Download Identification of seven new cut genes involved in

Journal of Cell Science 105, 135-143 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
135
Identification of seven new cut genes involved in Schizosaccharomyces
pombe mitosis
Itaru Samejima1, Tomohiro Matsumoto2, Yukinobu Nakaseko1, David Beach2 and Mitsuhiro Yanagida1,*
1Department of Biophysics, Faculty of Science,
2Howard Hughes Medical Institute, Cold Spring
Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
*Author for correspondence
SUMMARY
Fission yeast cut mutants cause cytokinesis in the
absence of normal nuclear division. These mutants show
abnormal uncoupled mitosis and are known to be the
result of mutations in the genes encoding DNA topoisomerase II, proteins related to spindle pole duplication,
and a kinesin-related mitotic motor. We have screened
717 temperature-sensitive (ts) mutants by individually
observing their cytological phenotypes at the restrictive
temperature, and have newly isolated 25 cut mutants.
Genetic analyses indicate that 14 of them fall into five
previously identified loci, namely, top2, cut1, cut5, cut7
and cut9, whereas nine have been mapped onto seven
new loci, designated cut13 to cut19. The cytological phenotypes of the newly identified cut mutants can be classified into three groups. One group consists of mutants
in which a portion of the nuclear chromatin is stretched
by the elongated spindle but the entire nucleus is not
INTRODUCTION
A set of duplicated chromosomes is separated and distributed to each of two daughter cells during mitosis and subsequent cytokinesis. Nuclear division consists of a number
of processes that include chromosome condensation, spindle formation, kinetochore attachment to the spindle, sister
chromatid separation and chromosome movement to the
poles, followed by spindle elongation and chromosome
decondensation. To ensure high-fidelity transmission of
genetic information to daughter cells, these processes must
be executed in a precise and coordinated fashion. Failure
of any of these processes would lead to a disorganized mitosis and daughter cells with altered genetic information. Such
a situation is generally a lethal event.
One approach to the investigation of the mitotic process
is to isolate and characterize mutants. Many mitotic mutants
of the fission yeast Schizosaccharomyces pombe have been
isolated and have proved to be useful for understanding the
control mechanism of mitosis (Nurse, 1990; Yanagida,
1989). A group of temperature-sensitive (ts) mutants called
cut (cell untimely torn; Hirano et al., 1986) are of particular interest with regard to coordination of different mitotic
separated, reminiscent of, but not identical to, the phenotypes of top2 and cut1; mutants cut14-208, cut15-85,
cut16-267 and cut17-275 display such a phenotype.
Another group exhibits non-disjunctioned and condensed chromosomes in the presence of the spindle;
cut13-131 belongs to this group. The cut19-708 mutant
has also been found to have condensed chromosomes.
The remaining group has a mixed phenotype of the
above two groups; namely, stretched chromatin and
condensed chromosomes; cut18-447 exhibits such a
phenotype. The isolation and characterization of the
mutated genes will be the subjects of future investigations.
Key words: mitosis, cut mutant, fission yeast, DNA topoisomerase
II, motor protein, spindle movement
events; cut mutants do not arrest at a specific stage at the
restrictive temperature (36°C), but undergo uncoordinated
mitosis, i.e. cytokinesis takes place without prior completion of nuclear division, resulting in uncoordinated cleavage of an undivided nucleus by the septum (Fig. 1). In some
cut mutants, cells containing the septum and the displaced
nucleus also appear, in addition to the cells with the cut
phenotype.
Mutations in a variety of genes are known to produce the
cut phenotype (Fig. 1). top2 mutants, defective in DNA
topoisomerase II (Uemura and Yanagida, 1984), show a
typical uncoordination between nuclear division and cytokinesis, so causing this phenotype (Fig. 1; Uemura and
Yanagida, 1986). A small portion of the nuclear chromatin
is pulled by the transient mitotic spindle in top2 mutant
cells at 36°C. In cut1 and cut2 mutants, a large portion of
the nuclear chromatin is extended by apparently normal
spindle dynamics, but the nuclear chromatin moves back to
the middle of the cell after the spindle has broken down
(Uzawa et al., 1990); cytokinesis follows the cut across the
undivided nucleus. Polyploid chromosomes are formed in
cut1 and cut2 if septation is blocked (Uzawa et al., 1990;
Creanor and Mitchison, 1990). The product of the cut1+
136
I. Samejima and others
Fig. 1. Schematic representation of the cut
mutant phenotype at the restrictive
temperature. Wild-type nuclear division is also
shown. Mutant cells enter cytokinesis without
completing normal nuclear division. Abnormal
mitoses seen in top2, cut1 or cut9 mutants
prior to cytokinesis are illustrated. Partially
separating DAPI-stained region resembling an
archery bow (archery-bow phenotype) is seen
in cut1 mutant. top2 mutant shows an
interphase-like non-condensed chromatin with
protrusion of a string of chromatin. cut9
mutant is transiently arrested at the midmitosis stage with the metaphase plate-like
structure.
gene partly resembles that of the budding yeast ESP1 gene
(Baum et al., 1988). The cut7+ gene was isolated by transformation and its predicted product has an amino acid
sequence similar to that of the microtubule-dependent
motor, kinesin (Hagan and Yanagida, 1990). cut7 mutants
are defective in spindle formation, probably caused during
the process of interdigitation of two half-spindles (Hagan
and Yanagida, 1992). cut9 mutant cells show a temporal
arrest in metaphase at 36°C (Fig. 1; I. Samejima and M.
Yanagida, unpublished result). The cut9+ gene was cloned
by transformation and the gene product has been characterized (I. Samejima and M. Yanagida, unpublished).
Nine cut genes (cut1+-cut9+) were originally identified
(Hirano et al., 1986) and two other genes (cut11+ and
cut12+) were recently found (R. Bartlett and P. Nurse, personal communication) so that 11 genes showing the cut
phenotype have so far been identified (the cut10 locus was
found to be identical to cut1; Uzawa et al., 1990, and S.
Uzawa, unpublished result). With regard to the phenotypes
that cause a fatal event in uncoupled mitosis, potentially
these genes have the ability to monitor a checkpoint event
in cell cycle control (Weinert and Hartwell, 1988; Hartwell
and Weinert, 1989). None of the cut+ genes, including
top2+ has, however, been shown to have such a function.
Interestingly, fission yeast checkpoint-deficient mutants,
isolated because of their inability to grow in the presence
of hydroxyurea, a DNA synthesis inhibitor, display the cut
phenotype (Enoch et al., 1991); these mutants enter mitosis without completing DNA synthesis, in the presence of
hydroxyurea. Four checkpoint mutants (rad1, rad3, rad9
and rad17) have been identified; these mutants also enter
abnormal mitoses when cells are either irradiated with
gamma-rays or blocked in DNA synthesis (Rowley et al.,
1992; Al-khodairy and Carr, 1992).
In the present study, a new search for cut mutants has
been made using a ts mutant collection, which was used for
the isolation of mutants that produce condensed chromosomes at the restrictive temperature (Matsumoto and Beach,
1991). Genetic analyses of the isolated mutants have identified seven new cut loci that produce distinct phenotypes.
MATERIALS AND METHODS
Schizosaccharomyces pombe strains used were 975h+, JY6 (h+
leu1 his2), SP6/HM123 ( h leu1), cut1-cut9 (Hirano et al., 1986),
top2 (Uemura and Yanagida, 1984), sad1-549 (Hagan and
Yanagida, 1990), dip7, cut11, and cut12 (gifts from R. Bartlett
and P. Nurse). Standard genetic procedures for tetrad dissection,
random spore and heterozygous diploid analyses, as described by
Gutz et al. (1974), were followed.
Media used were YEA (0.5% yeast extract, 3% glucose), YPD
(1% yeast extract, 2% polypeptone, 2% glucose), SD (minimal
medium; 0.67% yeast nitrogen base without amino acids, 2% glucose; 1.7% agar was added for plates), and EMM2 (minimal
medium; Mitchison, 1970). Media containing 1.6% agar were used
for plating, unless indicated otherwise. SPA medium contained
(per l): 10 g dextrose, 1 g KH2PO4, 10 mg biotin, 1 mg calcium
pantothenate, 10 mg nicotinic acid, 10 mg meso-inositol and 30
g agarose and was used for sporulation. Escherichia coli was
grown in LB (0.5% yeast extract, 1% polypeptone, 1% NaCl,
(pH7.5); 1.5% agar was added for plates).
The procedure for mutagenesis was described in detail by Matsumoto and Beach (1991). Mutant screening for the cut phenotype was carried out by growing individual ts strains in YEA or
YPD at the permissive temperature, and then incubating at the
restrictive temperature (Matsumoto and Beach, 1991); cells were
observed under a fluorescence microscope following DAPI staining, to determine whether they showed the cut phenotype at the
restrictive temperature. All the ts strains isolated and used for
backcrossing showed the ts phenotype in both YPD and YEA
media, except for strains 599 and 708 where the phenotype was
not as evident in YPD.
Fluorescence microscopy
The procedure for DAPI (4′,6-diamidino-2-phenyl-indole) staining of S. pombe cells, described by Adachi and Yanagida (1989)
was followed. Immunofluorescence microscopy using anti-tubulin
antibody (TAT1, a gift from Dr. K. Gull; Woods et al., 1989) and
New cut genes required in fission yeast mitosis
137
Bartlett and P. Nurse, personal communication) and sad1
(I. Hagan and M. Yanagida, unpublished). These were
crossed with the 21 new cut mutant strains, and a random
spore analysis for the ts phenotype was carried out. Unless
two ts− mutations in the crossed strains are tightly linked,
about a quarter of the progeny spores will be the wild-type
recombinants (ts+). The recombination frequency between
mutations in an identical gene will be less than 0.05%. As
shown in Table 1, twelve of the twenty-one mutants examined fell into the previously identified five loci: cut1 was
allelic to five strains (380, 474, 514, 686, and 693); top2
to three (4, 137, and 640); cut7 to two (138 and 611); and
two strains to cut5 (401) and cut9 (98), respectively. The
other nine strains (85, 122, 131, 208, 267, 275, 447, 599,
and 708) were linked to none of them.
To confirm the above assignments, plasmids carrying the
corresponding wild-type genes were introduced into mutant
cells and examined to determine whether the mutant phenotypes could be suppressed by a plasmid carrying the gene
tightly linked to the mutation site. The five genes used have
previously been cloned and characterized (top2+, Uemura
et al., 1986; cut1+, Uzawa et al., 1990; cut7+, Hagan and
Yanagida, 1990; cut5+, Y. Saka and M. Yanagida, unpublished result; cut9+, I. Samejima and M. Yanagida, unpublished result). These plasmids were introduced into five representative mutant strains by transformation. We found that
they were able to suppress the ts phenotype of corresponding mutants, therefore supporting the above genetic
identifications.
The four sterile strains were also transformed by the five
plasmids. Temperature sensitivity, but not sterility, of
strains 668 and 676 was rescued by plasmids carrying the
top2+ and cut1+ genes, respectively. This indicates that the
cut phenotypes of 668 and 676 are due to mutations in the
top2+ and cut1+ genes, respectively. None of the plasmids
rescued the ts lethal phenotype of the other two sterile
strains, 666 and 523. Therefore, we concluded that fourteen
newly isolated ts cut mutants were derived from five known
genetic loci.
aldehyde fixation was described by Hagan and Hyams (1988). The
secondary antibody used was FITC-conjugated goat anti-mouse
antibody (E.Y. Lab. Inc.). Each ts mutant cell was first grown in
YPD medium at the permissive temperature (26°C), and then
transferred to the restrictive temperature. Cells were taken at onehour intervals and observed under an epifluorescence microscope
after DAPI staining.
RESULTS
Isolation of cut mutants
A haploid S. pombe strain, SP6 (h leu1), was mutagenized
by nitrosoguanidine, and ts mutants were selected by
replica-plating at the permissive (26°C) and restrictive
(36°C) temperatures. One thousand ts mutants were thus
isolated, and 717 were subjected to cytological screening
(Matsumoto and Beach, 1991). Each strain grown at 26°C
was transferred to 36°C for 4 h prior to DAPI staining.
Cells were observed by fluorescence microscopy, which
revealed the nuclear chromatin structure within mutant cells
(Toda et al., 1981; Uemura and Yanagida, 1984; Hirano et
al., 1986; Ohkura et al., 1988). Twenty-five mutant strains,
which showed the cut phenotype (Hirano et al., 1986) in
more than 5% of their cell population at 36°C, were chosen
for further study.
Allelism test among cut mutants
Twenty-five newly identified cut mutants were first backcrossed with the wild-type h+ strain. Four (523, 666, 668,
676) were found to be sterile and failed to conjugate with
the wild type. In the remaining twenty-one strains, the ts
phenotype co-segregated 2+:2−, shown by tetrad analysis;
hence single mutations are responsible for the ts lethality
in these strains. One exception was the ts phenotype of
cut19-708, which was not segregated 2+:2−, so that it might
not be due to a single mutation.
Thirteen mutants that produced the cut phenotype were
previously identified, namely, top2 (Uemura and Yanagida,
1984), cut1-cut9 (Hirano et al., 1986), cut11 and cut12 (R.
Table 1. Random spore analyses of twenty-one newly isolated cut mutants
Strain
4
85
98
122
131
137
138
208
267
275
380
401
447
474
514
599
611
640
686
693
708
cut1
cut2
cut3
cut4
cut5
cut6
cut7
cut8
cut9
sad1
top2
cut11
cut12
+
+
+
+
+
+
+
+
+
+
–
+
+
–
–
+
+
+
–
–
+
+
+
+
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+
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+
+
+
+
+
+
+
–
+
+
+
+
+
+
–
+
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+
+
+
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+
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+
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+
+
+
+
+
–
+
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+
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+
+
+
+
+
–
+
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+
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+
+
+
+
+
+
–
+
+
+
+
+
+
–
+
+
+
+
+
+
Loci
top2
cut9
+
+
top2
cut7
+
+
+
cut1
cut5
+
+
+
cut1
cut1
+
+
+
+
+
+
+
+
cut7
top2
cut1
cut1
–
+
+
138
I. Samejima and others
Table 2. Pairwise crosses among nine new cut mutants
Strain
85
122
131
208
267
275
447
cut15-85
cut15-122
cut13-131
cut14-208
cut16-267
cut17-275
cut18-447
cut13-599
cut19-708
–
–
+
+
+
+
+
+
+
–
–
+
+
+
+
+
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+
+
–
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–
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–
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–
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–
+
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+
–
+
599 708
+
+
–
+
+
+
+
–
+
+
+
+
+
+
+
+
+
–
Identification of seven new cut genes
The remaining nine strains were crossed with JY6 (h+ leu1
his2), then subjected to complementation (criss-cross) tests
using the 81 possible pairwise crosses. As shown in Table
2, strains 85 and 122, and 131 and 599, respectively, were
found to belong to the same locus, but the other five showed
no linkage to any strains. Thus these nine mutations constituted seven new complementation groups.
The new cut loci were designated cut13 (131, 599), cut14
(208), cut15 (85, 122), cut16 (267), cut17 (275), cut18 (447)
and cut19 (708). Heterozygous diploids were made for each
cut mutant by crossing it with a mei1 mutant, and their ts
phenotype was examined. All of them were found to be ts+,
so these new cut mutations were recessive.
ts− segregants of the seven strains derived from crosses
with the wild type were grown at 26°C, and then shifted
up to 36°C. Samples of the cultures were withdrawn at
hourly intervals, and cells were fixed with glutaraldehyde,
stained with DAPI and observed under a fluorescence
microscope. All the ts segregant cells examined exhibited
the cut phenotype, demonstrating that the ts and cut phenotypes cosegregate in new cut mutants. In addition,
nuclear morphology of different cut mutant cells varied as
described below.
The frequencies of the cut phenotype in cut13 to cut19
mutants at the restrictive temperature were variable. In
cut13-131, cut14-208, and cut15-85, 35-60% cells showed
the cut phenotype at 36°C for 4 h. However, in cut16-267
and cut17-275, the frequencies were 10-20%.
Characterization of the cut13-131 mutant
The cellular and nuclear structures of cut13-131 at 36°C
were observed by fluorescence microscopy. Mutant cells
were first exponentially grown at 26°C, and then transferred
to 36°C. Samples of the culture were taken at one-hour
intervals, fixed by glutaraldehyde, stained by DAPI and
observed under an epi-fluorescence microscope. The wildtype interphase cells showed a single nucleus displaying the
hemispherical chromatin region, while mitotic cells displayed condensed chromosomes or dividing nuclei (Toda
et al., 1981). After nuclear division, cells underwent septation, followed by cell separation (Fig. 1).
As shown in Fig. 2A, a culture of cut13-131 initially contained a high proportion (80%) of normal interphase cells
(indicated by filled circles; also see Fig. 2B). The frequency
of the interphase cells decreased with the time of incubation at 36°C, down to a level of 10% after 4 h. One hour
after transfer to 36°C, cells displaying the condensed chromosomes (open triangles; Fig. 2C) peaked (maximal level,
12%). Cells with the cut phenotype (open circles; Fig. 2D)
increased after 2 h and reached 38% after 4 h. In addition,
septate cells containing an undivided nucleus (filled squares;
Fig. 2E) were seen in abundance. The frequency of such
cells began to increase after 3 h and attained 45% after 4 h.
Hence, approximately 83% cells of cut13-131 were abnormal in septation and cytokinesis at 36°C after 4 h.
To determine whether the spindle was formed in cut13131 cells at 36°C, cells were stained by the anti-tubulin antibody TAT1 (Woods et al., 1989) for immunofluorescence
microscopy. As shown in Fig. 2F, cells with a short spindle (average length 2 µm) showed condensed chromosomes
arranged in a metaphase plate-like structure (Hirano et al.,
1988). Aster microtubules (Hagan and Hyams, 1988) were
also seen. The frequency of such mid-mitotic cells peaked
after 1 h at 36°C. Cells with a longer spindle were also seen,
but infrequently. The length of the longer spindles was 4-6
mm, still much shorter than the wild-type elongated spindle
(maximum 15 µm). In those cells containing the longer spindle, condensed chromosomes were dissociated and scattered
along the spindle in the absence of sister chromatid separation (Fig. 2G). Thus, the spindle formed but failed to fully
elongate. However, the spindle structures were not visible
in cells after 3 h at 36°C; instead, the cytoplasmic microtubule arrays were abundant in septate cells.
None of the cut mutants so far isolated has shown this
mitotic defect. The short spindle phenotype and the absence
of sister chromatid separation somewhat resembled the phenotypes of cut4, cut8 and cut9 mutants (I. Samejima and
M. Yanagida, unpublished result).
Characterization of the cut14-208 mutant
The frequency of interphase cells in cut14-208 (indicated
by filled circles in Fig. 3A) decreased, while cells with the
cut phenotype (open circles) or septate cells (filled squares)
increased to 58% and 16%, respectively, when mutant cells
were incubated at 36°C for 4 h. In addition, cells displaying a portion of nuclear chromatin apparently pulled by the
mitotic spindle (open squares) gradually increased and
peaked at a level of 25% after 3 h (DAPI stained cells; Fig.
3B, top). The frequency of cells containing such an abnormal mitotic nuclear structure decreased to 4% after 4 h.
Cytokinesis was often seen to cut across the undivided
nucleus.
Anti-tubulin antibody TAT1 staining showed that the
mitotic spindle fully elongated in cut14-208 (Fig. 3C). A
small amount of fibrous chromatin DNA, apparently pulled,
extended along the elongated mitotic spindle. The main portion of the nuclear chromatin, which still remained in the
center of cells, also appeared to have been pulled, as both
sides were conical. This phenotype was distinct from that
of cut13-131. At least a portion of the chromosomes was
separated and the spindle fully elongated in cut14-208. This
phenotype resembles those of top2 (Uemura et al., 1986,
1987) and cut1 mutants (Hirano et al., 1986; Uzawa et al.,
1990). In cut1 cells, however, much larger amounts of chromatin DNA were separated, displaying the characteristic
archery-bow phenotype (Fig. 1). The phenotype of top2
mutant cells is very similar to that of cut14-208. In top2
mutants, the chromatin DNA pulled by the spindle was
recently demonstrated to contain the centromere DNA, by
New cut genes required in fission yeast mitosis
139
A
100
Frequency
cut13-131
50
0
1
2
3
4
(h)
Fig. 2. Phenotype of cut13-131 at the
restrictive temperature. Cells of cut13131 exponentially grown at 26°C were
transferred to 36°C and incubated for 4 h.
(A) Samples of the culture were taken at
one-hour intervals, stained with DAPI
and observed by fluorescence
microscopy. Frequency of cells showing
the interphase chromatin structure
(indicated by filled circles), the cut
phenotype (open circles), and the
condensed chromosomes (open triangles).
The septate cells with the interphase
chromatin to one side are indicated by
filled squares. (B-E), DAPI-stained
fluorescence micrographs of an
interphase cell at the permissive
temperature (B), a cell with condensed
chromosomes (C), a cell showing the cut
phenotype (D) and a septate cell with the
nucleus in one half only (E) at the
restrictive temperature.
(F-G) Immunofluorescence micrographs
using anti-tubulin antibody TAT1.
(F) Two cells showing the short mitotic
spindle (top) and the same cells stained
with DAPI (bottom). (G) Two cells
showing elongated spindle (top) and the
condensed chromosomes (bottom). Bar,
10 µm.
fluorescence in situ hybridization (FISH). In order to examine the functional relationship between cut14+ and top2+
genes, we tested whether cut14-208 could be suppressed by
a multicopy plasmid carrying the top2+ gene; however, no
ts+ transformant cells were obtained by transformation. The
double mutants cut14-top2 and cut14-top1 showed the cut
phenotype at 36°C.
Characterization of cut15-85
The phenotype of cut15-85 was similar to that of cut14208. Cells with the cut phenotype (open circles) and septate cells (filled squares) increased to the levels of 56% and
25%, respectively, at 36°C after 4 h (Fig. 4A). Cells displaying pulled nuclear chromatin were observed. The fre-
quency of such aberrant mitotic cells made a peak (20%)
at 2 h, but only 2% of the total cells showed such figures
after 1 or 4 h. This pulled chromatin phenotype is similar,
but not identical, to that of cut14-208. The overall structure of the nuclear chromatin was quite variable, often
highly extended and deformed (Fig. 4B). In cut14-208,
however, only a small portion of chromatin DNA was
pulled. The amount of extended chromatin in cut15-85 was
larger than that of cut14-208, and intermediate between
top2 and cut1 mutants.
Characterization of other cut mutants
In cut16-267 mutants at 36°C, small portions of cells
showed the nuclear chromatin stretched by the spindle (data
140
I. Samejima and others
A
A
100
cut14-208
Frequency
80
60
40
20
0
0
1
2
3
4
5
Time (h)
Fig. 3. Phenotype of cut14-208 at the restrictive temperature. Cells of cut14-208 were grown at 26°C and then transferred to 36°C.
(A) Frequency of cells with the interphase chromatin (filled circles), the cut phenotype (open circles), the nuclear chromatin pulled by the
spindle (open squares), and the septum (filled squares). (B-C) Immunofluorescence micrographs of cut14-208 incubated at 36°C for 2 h.
Anti-tubulin stain (above) and DAPI (below). Bar, 10 µm.
not shown). The frequency of cut17-275 cells with the cut
phenotype was lower than that of septate cells (Fig. 5A);
66% of cells were septate, while 18% displayed the cut phenotypte after 4 h at 36°C. DAPI stained cut17-275 cells
after 2 h at 36°C (Fig. 6A) showed interphase chromatin in
one side of the septate cells and no nucleus in the other,
and the nuclear chromatin was pulled. The extended chromatin was often asymmetrical, extending in only one direction, along the spindle axis. In addition to the septate cells,
cells without a nucleus were frequently seen. Such anucle-
ate cells might be produced by cytokinesis of septate cells
with a single nucleus.
The frequency of the cut phenoypte in cut18-447 (Fig.
5B) was approximately 40% after 4 h at 36°C. Cells with
stretched or condensed nuclear chromatin were infrequently
noted prior to cytokinesis. Cells incubated at 36°C for 3 h
showed DAPI-stained nuclear chromatin that was mostly of
the interphase type. Nuclear chromatin in mitotic cells,
however, was highly variable in morphology (Fig. 6B).
Stretched nuclear chromatin very similar to that found in
A A
100
cut15-85
Frequency
80
60
40
20
0
0
1
2
3
4
5
Time (h)
Fig. 4. Phenotype of cut15-85 at the restrictive temperature. (A) Cells grown at 26°C were transferred to 36°C. Frequency of cells
showing the interphase chromatin (filled circles), the cut phenotype (open circles), the pulled nuclear chromatin (open squares) and the
septum with the nucleus in one side of the cell (filled squares). (B) DAPI-stained cells incubated at 36°C for 2 h. Deformed or pulled
chromatin in the septate cells are shown. Bar, 10 µm.
New cut genes required in fission yeast mitosis
A
100
cut17-275
Frequency
80
60
40
20
0
0
1
2
3
4
5
Time (h)
B
100
cut18-447
Frequency
80
60
40
20
0
0
1
2
3
4
5
Time (h)
Fig. 5. The phenotypes of cut17-275 (A) and cut18-447 (B) at the
restrictive temperature. Frequency of cells showing the interphase
nuclear chromatin (filled circles), the cut phenotype (open circles),
the septum with the nucleus in one side (filled squares) and the
condensed or pulled nuclear chromatin (open triangles).
cut1 or cut2 was seen. Seven per cent of the cells had condensed chromosomes, usually clustered, after 3 h.
Cells with condensed chromosomes were also observed
in cut19-708 and two sterile strains. Strains 523 and 666
displayed such chromosomes in 14% and 17% of cells,
respectively, after 2-3 h at 36°C. The frequency of the septate cells was high, 45%-60%, after 4 h at 36°C, while cells
showing the cut phenotype were less frequent, 14%-22%.
523 was auxotrophic (poor growth in EMM medium containing leucine), but the segregation of ts and auxotrophy
was not examined because of its sterility. ts nuc2-663 is
sterile and produces a high frequency of condensed chromosomes (Hirano et al., 1988). The cloned nuc2+ gene did
not complement the ts phenotype of 666.
DISCUSSION
Fission yeast ts mutants were previously screened for those
exhibiting the cut phenotype at the restrictive temperature
(Hirano et al., 1986). After examining 587 ts strains, 18
were obtained which made up 10 complementation groups,
including top2. In the present study, a new collection of
141
717 ts mutants (Matsumoto and Beach, 1991) was
screened, and 25 cut mutants, making up 12 complementation groups, were isolated. Five genetic loci, top2, cut1,
cut5, cut7 and cut9, were identified in the previous
and the present cut mutant isolation. Thus, a total of 17
genetic loci producing the cut phenotype were obtained
from approx. 1300 ts strains, making up roughly 1.3% of
the ts strains examined. Because 12 of the loci consist of
only one allele, the saturation point for cut mutants has not
yet been reached, and many more cut mutants may be isolated. The S. pombe genome consists of approximately
1.4×107 bp (Fan et al., 1988), probably containing 7,00010,000 genes. Assuming that 30% of the genes can generate ts mutants, approximately 25-40 loci may be expected
to produce the ts cut phenotype. This number is not surprisingly large, considering the complexity of cell division
mechanisms. cut mutants were also isolated from a collection of cold-sensitive (cs) mutants (Uemura et al., 1987).
Among 982 cs mutants cytologically examined at the
restrictive temperature, only three showed the cs cut
phenotype, and one was top2 defective. The reason for the
low occurrence of cs cut mutants is not yet known. Higher
temperature or higher growth rate may facilitate mitotic
uncoupling in ts mutant cells, which results in the cut
phenotype. Alternatively, the products of many cut+ genes
might be enzymes, so that denaturation of mutant enzymes
at low temperature might occur only infrequently. cs mutations may frequently be found in genes, whose products
are needed for making higher-order structures by proteinprotein interactions.
The cut+ gene products so far identified are cut1+ and
cut2+ (Uzawa et al., 1990), cut7+ (Hagan and Yanagida,
1990), cut8+ and cut9+ (I. Samejima and M. Yanagida,
unpublished, quoted by Goebl and Yanagida, 1991) in
addition to top2+ (e.g. see Shiozaki and Yanagida, 1991,
1992). Among those, only two gene products (top2+ and
cut7+) are understood in terms of molecular functions in
mitotic chromosome disjunction (e.g. chromosomal DNA
topology alteration and kinesin-related motor activity for
spindle formation). cut1+ and cut2+ genes are possibly
involved in the regulation of spindle pole body duplication
(Uzawa et al., 1990); the cut1 protein is large (210 kDa),
containing a putative nucleotide binding sequence and a
COOH domain similar to that of the budding yeast ESP1.
The cut9+ gene product is similar to that of the budding
yeast CDC16 gene (Icho and Wickner, 1987), containing
the TPR (tetratricopeptide repeat) repeats (Goebl and
Yanagida, 1991), and appears to be required for spindle
elongation in the metaphase/anaphase progression (I. Samejima and M. Yanagida, unpublished result).
Nine strains among the 25 ts cut mutants isolated in the
present study were allocated to seven new genetic loci
(designated cut13 to cut19). Their cytological phenotypes
have been classified into three groups. One is similar to
that of top2 or cut1 mutants, displaying a portion of nuclear
chromatin pulled by the spindle. In a top2 mutant most of
the chromosomal DNA remained unseparated, while in a
cut1 mutant a large part of the DNA was temporarily separated. The cytological phenotype of cut14-208 cells resembled that of top2, while that of cut15-85 was similar to that
of cut1. cut16 and cut17 mutant cells also displayed
142
I. Samejima and others
Fig. 6. DAPI-stained micrographs of cut17-275 (A) and cut18-447 (B) at the restrictive temperature for 2 h. Bar, 10 µm.
stretched chromosomes. Another type of mutant showed
condensed chromosomes before abnormal cell separation.
Condensed chromosomes were seen in cut7 and cut9
mutants with half-spindles and a short spindle, respectively. cut13-131 belongs to this group, displaying a
metaphase-like short spindle or somewhat elongated spindle with non-separated condensed chromosomes. The cytological phenotypes of the remaining cut16 and cut19
mutants were not well characterized. In cut18, cells
revealed stretched chromatin as well as condensed chromosomes. Judging from the heterogeneous nature of the
gene products and the variability of the phenotypes, the
cut gene products appear to be involved in various aspects
of cell division. A common characteristic of the mutant
phenotypes is that the loss of any cut genes causes abnormal cell separation, uncoupled from nuclear division. We
are currently engaged in identifying their gene products
and investigating their participation in mitosis.
We thank K. Gull for antibody TAT1, R. Bartlett and P. Nurse
for cut11 and cut12 strains and I. Hagan for helpful discussions.
This work was supported by a grant from the Ministry of Education, Science and Culture of Japan to M. Y. and National Institutes of Health grant GM34607 to D. B.; D. B. is an Investigator
of the Howard Hughes Medical Institute.
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(Received 21 December 1992 - Accepted 1 February 1993)