{'fraction_non_alphanumeric': 0.03618744440803333, 'fraction_numerical': 0.03351903000795843, 'mean_word_length': 4.497169325784869, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
A Temperature-Sensitive dcw1 Mutant of Saccharomyces cerevisiae Is Cell Cycle Arrested with Small Buds Which Have Aberrant Cell Walls

ABSTRACT Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Δdcw1 Δdfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Δdfg5). When DC61 cells were incubated at 37°C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37°C. At 37°C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37°C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37°C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Δdcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

Fungal cells including yeast cells have sturdy shells, i.e., cell walls, to endure osmotic stress in their environment. Without the cell wall, they easily burst and die. The cell wall also must be plastic so that it can expand outside the lipid bilayer during growth (6). Maintaining both plasticity and rigidity during bud cell wall construction during the cell cycle is a complex process that involves many factors. Saccharomyces cerevisiae is a good model for studying this process. In S. cerevisiae, when cells reach a critical size in late G 1 , they simultaneously start budding. In the very first stage of budding, when the shape of the bud is nearly spherical, the newly synthesized mannoproteins and glucan are uniformly incorporated over the whole surface of the emerging bud (10). During the S phase, DNA replication occurs and the polarized tip growth becomes predominant as the bud becomes larger (5,7). When the size of the bud is approximately one-third the size of the mother cell, which coincides with the completion of the S phase, separation of spindle pole bodies (SPBs) occurs and bipolar spindles are formed (3). When the size of the bud is two-thirds the size of the mother cell, the maturation phase of the bud growth starts and the cell wall components are again incorporated uniformly over the whole bud surface (5,7,10). One of the gene products involved in the construction of the cell wall of the growing bud is Fks1p, which functions in the biosynthesis of ␤-1,3-glucan (9), the major constituent of the cell wall. Still, much is un-known about the construction of other constituents of the bud cell wall.
We previously showed that Dcw1p and Dfg5p are required for cell growth and cell wall biogenesis and that Dcw1p is a glycosylphosphatidylinositol-anchored membrane protein (16). Subsequently, another group found that Dfg5p of Candida albicans is required for hypha formation at alkaline pH and is found in the cell membrane and cell wall extract fractions (31). To investigate the roles of Dcw1p and Dfg5p, a double disruptant of dcw1 and dfg5 was generated. The double disruptant was transformed with a plasmid containing a functional DFG5 gene under the control of the GAL1 promoter, so that its expression could be shut off by transferring the cells to a glucose-containing medium. When DFG5 expression was repressed, the cells were round and large, and Cwp1p, a major cell wall protein (28), was secreted into the medium. In the absence of DFG5 expression, chitin was delocalized and the amount of chitin was increased. However, even after DFG5 expression is turned off, the cells are considered to continue to have Dfg5p activity until all of the residual Dfg5p is degraded, a process which appears to take about 18 h. In order to see the phenotype of the double disruptant of DCW1 and DFG5, in which the enzymatic activities of Dcw1p and Dfg5p are rapidly destroyed, i.e., to obtain further insight into the enzymatic activities of Dcw1p and Dfg5p, we constructed mutant alleles of DCW1 and DFG5 whose gene products function normally at 25°C but immediately lose most of their activity at 37°C. The mutant containing the dcw1 allele was defective in bud growth, the cell cycle was arrested after DNA replication and prior to the separation of SPBs, and the buds had aberrant cell walls, when incubated at 37°C. The mutant containing the dfg5 allele showed similar phenotypes. These results indicated that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

MATERIALS AND METHODS
Yeast strains, culture conditions, and synchronization procedure. The yeast strains used in this work are summarized in Table 1. The yeast strains were grown at 25 or 37°C in YPAD medium, which contained 1% yeast extract, 2% Bacto Peptone, 0.01% adenine sulfate, and 2% glucose with or without 1 M sorbitol. For selection of 5-fluoroorotic acid (5-FOA)-resistant clones, we used medium containing 0.67% Bacto Yeast Nitrogen Base, 0.08% complete supplement mixture without uracil (Bio 101), 2% glucose, 50 g of uracil/ml, 0.01% adenine sulfate, 0.1% 5-FOA, and 2% agar. Escherichia coli strain JM109 was used for the preparation of plasmid DNA. E. coli strains were grown at 37°C in Luria-Bertani broth containing 100 g of ampicillin/ml for the selection of transformants. The cell cycle was synchronized as previously described (18) with slight modifications. After wild-type cells were grown to an optical density at 660 nm (OD 660 ) of 0.3 in YPAD, hydroxyurea was added to a final concentration of 0.15 M. The cells were incubated at 30°C for 2 h, washed with chilled water three times, and incubated in YPAD at 30°C for the indicated times.
PCR mutagenesis and plasmid construction. Random mutations in the DCW1 and DFG5 genes were generated with a Gene Morph PCR mutagenesis kit (Stratagene). A DCW1 or DFG5 fragment was generated by mutagenic PCR with primers BamDCW1-1 and BamDCW1-2 or BamDFG5-1 and BamDFG5-2 (Table 2), cut with BamHI, and ligated into BamHI-cut pRS415 (pRS415-dcw1 or -dfg5). The plasmids were amplified in E. coli, purified with Wizard Midiprep (Promega), and transformed into strain 56FCF (⌬dcw1 ⌬dfg5 pYC2-DFG5) (16). Clones which formed colonies at 25°C but did not form colonies at 37°C on a glucose-containing medium were selected and plated on 5-FOA medium to counterselect for pYC2-DFG5 (16), which originally kept 56FCF alive. The plasmids were extracted from the selected clones and amplified in E. coli. The amplified plasmids were cut with BamHI and ligated into BamHI-cut pRS405 (pRS405-dcw1 or -dfg5). The plasmids were transformed into 56FCF and plated on 5-FOA medium to counterselect for pYC2-DFG5. Construction of pRS416Y was described in a previous report (16). (Plasmid pRS416 contains wild-type DCW1.) A plasmid harboring SPC42-GFP (1), which encodes green fluorescent protein (GFP)-tagged Spc42p, a component of SPB (8), was a kind gift from John V. Kilmartin.
Fluorescence-activated cell sorting (FACS) analysis. Cells were grown in YPAD to a density of 5 ϫ 10 6 to 10 ϫ 10 6 cells/ml at 25°C and incubated at 37°C for 3 h. Culture samples corresponding to 10 7 cells were washed in 1 ml of 0.2 M Tris-HCl (pH 7.5), fixed in 1 ml of 0.2 M Tris-HCl (pH 7.5) containing 70% ethanol, washed in 1 ml of 0.2 M Tris-HCl (pH 7.5), dissolved in 1 ml of 0.2 M Tris-HCl (pH 7.5) containing 0.25 mg of RNase A (Wako)/ml, incubated at 50°C for 1 h, mixed with 20 l of 50-mg/ml proteinase K (Wako), incubated at 50°C for 1 h, pelleted by centrifugation at 10,000 ϫ g for 2 min, dissolved in 1 ml of 0.2 M Tris-HCl (pH 7.5) containing 16 g of propidium iodide/ml, incubated at room temperature for 30 min in a dark room, sonicated, and analyzed by flow cytometry (Coulter; Epics Elite ESP) (13).
Northern analysis. Total RNA was isolated, transferred to Hybond XL (Amersham Pharmacia Biotech), and probed with a DNA fragment that was PCR amplified with primers DCW1Probe-1 and -2, DFG5Probe-1 and -2, and ACT1-1 and -2 or an 0.86-kb XhoI-HindIII product of the fragment which was PCR amplified with primers CLN2-1 and CLN2-2.
Statistical analysis. The significance of differences was determined by using the t test with adjustment for unequal variances (Welch test).

RESULTS
Isolation of DCW1 and DFG5 mutants defective in growth at 37°C. To obtain a mutant in which the enzymatic function shared by Dcw1p and Dfg5p is normal at 25°C but is rapidly destroyed at 37°C, we attempted to generate a mutant that contains a mutagenized dcw1 gene and a disrupted dfg5 gene, or a disrupted dcw1 gene and a mutagenized dfg5 gene. To do this, we constructed mutant alleles of DCW1 or DFG5 by mutagenic PCR. The PCR-amplified fragment was ligated into a plasmid, the plasmid was introduced into a ⌬dcw1 ⌬dfg5 background (56FCF) (16), and 50 colonies were obtained from both transformations. From the obtained transformants, we selected five clones that grew well at 25°C but did not grow at 37°C (Fig. 1A and B). Sequence analysis revealed that the open reading frames (ORFs) of DCW1 and DFG5 of the five selected mutants contained three to six point mutations, all of which were predicted to lead to amino acid substitutions in Dcw1p or Dfg5p (Table 3). Site-directed mutagenesis of DCW1 and DFG5 confirmed that none of the single amino acid substitutions of the mutants contributes to the temperaturesensitive defects of Dcw1p and Dfg5p (data not shown). This suggests that more than one mutation is involved in the temperature-sensitive phenotype, as was found in other reported mutants (17,23,27). Of these five mutants, the mutants that contained alleles of dcw1-3 and dfg5-29 (designated DC61 and DF66, respectively) were selected for further examination be-  cause their growth was clearly shut off at the nonpermissive temperature (37°C) (Fig. 1A and B). We confirmed that they formed colonies at 25°C but not at 37°C ( Fig. 2A and B). The temperature sensitivity of these two mutants was complemented by transforming a plasmid containing wild-type DCW1. Both DC61 and DF66 formed colonies at 37°C in YPAD agar containing 1 M sorbitol, although DF66 grew rather slowly (Fig. 2C). This result indicates that DC61 and DF66 are unable to form colonies at 37°C because of a cell wall defect. DC61 cells incubated at 37°C for 3 h could grow again at 25°C (Fig.  1C). DC61 and DF66 cells arrest growth with small buds at 37°C. To determine the optimal time for microscopic observations, we measured the viability of these cells as estimated by CFU per milliliter. DC61 cells remained viable for 3 h after upshift to 37°C although they showed increasing loss of viability after 5 h at 37°C (Fig. 1C). DF66 showed a similar viability transition (data not shown). From these data, we selected an incubation time of 3 h at 37°C for further investigation, because, at this time, the DC61 cells were still viable and the effect of depletion of the enzymatic activity of Dcw1p and Dfg5p on living cells could be clearly observed. At this time, DC61 and DF66 cells incubated at 37°C had significantly more buds than did the ones incubated at 25°C (Fig. 3A and B) (P Ͻ 0.05). Under these conditions, most of the cells had only a single bud. At 25°C, the areas of most of the buds were between 20 and 80% of the area of the mother cells (Fig. 3C, upper right panel). However, at 37°C, the areas of most of the buds of DC61 cells  were less than 20% of the area of the mother cells (Fig. 3C, lower right panel). For the wild type (DC63), the bud area distributions were not significantly different between 25 and 37°C (Fig. 3C, left panels). Measurement of the bud areas of DF66 gave similar results (data not shown). These results indicate that, in cells depleted of Dcw1p or Dfg5p, bud emergence is normal but bud growth is defective.

DC61 cells are arrested after DNA replication and prior to the separation of SPBs and show actin delocalization.
The preceding results suggested that, when DC61 cells are incubated at 37°C, their cell cycle is arrested. This was confirmed by a FACS analysis, which showed that the ratio of DC61 cells with 1N DNA content to cells with 2N DNA content was lower in cells incubated at 37°C (Fig. 4D) than in cells incubated at 25°C (Fig. 4C). This was not the case with DC63 ( Fig. 4A and  B). This result indicates that the cell cycle progression of DC61 is arrested after DNA replication when incubated at 37°C.
We examined the cell cycle of DC61 cells in more detail. The nucleus of many DC61 cells incubated at 25°C was close to the bud neck, while the nucleus of almost all DC61 cells incubated at 37°C was not localized near the bud neck (Fig. 5A). The percentage of DC61 cells with an elongated distribution of tubulin was 35.8% at 25°C but only 9.8% at 37°C (Fig. 5B). This suggested that, in DC61 cells incubated at 37°C for 3 h, bipolar spindles are not formed. This was confirmed by examining the state of SPBs by using GFP-tagged Spc42p (1), which is a component of SPB (8). The percentage of DC61 cells with double dots was 44.6% at 25°C but only 11.6% at 37°C (Fig.  5C). These results clearly indicate that, when incubated at 37°C for 3 h, DC61 cells are arrested prior to SPB separation. In addition, actin patches were delocalized in DC61 cells incubated at 37°C for 3 h (Fig. 5D), showing that the polarity of the cells is lost.
Small buds of DC61 accumulate chitin in the bud cortex and are lysed at 37°C. To verify that the small buds of DC61 incubated at 37°C have aberrant cell walls, we examined the distribution of chitin in the buds. Calcofluor white, a chitin stain, stained only the bud neck in the DC61 cells incubated at 25°C and wild-type DC63 cells (Fig. 6A and B). However, in the DC61 cells incubated at 37°C, it stained the bud cortex, especially the tips of the small buds. These results indicate that chitin is deposited in the bud cortex, especially in the tips of the small buds of DC61 cells incubated at 37°C. We conclude that the abnormal distribution of chitin is due to a cell wall defect. In addition, alkaline phosphatase activity, which is normally confined to the vacuole, was detected in some of the small buds of DC61 incubated at 37°C, indicating that alkaline phosphatase leaked from the vacuole as a result of a defect of the cell wall of these buds (Fig. 6C). These results show that the small buds of DC61 incubated at 37°C have aberrant cell walls.
Accumulations of DCW1 and DFG5 mRNAs are cell cycle and growth phase regulated. The preceding results indicate that Dcw1p and Dfg5p have important roles in cell cycle progression. Therefore, we examined whether mRNA levels of these genes are cell cycle regulated. The mRNA levels of DCW1 and DFG5 peaked at the G 1 and S phases, respectively (Fig. 7B). These results clearly show that the mRNA levels of DCW1 and DFG5 are regulated according to the cell cycle. We next investigated how the mRNA levels of DCW1 and DFG5 vary among the different growth phases. Genes involved in cell wall synthesis are differentially transcribed at different growth phase. Among the genes involved in cell wall biosynthesis, those required for growth and proliferation are transcribed in the exponential phase (35), while those required for protection against the environment and for long-term survival are transcribed in the stationary phase (29,34). We found that DCW1 and DFG5 mRNAs, like ACT1 mRNA, were abundant in the exponential phase (Fig. 7B). ACT1 transcription is elevated during periods of growth (19). This shows that DCW1 and DFG5 are involved in exponential growth, which is consistent with the fact that Dcw1p and Dfg5p are required for normal bud formation.

DISCUSSION
Although it has been indicated that Dcw1p and Dfg5p are involved in cell wall biogenesis (16,33), the concrete step in which these proteins are involved was not known. In this report, we showed that cells with temperature-sensitive alleles of dcw1 (DC61) and dfg5 (DF66) at the nonpermissive temperature exhibited three remarkable phenotypes: (i) cessation of bud growth as shown by small buds, (ii) cell cycle arrest after DNA replication and prior to SPB separation, and (iii) defective bud cell walls as shown by chitin accumulation and by alkaline phosphatase leakage. These phenotypes indicate that Dcw1p and Dfg5p are required for formation of the cell wall in growing buds. These results are consistent with the finding that mRNA levels of DCW1 and DFG5 are abundant at the G 1 and S phases, respectively, and in exponentially growing cells. Since the cell wall is a large complex structure comprised of many constituents including glucan, chitin, and proteins, many gene products are considered to be involved in bud cell wall formation. We believe that Dcw1p and Dfg5p are two of these gene products.
We previously showed that the promoter shutoff of DFG5 in the background of the double disruptant of dcw1 and dfg5 causes a large and round cell morphology, delocalization of chitin, and an increase in the amount of chitin. On the other hand, the dcw1 ts (DC61) cells form small buds and cease growing. The difference in the phenotypes in these two strains can be explained by the rate at which the enzymatic function of Dcw1p or Dfg5p disappears. In promoter shutoff cells, depletion of the enzymatic function of Dfg5p depends on the turnover of Dfg5p, because only transcription of DFG5 ceases when cells are transferred to a medium containing glucose. In contrast, in the dcw1 ts cells, the enzymatic activity of Dcw1p is thought to be rapidly destroyed when the incubation temperature is increased. Therefore, the effect of the loss of activity of Dcw1p should be clearly observed in dcw1 ts cells. The dcw1 ts and dfg5 ts (DF66) cells could grow at the nonpermissive temperature under osmotic support (i.e., in YPAD agar containing 1 M sorbitol). This is also in contrast to the promoter shutoff cells, which could not grow even under osmotic support. A possible explanation for this phenotype is that the enzymatic activities of Dcw1p or Dfg5p in the DC61 and DF66 cells did not completely disappear at the nonpermissive temperature, whereas the transcription of DFG5 completely disappeared after shutoff of the promoter.
The mRNA levels of DCW1 and DFG5 peaked at the G 1 and S phases, respectively (Fig. 7B). The genes involved in the G 1 -S transition possess SCB (Swi4p-dependent cell cycle box) or MCB (MluI cell cycle box) elements upstream of the ORFs (15). The G 1 -S transition events include bud growth, cell wall biosynthesis, and DNA replication (12,14,20,24). One of these genes is FKS1, which has SCB and MCB elements. FKS1 mRNA accumulates at the G 1 phase, and Fks1p is involved in the construction of the bud cell wall (22,26). DCW1 also has two SCB elements and two MCB elements upstream of its ORF, and DFG5 has one SCB element upstream of its ORF (Table 4). These findings support our hypothesis that Dcw1p and Dfg5p are involved in bud growth, which is a G 1 -S transition event. Because the temperature-sensitive mutants of dcw1 and dfg5 show similar phenotypes, they are considered to have similar roles in bud growth. However, Dcw1p is thought to exert its function during initial bud growth late in the G 1 phase, while Dfg5 is thought to exert its function when the buds become larger in the S phase. Suzuki et al. described a new cell cycle checkpoint that ensures coupling of cell wall synthesis and mitosis (32). This checkpoint monitors the progress of cell wall synthesis and causes a cell cycle arrest after DNA replication and prior to SPB separation in response to the defect of cell wall synthesis. This checkpoint was found in an fks1 ts mutant in which an enzyme that catalyzes ␤-1,3-glucan biosynthesis (9) is deficient at the nonpermissive temperature. The cell cycle arrest of this fks1 ts mutant is strikingly similar to that of dcw1 ts cells in this report, suggesting that dcw1 ts cells are cell cycle arrested by this cell wall integrity checkpoint. In addition to the cell cycle arrest, fks1 ts cells share several other characteristics with dcw1 ts cells, including cell lysis, chitin distribution in the bud tip, and arrest with a small bud (11). Because Dcw1p is localized mainly in the membrane and partly in the cell wall (16), Dcw1p and Dfg5p might be involved in construction of bud cell wall components like Fks1p. Because Dcw1p and Dfg5p are homolo-gous to bacterial glycanases (16,21), they might participate in remodeling of newly synthesized cell wall components. However, the exact enzymatic functions of Dcw1p and Dfg5p are still unknown. Closer examination of the cell walls of the dcw1 ts or dfg5 ts cells should clarify their functions.