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12: 983-990 (1996)
Yeast Sequencing Reports
Characterization of cwZl+, a Gene from
Schizosaccharomyces pombe Whose Overexpression
causes Cell Lysis
Znstituto de Microbiologia Bioquimira, Consejo Superior de Investigaciones Cientijicas and
Universidad de Salamanca, 37007 Salamanca, Spain3
Received 6 February 1996; accepted 26 April 1996
From a Schizosaccharornyces pombe genomic library we have isolated the gene cwllf that causes cell lysis when it is
overexpressed in the absence of an osmotic stabilizer. Southern hybridization showed that cwlI+ exists as a single
copy in the S. pombe genome. The cwll+ gene nucleotide sequence revealed a putative open reading frame of 924 bp
encoding a polypeptide of 308 amino acids with a calculated M, of 27 000. The cwll+ DNA hybridizes to a major
RNA transcript of 1.5 kb whose 5' end maps at a position 452 bp upstream from the predicted translation start.
Comparison of the amino acid sequence with those included in the current databases, showed no significant
similarity to any known sequences. Cells overexpressing the cwll' gene under the control of the S. pombe nmt
inducible promoter displayed a reduced cell wall content, were unable to separate after division and lysed drastically
in the absence of osmotic stabilizer. Disruption of the cw11+ gene caused no noticeable phenotype, The sequence has
been deposited in the EMBL data library under Accession Number X9445.
fission yeast; dominant genetics; cell wall regulation
The major structural polymer of budding and
fission yeast cell wall is a linear 1,3-P-glucan
branched with some 1,6-P-glucan (Fleet, 1991). In
Saccharomyces cerevisiae, several genes involved
in 1,3-P-glucan biosynthesis have been isolated
by different approaches. Based on the sequence of
internal peptides derived from a protein band
enriched in the process of 1,3-p-glucan synthase
purification, two 1,3-P-glucan synthase-related
genes, GSCl and GSC2, have been isolated (Inoue
et al., 1995). Genes defined by complementing
mutations of resistance to 1,3-P-glucan synthesis
inhibitors in S. cerevisiae such as echinocandin
*Corresponding author.
CCC 0749-503)3/96/100983-08
0 1996 by John Wiley & Sons Ltd
analogs (Douglas et al., 1994; El-Sherbeini and
Clemas, 1995), papulacandin B (Castro et al.,
1995) or Hansenula mrakii killer toxin (Yamamoto
et al., 1986; Hong et al., 1994; Kusuhara et al.,
1994) have also been described. A third genetic
strategy has been to isolate mutants hypersensitive
to different agents known to affect the cell wall
assembly, such as calcofluor white (Ram et al.,
1994). With such techniques, several genes have
been described. FKS1IETG1ICWH53ICND1I
GSCIIPBRI (Douglas et al., 1994; Eng et al., 1994;
Ram et al., 1995; Inoue et al., 1995; Castro et al.,
1995) is a gene encoding a large polypeptide with
16 potential transmembrane domains that might
be a structural component of 1,3-P-glucan synthase (Inoue et al., 1995). A second gene, FKS2I
GSC2, highly homologous to FKSIIGSCI, has
been described (Inoue et al., 1995; Mazur et al.,
1995) as a subunit of another yeast 1,3-p-glucan
synthase. GNSl encodes a 40 kDa protein that is
also predicted to be a membrane protein. It has no
significant homology with any known protein and
might constitute either a subunit or a regulator of
1,3-P-glucan synthase activity (El-Sherbeini and
Clemas, 1995). H K R l is an essential gene that
encodes a presumptive calcium-binding type I
membrane protein. Its overexpression promotes
resistance to H. mrakii killer toxin and increases
the a-glucan content of the cell wall (Kasahara
et al., 1994). Finally, KNR4ISMZl codes for a
nuclear protein that might regulate cell wall
synthesis (Hong et al., 1994).
A different genetic approach to identify genes
involved in the maintenance of cell wall integrity
and cell viability has been the isolation of mutants
displaying a thermosensitive lytic phenotype
(Venkov et al., 1974; Cabib and Duran, 1975;
Ribas et al., 1991). Mutants of this type have
allowed the identification of the SLT2 gene from
S. cerevisiae, encoding a serinehhreonine protein
kinase from the family of MAP kinases (Torres
et al., 1991), or the cwg2' gene from Schizosaccharomyces pombe, coding for the P-subunit of
a type I geranylgeranyltransferase (Diaz et al.,
1993). The plethora of mutants that can be isolated
by these methods reflects the complexity of
functions involved in cell wall integrity and cell
As a new approach in the search for genes
involved in cell wall biosynthesis, we searched
for genes that caused overexpression-mediated
cell lysis in the absence of an osmotic stabilizer.
Dominant genetics has been used for the identification of regulatory genes in different physiological events (Liu et al., 1992; Ramer et al., 1992). In
this paper we describe the characterization of the
S. pombe cwll+ gene whose overexpression causes
osmotic lysis.
Strains, growth conditions and genetic methods
The S. pombe auxotrophic strains leul-32 (Kohli
et al., 1977) and ura4-dl8 (Grimm et al., 1988)
were used in this study. They were grown in YED
medium (1% yeast extract, 1% glucose) or minimal
medium, (1% glucose, 0.7% yeast nitrogen base
without amino acids, 0.9 g/l KCl, 1 mg/l citric
acid, 10 pg/l biotin, 1 mg/l calcium pantothenate,
10 mg/l nicotine acid and 10 mg/l m-inositol)
supplemented with the necessary requirements.
Incubations were carried out at 28°C. Growth was
monitored either by A600measurements or by cell
number counting.
Escherichia coli JM101, JM109 or DH5a were
used. These were grown in LB medium (1%
bactotryptone, 0.5% yeast extract, 1% NaC1) supplemented with 50 pg/ml ampicillin or 15 pg/ml
tetracycline when appropriate. Solid medium
plates contained 2% agar.
Yeast extract, yeast nitrogen base, bactotryptone and agar were from Difco Laboratories,
Detroit, Michigan, U.S.A.
Plasmids and recombinant D N A methods
The S. pombe genomic library was constructed
in pDB262 (Wright et al., 1986). This vector
contains the S. cerevisiae LEU2 gene which
complements S. pombe leul-32, and part of the
2 pm circle, which allows high frequency transformation. The S. pombe expression plasmid pREP4
(Maundrell, 1993) was used for the overexpression
of the cwll' gene under the control of the nmt
inducible promoter. S. pombe was transformed by
the protoplast method (Beach and Nurse, 1981) or
the lithium chloride method (It0 et al., 1983).
Other DNA manipulations were carried out
by standard techniques (Sambrook et al., 1989).
Southern analysis was performed using a radioactively labelled probe prepared as described
(Rigby et al., 1982), or by the random primed
method (Feinberg and Vogelstein, 1984), using a
non-radioactive digoxigenin-dUTP labelled probe.
Northern analysis was performed using total
RNA extracted as described (Percivall-Smith and
Segal, 1984). The electrophoresed RNA was
transferred to nitrocellulose membranes and
hybridized with a probe radioactively labelled by
nick translation.
The nucleotide sequence was determined by
subcloning the 4 kb fragment bearing the cwll+
gene in to both KS - and KS' Bluescript vectors
(Stratagene Inc., La Jolla, CA). A series of overlapping deletions in both orientations were created
by unidirectional exonuclease I11 digestion. Singlestranded DNA was produced using the M13K07
helper phage and both strands were sequenced
using the dideoxy chain-termination method
(Sanger et al., 1977). The DNA sequence was
translated to the predicted amino acid sequence
using the DNAsis program. The amino acid
sequence was thereafter compared with the
Figure 1. Micrographs of S. pombe led-32 cells grown in
minimal medium at 28°C. (A) Wild type; (B) pCGl transformants: (C and D) pCG4 transformants grown in the absence of
thiamine for (C) 14 h and (D) 18 h. The bar corresponds to
5 pm.
sequences available in the EMBL data bases using
the FASTA program.
Analysis of mRNA 5' termini was carried out
by primer extension essentially as described
(Sambrook et al., 1989). Briefly, a synthetic oligonucleotide (sequence 5'-GATTTTTGGATTTT
GCTGTAGG-3') complementary to nucleotide
positions -262 to -284 of the cwll sequence was
5' end-labelled with [ Y - ~ ~ P ] A T(5000
P Ci/mmol,
Amersham) and polynucleotide kinase. Poly (A)
RNA from strain 972h- was denatured and
hybridized with an excess of the labelled complementary oligonucleotide. Reverse transcriptase
RAV-2 (Amersham) was then used to extend the
primer, producing a labelled cDNA complementary to the RNA template. The cDNA was
analysed by electrophoresis on a sequencing gel
adjacent to the sequencing reactions of the c w l l c
gene primed with the same oligonucleotide.
Other methods
S. pombe chromosomes were separated by alternating electric field electrophoresis with a Bio-Rad
CHEF DR-I1 system (Carle and Olson, 1985) at
50V, switching the electric field every 30 min for
72 h. The separated chromosomes were transferred
to a nitrocellulose membrane and hybridized with
the cwll+ probe. Cell wall quantitation and fractionation was done by measuring [U-'4C]glucose
Figure 2. (A) Restriction map and sequencing strategy used in
the determination of the nucleotlde sequence of the mpCGl
insert. The position of the cwlZ+ gene is indicated with an arrow
showing the direction of transcription. Thin arrows indicate the
direction and the extent to which the sequence was determined.
(B) Hybridization of the radioactively labeled pCGDl insert
with genomic S. pombe DNA cut with: (1) X b d ; (2) PstI;
(3) HindsII1. (C) Chromosomal localization of the cwll+ gene.
The migration of the three S. pombe chromosomes is indicated. Southern blot was done using the same probe as in
Figure 2B.
(240 mCi/mmol, Amersham) uptake as described
(Ribas et al., 1991).
Isolation of a plasmid containing the cwll+ gene
S. pombe leul-32 h - was transformed with a
S. pombe genomic DNA library constructed in the
plasmid pDB262 (Wright et al., 1986). Assessment
of cell lysis in the presence or absence of 1.2 Msorbitol was carried out by detection of red colonies in the presence of Phloxin B (Ribas et al.,
1991). When autolysis occurred the colonies
progressively stained red whereas they remained
white when the cells did not lyse. Five leucine
- z-
- -5-
- 2
region responsible for the lytic activity, the insert
was shortened using the HindIII restriction sites
present in the fragment (Figure 2A). The two
plasmids obtained were unable to cause lysis in the
absence of osmotic stabilizer and therefore the
whole insert was sequenced on both DNA strands
according to the strategy outlined in Figure 2A.
A single open reading frame (ORF) was found
between bases 2391-3315 of the insert and this
putative gene was called cwll+ for ‘cell wall lysis’.
Southern blot analysis of S. pombe genomic DNA
cut with XbaI, Pstl, or HindIII using the whole
insert as probe indicated no rearrangement of
the DNA structure during cloning and that the
S. pombe genome contains a single copy of
the cloned gene (Figure 2B). The cwIl+ gene
was located on chromosome 11, as shown in
Figure 2C.
cwII nucleotide sequence and transcription
Two kilobases from the pCGDl insert nucleotide sequence containing the cwll+ gene are shown
in Figure 3A. There is a single O R F 924 bp long
and no consensus signals for intron splicing were
observed. The predicted encoded protein has 308
amino acids with a Mr of 27000. The protein is
relatively hydrophilic with a similar number of
acidic and basic amino acids and a calculated
isoelectric point of 6.4. The Kyte and Doolittle
hydropathy plot of the product (Kyte and
Doolittle, 1982) revealed no regions of marked
hydrophobicity characteristic of N-terminal signal
peptide sequences (Figure 3B). There is a potential
membrane-spanning domain around amino acid
250, four potential N-glycosylation sites at amino
acids 65, 113, 135 and 303, five potential phosphorylation sites for protein kinase C and six for
casein kinase 11. A search of the GenBank and
EMBL data banks did not reveal any significant
resemblance of the cwll+ encoded protein to any
described protein.
Expression of the O R F was assessed by
Northern blot analysis of S. pombe total mRNA
using the HindIII-Hind111 fragment containing
cwll+ as a probe. A single 1.5 kb transcript
was detected in both the wild type and the
pCG-transformed S. pombe (Figure 4A).
The 5‘ flanking sequence of the coding region
has a TATA element of 480 bp upstream from the
translation start. In order to determine whether
this could be part of the promoter, we identified
the transcription initiation site by primer extension
of an oligonucleotide complementary to the -262
- 2.3 Kb
-1.3 Kb
Figure 4. (A) Northern analysis of the w l I + transcript. Total
RNA from S. ponzhe wild type (1) and transformed with pCGl
(2) was resolved by agarose gel electrophoresis, transferred to a
nylon membrane and hybridized to the radioactively labeled
pCGDl insert. (B) Determination of the 5’ end of the c w l l f
mRNA transcript by primer extension. The DNA sequence
shown corresponds to the minus strand of the genomic DNA
and was obtained using as primer the same oligonucleotide used
to extend the mRNA with reverse transcriptase. The asterisk
indicates the nucleotide matching the RNA 5’ end.
to -284 nucleotides upstream from the ATG of
the O R F in the sequence. One major transcript
(Figure 4B) was detected at 452 bp upstream from
the ATG. It is therefore likely that the mentioned
ATATAA sequence at -480 bp would be part of
the cwllf promoter.
The 3‘ non-coding region contains two of the
proposed (Miyake and Yamamoto, 1990) polyadenylation consensus sequences, AATATA and
ATAAATA, 118 bp and 259 bp downstream from
the O R F stop codon respectively. Sequences
matching the S. cerevisiue termination motif (TAG
. . . TATGT . . . TTT) might also be active in
S. pombe (Hayles and Nurse, 1992) and are found
before the second polyadenylation sequence.
cwll+ gene overexpression und disruption
On the basis of the observations with the
plasmid pCGl, we cloned the cw11+ gene in the
plasmid pREP4 under the control of the nmtl
thiamine repressible promoter (Maundrell, 1993).
When S. pombe was transformed with this plasmid
(pCG4) and grown in minimal medium without
thiamine, the cells did not separate after dividing
(Figure 1C) but formed chains of cells that subsequently lysed (Figure 1D). The lysis first affected
the mother cell and was prevented by sorbitol. In
the presence of thiamine, pCG4 transformants
grew as the wild type. We analysed the cell wall
probe b
w t 1 2 3 4 5
w t 1 2 3 4 5
- 3 Kb
- 3 Kb
- 2 Kb
- 2 Kb
probe a
probe b
Figure 5. (A) Scheme of the cwll+ gene disruption. (B) Hybridiration of the HindIII-cut genomic DNA from the S. pomhe wild
type and five selected disruptant clones to the probes indiated in
(A). The probes were labeled with digoxigenin-dUTP by the
random primed method.
composition of pCG4 transformants to ascertain
whether the (I-3)P-glucan content or any other
major cell wall polymer might be affected by the
cwll+ overexpression. No changes in cell wall
composition were observed (data not shown) but
there was a 20% decrease in the total uptake of
['4C]glucose into the whole cell wall fraction as
compared with the S. pornbe. wild type.
In order to determine whether the cw11+ gene
is essential, a disruption mutation was generated, as shown in Figure 5A. The AsuII-AsuII
2.8 kb fragment containing the cw11+ gene
(Figure 2A) was cloned into the pGEM3
(Promega), generating the plasmid pCG5. The
HindIII-SulI fragment from this plasmid was
replaced by the uru4+ gene cut with the same
restriction enzymes. The plasmid thus obtained
(pCG6) was BurnHI-EcoRI cut and the linear
fragment was used to transform S. pombe
leul-32 uvu4-Dl8 h . Integration and disruption
at the cwll+ locus was confirmed by Southern
blot analysis using two different probes (Figures
5B). None of the disruptant clones analysed
showed a phenotype different from that of the
wild type when they were grown either at 28°C or
The morphogenesis and growth of yeast cells are
necessarily related to the biosynthesis and degradation of the cell wall. Therefore, these processes
must be strictly controlled and linked to the general signal transduction pathways of the cell (Cid
et ul., 1995). We employed dominant genetics in an
attempt to identify new genes directly or indirectly
involved in cell wall biosynthesis. The gene cloned
in this study, cwll+, showed no known homologs
and the gene product contained no obvious motifs
which might provide further insight into its possible function. An interesting feature of the cwll'
gene is that transcription starts 452 nucleotides
upstream from the ORF, while in S. pombe transcription generally starts within 200 nucleotides
upstream from the O R F (Russell, 1989). However,
there were no short ORFs in the cwlI+ leader
sequence that might imply translational control of
the gene.
The morphological phenotype before lysis of
cells overexpressing cwll+ under its own promoter
is quite similar to that exhibited by S. pombe cells
in which (1-3)P-~-glucan synthesis is inhibited
(Varona et a/., 1983). When a stronger promoter
such as nmt is used, cells lyse more dramatically in
the absence of an osmotic stabilizer, have a lower
cell wall content and show a defect in cell separation after division. therefore, cwU+ might be
directly involved in the regulation of the cell
separation process. In budding yeasts, a burst of
chitin synthesis at the end of mitosis deposits the
primary septum on the mother cell side within
the previously formed chitin ring. Deposition of
additional cell wall material from both sides of the
primary septum produces the secondary septum.
An asymmetric degradation of the septum is
required for cell separation and the chitinous
septum remains with the mother cell as part of the
bud scar. Cell separation depends on the CTSIcoded chitinase (Kuranda and Robbins, 1991)
differentially regulated in mother and daughter
cells by A CE2 (Dohrmann et al., 1982). It has been
proposed that the chitin synthase Chslp exerts a
repair function at the end of cytokinesis, counterbalancing the lytic effect of the chitinase (Cabib
et al., 1992). Unfortunately, little is known about
septum formation in fission yeast or about the lytic
enzymes required to separate the two cells once
the septum has been formed (Fankhauser and
Simanis, 1994). On the other hand, disruption of
the cwll+ gene caused no noticeable phenotype
in S. pombe and hence further studies will be
necessary to elucidate the possible role of cwlZ+ in
morphogenesis and cell wall assembly.
We thank Dr Y. Sanchez and Dr J. C. Ribas for
critical reading of the manuscript, C. Belinchon for
photographic work and N. Skinner for correcting
the English. M. Arellano acknowledges support
from a fellowship granted by the University of
Salamanca. This work was supported by grant
B109 1-0437 from the Comision Interministerial de
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