni-agarose
beads. The constructed pRSET-cyclin B plasmid was transformed into
E. coli strain BL-21 and the expression of the gene was maximally
induced by the addition of 1 mM IPTG at a culture density of
A600=0.7-0.9 for 5 hours at 37°C.
The cells of 3 liter culture were harvested by centrifugation at 4000
g for 20 minutes, and the cell pellets were frozen and thawed. Then
the pellets were resuspended in 60 ml of buffer A (6 M guanidine
hydrochloride, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 8.0),
and shaken for 1 hour at room temperature. The suspension was centrifuged at 10,000 g for 15 minutes at 4°C and the supernatant was
collected. A 10 ml sample of a 50% slurry of Ni-agarose beads, previously equilibrated in the lysis buffer was added to the supernatant
and the mixture was rocked at room temperature for 2 hours. The
beads were placed in a column, washed with 100 ml of buffer A and
50 ml of buffer B (8 M urea, 0.1 M sodium phosphate, 0.01 m TrisHCl, pH 8.0), and then washed extensively with buffer C (8 M urea,
0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 6.3) until 280 nM
absorption of the elute was less than 0.01. The recombinant protein
was eluted with 15 ml of buffer D (8 M urea, 0.1 M sodium phosphate,
0.01 M Tris-HCl, pH 5.9), followed by 15 ml of buffer E (8 M urea,
0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 4.5). The eluted
fractions were subjected to SDS-PAGE analysis and the fractions containing the His-cyclin B fusion protein were pooled. Since the fusion
protein was not soluble in the absence of 8 M urea, urea-solubilized
protein was used to generate the antibody.
Rabbits were injected with 150 µg of the urea-solubilized Hiscyclin B fusion protein mixed with Hunter’s Titer MAX™ as adjuvant
(CyRx Co.) and then boosted twice after 2-week intervals with the
same amount of antigen in the same adjuvant. The cyclin B antiserum
was purified by 50% ammonium sulfate precipitation as described by
Maniatis et al. (1982) and then affinity purified using a column of Hiscyclin B-Affi gel agarose beads. Non-specifically bound proteins were
removed with 10 column volumes of antibody loading buffer (Pierce)
and the specific antibody was eluted with antibody elution buffer
(Pierce). The fractions containing the antibody were combined,
dialyzed against water for 16 hours, dialyzed against PBS for 8 hours
and then concentrated to a small volume by centrifugation through a
Centricon 30 (Amicon) at 1,000 g for 20 minutes.
Polyacrylamide gel electrophoresis and immunoblotting
SDS-polyacrylamide gel electrophoresis and immunoblotting were
performed essentially as previously described (Harlow and Lane,
1988). Cell extracts were added to an equal volume of sample buffer
(100 mM Tris-HCl, pH 6.8, 2% SDS, 0.2% Bromophenol Blue, 20%
glycerol, 5% β-mercaptoethanol, 4 M urea). The urea was required to
denature the cyclin B protein in the cell lysate, since it was found that
the antibody did not recognize the protein in the absence of urea
treatment. A 20 µg sample of cell lysate protein was fractionated on
12% gels and blotted onto a nylon membrane. The blot was blocked
with 5% skim milk powder (Carnation) for at least 1.5 hours and then
treated with the antigen-purified cyclin B antibody at a dilution of
1:1,000. Goat anti-rabbit IgG conjugated to horseradish peroxidase
was used at 1:10,000 as a secondary antibody and the signal generated
was detected by enhanced chemiluminescence (Amersham).
Giemsa staining
Cells were washed and fixed as described by Brody and Williams
(1974). The cells were treated with ribonuclease A (Sigma) at 0.2
mg/ml in 0.067 M Sorensen’s buffer, pH 6.8, at 37°C for 5 minutes
and then stained with 10% (v/v) Giemsa stain (Improved R66 from
BDH) in 0.067 M Sorensen’s buffer (pH 6.8) for 40 minutes (ZadaHames, 1977). The cells were examined by phase-contrast
microscopy using ×1,000 magnification and green bright-field illumination, and then photographed.
RESULTS
Amplification of a cyclin B homologous sequence
from Dictyostelium by PCR
Two degenerate oligonucleotides corresponding to two highly
conserved regions within the cyclin box of B type cyclins were
used as primers for PCR with cDNA derived from vegetative
mRNA as template. The reaction yielded several products of
variable sizes, which were cloned into Bluescript KS+ vector.
Forty four clones were isolated with inserts ranging from 150
to 600 bp and one clone with a 450 bp insert encoding an amino
acid sequence that exhibited a high percentage of identity to
the cyclin box region of cyclin B proteins from other species.
Fig. 1. Restriction map of Dictyostelium cyclin B genomic clone
(clb1-g) with a schematic alignment of cDNA clones (clb1-c1 and
clb1-c2) and the initially isolated PCR product. The open boxes
represent coding sequences, the hatched box represents the 128 bp
intron and the lines represent the 5′ and 3′ non-coding sequences.
Only restriction sites that are relevant to the construction of the
various subclones and constructs are shown. Bar, 500 bp.
3108 Q. Luo, C. Michaelis and G. Weeks
Fig. 2. Nucleotide and deduced amino acid sequence of the Dictyostelium cyclin B gene (clb1). The 5′, 3′ and intron non-coding nucleotide
sequences are designated by lower case letters and the coding regions are in capital letters. The numbers indicate the nucleotide positions in the
sequence starting from the beginning of the isolated genomic clone. The asterisk indicates the termination codon.
Truncated cyclin B in Dictyostelium cell division 3109
The PCR product was 32P-labeled and used to probe a
Southern blot of restriction-digested DNA to confirm that it
corresponded to a Dictyostelium gene. At high stringency a
single hybridization fragment was observed (data not shown).
Isolation and characterization of the cyclin B gene
The 32P-labeled 450 bp PCR product was used to screen phage
libraries derived from mRNA of vegetative and early developmental cells. Three positive clones were isolated from the
vegetative cell cDNA library and one of these (clb1-c1) had an
insert of 1197 bp. Sequencing revealed that it only encoded
part of the expected cyclin B gene (Fig. 1). An additional clone
(clb1-c2) with a 2.1 kb insert was isolated from the early development library and sequencing revealed two open reading
frames, but only the downstream one had sequence overlaps
with the clb1-c1 clone. This downstream sequence had a
potential methionine initiation codon and upstream of the
methionine there were three termination codons, suggesting
that the cDNA might contain the complete coding sequence.
However, since the clone was a hybrid it was possible that the
5′ sequence was part of the second, upstream gene.
In order to confirm that the clb1-c2 clone contained the
complete cyclin B sequence, the labeled 514 bp BglII-EcoRI
fragment of clb1-c2 cDNA was used to screen a genomic
library. One positive clone (clb1-g) was isolated that contained
a 2.5 kb insert. The DNA was digested with several restriction
enzymes to generate a restriction map of the gene (Fig. 1). Subsequent DNA sequence analysis and comparison of the
genomic sequence with that of the clb1-c2 cDNA revealed
three things. Firstly, the isolated cyclin B genomic DNA has
432 bp of 5′ upstream sequence that does not contain a
complete functional promoter (G. Weeks, unpublished observation). Secondly, the genomic sequence contains a single 128
bp intron close to the 5′ end of the coding sequence (Fig. 2)
and this intron has conserved short consensus sequences at the
splice junctions (Mount, 1982). Thirdly, the sequence of the
genomic clone from 418-672 bp and 801-2018 bp, which
includes 15 bp of 5′ non-coding sequence and 151 bp of 3′ noncoding sequence, is identical to the cDNA sequence of cDNA
clone clb1-c2 (Fig. 2). The three stop codons within the 15 bp
upstream sequence confirmed that the presumptive start codon
identified initially in the clb1-c2 sequence was correct. These
data support the idea that the clb1-c2 clone did contain a fulllength cDNA. The complete nucleotide sequence and the
derived amino acid sequence of the gene are shown in Fig. 2.
An additional 450 bp of 3′ non-coding sequence of the gene
has not yet been determined.
The 1308 bp open reading frame from the full-length clb1c2 cDNA predicts a protein of 436 amino acids with a
molecular mass of 49.5 kDa. Previous comparisons of the
amino acid sequences of cyclins have identified a large region
of homology termed the cyclin box (Draetta, 1990). The
cyclin box begins around residue 200 (using numbers from
human cyclin B1) and extends for 150 amino acids. Fig. 3
shows an alignment of the cyclin box of the encoded Dictyostelium protein with the cyclin boxes of the Xenopus B2,
Fig. 3. Alignment of the amino acid sequence of the cyclin box of the Dictyostelium cyclin B protein with those of selected cyclins of other
organisms: Xenopus cyclin B2 (Minshull et al., 1989), starfish cyclin B (Labbé et al., 1989), human cyclin B1 (Pines and Hunter, 1989) and
human cyclin A (Wang et al., 1990). Amino acids that are identical to those of Dictyostelium cyclin B are indicated by a dash (-). The B-type
cyclin consensus sequence (FLRRxSK) is boxed and the A type consensus sequence EVxEEY/D is indicated (m). Two gaps have been
introduced into the cyclin B sequences to obtain the best alignment with human cyclin A.
3110 Q. Luo, C. Michaelis and G. Weeks
kb
Fig. 4. Southern blot analysis of the Dictyostelium clb1 gene. A 5 µg
sample of genomic DNA from D. discoideum strain V12-M2 was
digested with EcoRI (lane 1), ClaI (lane 2), HindIII (lane 3), HincII
(lane 4). The digested DNA was size fractionated on a 0.8% agarose
gel and then transferred to a nylon membrane. The filter was probed
with the 32P-labeled 1.5 kb fragment of clb1-c2 cDNA and washed
under low stringency conditions (A) and high stringency conditions
(B) as described in Materials and Methods. Molecular size standards
(kb) are indicated.
starfish B, human B1 and human A cyclins. Within this
sequence the presumptive Dictyostelium cyclin B exhibits
50% identity with Xenopus cyclin B2, 51% identity with
starfish cyclin B and 48% identity with human cyclin B1. It
exhibits considerably less identity to human cyclin A (40%)
and is even more diverged from other types of human cyclin:
cyclin E (33%), cyclin D1 (26%) and cyclin C (16%)
(Minshull et al., 1989; Labbé et al., 1989; Pines and Hunter,
1989; Wang et al., 1990; Surana et al., 1991; Lew et al.,
1991). Furthermore, the Dictyostelium protein has a sequence
motif (FLRRxSK), which is conserved in B-type cyclins but
not in A-type cyclins (Fig. 3), but does not contain the
EVxEEY/D motif, which is conserved in A-type cyclins but
absent from B-type cyclins. Dictyostelium cyclin B also has
the conserved destruction box (RxxLxxIxN), characteristic of
other B-type cyclins (Glotzer et al., 1991; Hunt, 1991). In
view of these results we have designated the Dictyostelium
protein cyclin B1 and the encoding gene clb1. When a
Southern blot was probed with the 32P-labeled 1.5 kb HincIIEcoRI fragment of clb1-c2 cDNA, only one hybridization
fragment was found in each of four Dictyostelium genomic
DNA restriction digests under both low- and high-stringency
conditions (Fig. 4). This result indicates that the clb1 is a
single copy gene and also suggests that there are no genes that
are highly related to clb1 in Dictyostelium.
Expression of the clb1 gene during cell growth
Northern blot of RNA isolated from Ax-2 cells at various
Fig. 5. Northern blot analysis of RNA isolated from Dictyostelium
Ax-2 cells at various stages of growth. Total RNA was extracted
from cells at the following cell densities: 9×105 (lane 1), 2×106 (lane
2), 5×106 (lane 3), 8×106 (lane 4), 1×107 (lane 5), 2×107 (lane 6),
2×107 (lane 7) and 20 µg was fractionated, blotted and probed with
the 514 bp fragment of clb1-c2 cDNA as described in Materials and
Methods. The approximate size (kb) of the detected transcript is
indicated by the arrow and was determined by the mobility of the
transcript relative to high molecular size RNA standards (BRL).
stages of growth, revealed a single mRNA of approximately
2.3 kb (Fig. 5). The clb1 mRNA was present at constant levels
during the exponential growth of an asynchronously growing
population but then decreased dramatically when cells passed
from the exponential phase into the stationary phase. Synchronized cell growth was obtained by diluting stationary
phase Ax-2 cells into fresh axenic medium (Yarger et al.,
1974). Since cell division took 5 hours to complete, the
synchrony obtained was relatively poor (Fig. 6A). Nevertheless, northern blot analysis revealed cell cycle-dependent
expression of the clb1 gene (Fig. 6B). The clb1 mRNA was
barely detectable in the initial cell population (Fig. 6B), a result
consistent with that previously obtained from stationary phase
cells (Fig. 5), but began to increase after only 1 hour in fresh
medium. The mRNA level continued to increase to a maximum
at 3-4 hours just prior to the initiation of cell division. Levels
than declined to a minimum at 8 hours just before cell division
was finished and then increased during the remainder of the
experiment (Fig. 6B).
When cell-free extracts were analyzed for levels of cyclin
B protein by western blot analysis using affinity-purified
cyclin B antibody, it was found that the antibody reacted with
only one protein of approximately 50 kDa, the predicted size
of the encoded clb1 product (Fig. 6C). The protein level was
very low in stationary-phase cells (0 hour) and then increased
to a maximum between 5 and 6 hours at which time cells were
beginning to divide. Protein levels then decreased until the end
of cell division. These results indicated that the Dictyostelium
clb1 mRNA and the encoded cyclin B protein are expressed
at specific stages of the cell cycle, a characteristic feature of
the cyclins of other species. It is of note that mRNA
expression precedes protein expression by approximately two
hours, a result similar to that seen previously for developmentally expressed Dictyostelium genes (Kimmel and Firtel,
1982).
Truncated cyclin B in Dictyostelium cell division 3111
Fig. 7. Southern blot
analysis of clb1∆105
transformants. A 10 µg
sample of genomic DNA
isolated from two
independent
transformants (lanes 2
and 3) and Ax-2 (lanes 1
and 4) was digested with
XbaI and SstI, and
fractionated and blotted
as described in Materials
and Methods. The blot
was washed under high
stringency conditions and
exposed against X-ray
film for either 30 minutes
(lanes 1-3) or overnight
(lane 4). The letters (A)
and (B) indicate the
fragments containing the
endogenous clb1 gene
and the 1.2 kb clb1∆105
transgene, respectively.
Fig. 6. clb1 mRNA and cyclin B protein levels in Ax-2 cells
synchronized by stationary phase release. Stationary-phase cells
were diluted into fresh medium at a density of 1×106 cells/ml and:
(A) cell number, (B) clb1 mRNA and (C) cyclin B protein were
determined at the indicated times (hours), as described in Materials
and Methods. The northern blot (B) was probed with the labeled 1.5
kb fragment of clb1-c2 cDNA.
Effects of overexpression of a N-terminal truncated
cyclin B on cell growth
The 5′ portion of the clb1 cDNA encoding the N-terminal 105
amino acids of the protein, which included the destruction box,
was deleted and the truncated clb1 gene was inserted into a
vector pVEII under the control of the regulatable discoidin-Iγ
promoter. The pVEII-cyclin B construct was transformed into
Ax-2 cells and cells were selected for G418 drug resistance in
the presence of 1 mM folate to suppress the activity of the
discoidin promoter. Nine transformants were isolated. Two of
the transformants were analyzed for the presence of the
transgene. The Southern blot analysis showed the predicted 1.2
kb XbaI/SstI fragment in both transformants, indicating no
gross rearrangement of the transgene. The fragment was
present in a very high copy number, since the endogenous gene
was barely detectable after overnight exposure of the Southern
blot, while the transgene required only a 30 minute exposure
(Fig. 7).
Transformants were grown to 2×106 cells/ml in HL-5
medium containing folate and then reinoculated into fresh HL5 medium either with or without folate at a density of 1×105
cells/ml. The growth of one of the transformants is shown in
Fig. 8A. In the presence of folate, growth continued to a
density of approximately 6×106 cells/ml, whereas in the
absence of folate, growth ceased at a density of approximately
9×105 cells/ml (Fig. 8A). The nine independently isolated
transformants exhibited exactly the same characteristics (data
not shown).
The expression of the truncated clb1 gene, was determined
by northern blot analysis. The clb1 probe hybridized to the 2.3
kb mRNA expressed from the endogenous gene and also to a
1.2 kb mRNA expressed from the truncated transgene (Fig.
8B). The truncated clb1 mRNA level increased dramatically
after growth for 18 hours in the absence of folate and levels
were still high 40 hours after cell growth had arrested. The
mRNA expressed from the endogenous gene was undetectable,
suggesting the possibility that the very high level of expression
from the truncated gene repressed the expression of the
endogenous gene. When the transformant was grown in
medium containing folate a much lower level of truncated clb1
was expressed.
Western blot analysis revealed that both the native and the
truncated protein were detectable in transformants grown in
the absence of folate, although the truncated protein had a
somewhat slower mobility than that anticipated from its
molecular mass (Fig. 9B). When cells were grown in the
absence of folate from a density of 1×105 cells/ml to a density
of 6×105 cells/ml the truncated protein was only present at
the same level as the native protein. However, by the time
cell growth had ceased at 1.2×106 cells/ml, the level of
truncated protein had increased substantially and it remained
3112 Q. Luo, C. Michaelis and G. Weeks
Fig. 8. Cell growth and northern blot analysis of a clb1∆105
transformant. Exponentially growing cells were transferred from HL5 medium containing 1 mM folate to HL-5 medium containing either
1 mM folate (indicated by the filled circles) or no folate (indicated by
the open circles). (A) Cell numbers were determined by
hemocytometer counts at the indicated times. (B) A 20 µg sample of
total RNA isolated from cells grown in the presence of folate (lanes
1 to 5) and in the absence of folate (lane 6 to 10) was fractionated,
blotted and probed with the 32P-labeled 514 bp fragment of clb1-c2
cDNA. Cells were harvested at 0 hour (lanes 1 and 6), 18 hours
(lanes 2 and 7), 49 hours (lanes 3 and 8), 63 hours (lanes 4 and 9)
and 89 hours (lanes 5 and 10) after transfer. The clb1 mRNA and
clb1∆105 mRNA are indicated by arrows.
high in the growth-arrested cells for the next 24 hours of incubation (Fig. 9A,B). When cells were grown in the presence
of folate, the truncated mRNA was present in slightly higher
levels than the mRNA expressed from the endogenous gene
(Fig. 8B) but the truncated protein was not detectable (Fig.
9B).
It has been reported that the presence of stable cyclin B
causes mitotic arrest (Luca et al., 1991; Murray et al., 1989;
Ghiara et al., 1991). Dictyostelium cells were Giemsa stained
to determine if the cells that ceased growing in the absence of
folate were arrested in M phase. Approximately 60% of the
cells that had ceased growing for 12 hours contained
condensed chromosomes. Most of the condensed chromosomes were insufficiently resolved to assess the stage of
mitotic arrest but, in a few instances, prophase, metaphase (Fig.
10) and anaphase (data not shown) structures could be
detected. By comparison, only 1-2% of cells from a randomly
growing parental AX-2 population have condensed chromosomes. These results indicate that cells overexpressing the
truncated cyclin B gene arrest in M-phase. We have not yet
determined whether the arrested cells have elevated histone H1
kinase activity.
Fig. 9. Cell growth and western blot analysis of a clb1∆105
transformant. Exponentially growing cells were transferred from the
HL-5 medium containing 1 mM folate to HL-5 medium containing
either 1 mM folate (indicated by the filled circles) or no folate
(indicated by the open circles). (A) Cell numbers were determined by
hemocytometer counts at the indicated times after transfer. (B) A 20
µg sample of protein from cells grown in the medium containing 1
mM folate (lane 1, 2 and 3) and no folate (lanes 4-8) was
electrophoresed, blotted and probed as described in Materials and
Methods. Cells were harvested in 2% SDS solution at the times
indicated in (A).
DISCUSSION
In this paper we have described the isolation of a gene from
Dictyostelium, whose encoded product contains a conserved
cyclin box sequence, characteristic of all cyclin proteins.
Alignment of the cyclin box sequences with those from other
species showed that the Dictyostelium gene product had a
higher degree of identity to B-type cyclins than to other
members of the cyclin family. We therefore classified the
protein as a B-type cyclin and designated the gene cyclin B1
(clb1).
The presence of multiple B-type cyclins has been reported
in several species (Gallant and Nigg, 1992; Minshull et al.,
1989; Surana et al., 1991). The cyclin B1 and B2 of Xenopus
have a high degree of sequence identity to each other within
the cyclin box (65%). This high sequence identity is sufficient
for them to cross-hybridize under low stringency wash conditions (Minshull et al., 1989). The CLB1 and CLB2 gene
products of Sacchanomyces cerevisiae show a similar high
degree of sequence identity to each other (82%) and also crosshybridize (Surana et al., 1991). In order to determine if Dictyostelium possessed other highly related cyclin B genes, low
stringency washes were performed on a Southern blot restriction digest (Fig. 4). No additional hybridizing fragment was
detected, suggesting that there are no genes that are highly
related to the clb1. Under the same low stringency wash con-
Truncated cyclin B in Dictyostelium cell division 3113
Fig. 10. Giemsa staining of a clb1∆105 transformant. Cells were
incubated in HL-5 medium containing no folate for 12 hours after
growth had ceased and then fixed and stained with Giemsa as
described in Materials and Methods.
ditions a whole family of Dictyostelium cdc2-related genes
cross-hybridized to the previously isolated cdc2 gene
(Michaelis and Weeks, 1993). We cannot rule out the possibility, however, that there are additional B-type cyclins with
lower levels of sequence identity within the cyclin box.
In vertebrate and invertebrate embryo and in Schizosaccharomyces pombe, levels of cyclin B protein oscillate during the
cell cycle, but the level of cyclin B mRNA remains constant.
However the levels of both cyclin B mRNA and protein in
human and S. cerevisiae cells are periodic during the cell cycle.
The levels peak late in the G2 phase of the cell cycle and are
at a minimum in G1 phase (Pines and Hunter, 1989; Ghiara et
al., 1991). In Dictyostelium the production of clb1 mRNA and
cyclin B protein show a similar overall pattern to that of S.
cerevisiae and the human tissue culture cells. When Dictyostelium cells were released from stationary phase by the
addition of fresh medium, partial synchronous growth was
induced. The completion of cell division required 4 to 5 hours
(Fig. 6A), whereas it has been estimated that the M phase in
Dictyostelium is of only 15 minutes duration (McDonald and
Durston, 1984). Nevertheless, the clb1 mRNA level increased
dramatically soon after refeeding and reached a maximum at
3 and 4 hours, just before cell division was initiated. The cyclin
B protein level was very low between 0 and 3 hours and then
reached a maximum between 5 and 6 hours, which is in the
middle of the cell division period. The cyclin B protein level
decreased more dramatically than the clb1 mRNA level when
cell division was completed at 8-9 hours.
Just as the rise in cyclin B is essential for cells to enter
mitosis, its destruction is required for cells to exit mitosis and
enter interphase. The destruction of cyclin B at the end of
mitosis is dependent on its ubiquitination, which is targeted by
the cyclin destruction box, a short consensus sequence located
within the N-terminal 90 amino acids of cyclin B (Glotzer et
al., 1991). It has been shown that both sea urchin and clam
cyclin B, which lack the N-terminal 90 and 97 amino acids,
are resistant to proteolysis. The introduction of these truncated
cyclin B protein into Xenopus eggs and egg lysates keeps Cdc2
protein kinase hyperactivited, maintains chromosomes in a
condensed state and blocks the completion of mitosis (Murray
et al., 1989; Luca et al., 1991). Similarly, in S. cerevisiae, when
a truncated cyclin B gene (CLB1) was transformed into cells
under the control of the GAL1 promoter, the expression of the
truncated cyclin B protein resulted in cessation of cell growth
due to mitotic arrest (Ghiara et al., 1991). In the present study,
we have transformed cells with a truncated Dictyostelium
cyclin B gene that lacks the coding sequence for the 105 Nterminal amino acids and which is under the control of the
folate-repressible discoidin promoter. In the absence of folate,
the truncated gene was highly expressed and cell growth
arrested in M phase. These results suggest that the destruction
of the clb1 gene product is essential for passage through M
phase and further confirm the classification of the isolated Dictyostelium cyclin as a B-type mitotic cyclin.
The demonstration that a truncated cyclin B gene arrests
Dictyostelium growth during mitosis provides a possible
approach for determining if continuation of the cell cycle is
necessary for Dictyostelium development. Thus it should be
possible to place the truncated cyclin B gene under the control
of developmental promoters to determine if cell cycle arrest at
a specific developmental stage blocks continued development.
In addition, it might be possible to alter the developmental
pattern by manipulating the levels of the truncated cyclin B
protein during growth.
We thank Dr J. Daniel for the λZapII, Dr J. Cardelli for the λgt11
cDNA libraries, Dr F. Dill for assisting us in the examination of the
Giemsa-stained cells and Dr G. B. Spiegelman for many stimulating
discussions. This research was supported by a grant from NSERC.
The nucleotide sequence of the clb1 gene has been submitted to the
Genbank database under accession number U11056.
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(Received 20 April 1994 - Accepted 1 July 1994)