Interspore bridges: a new feature of the Saccharomyces cerevisiae spore wall

Alison Coluccio and Aaron M. Neiman

Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, State University of New York, Stony Brook, 332 Life Sciences, Stony Brook, NY 11794-5215, USA

Correspondence
Aaron M. Neiman
Aaron.Neiman{at}sunysb.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Saccharomyces cerevisiae spore wall is a multilaminar coat that surrounds individual spores and protects them from environmental insult. Scanning electron microscopy reveals that the four spores of an ascus are connected by interspore bridges. Transmission electron microscopy of spores indicates that these bridges are continuous with the outer layers of the spore wall. In chs3 mutants, which lack the chitosan and dityrosine layers of the spore wall, bridges are absent. By contrast, in dit1 mutants, which lack only the dityrosine layer, bridges are present, suggesting that the bridges may be composed of chitosan. Interspore bridges are shown to be necessary to hold spores together after release from the ascus. A function for these bridges in the maintenance of heterozygous markers in a homothallic yeast population is proposed.


Abbreviations: SEM, scanning electron microscopy; TEM, transmission electron microscopy


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sporulation in baker's yeast, Saccharomyces cerevisiae, is triggered in MATa/MAT{alpha} diploid cells by the absence of a nitrogen source and the presence of a non-fermentable carbon source (Kupiec et al., 1997). Under these conditions, cells enter meiosis to produce four haploid nuclei. These nuclei are packaged into spores in two stages. In the first stage, secretory vesicles coalesce within the cytoplasm to form new double-membrane sheets, termed prospore membranes (Moens, 1971; Neiman, 1998). Each of the four haploid nuclei formed by the meiotic divisions becomes completely engulfed by a prospore membrane, resulting in the formation of four daughter cells, or prospores, within the cytoplasm of the mother cell. In the second stage, after capture of the nuclei within prospore membranes, formation of the spore wall proceeds by deposition of spore wall materials within the lumen of the prospore membrane (Lynn & Magee, 1970). The completed spore wall serves to protect the spore from environmental damage (Smits et al., 2001).

The spore wall is a more elaborate structure than the vegetative wall of S. cerevisiae. The four major components of the spore wall are arranged in layers formed consecutively, beginning with the innermost layer and working outward (Tachikawa et al., 2001). The two inner layers consist primarily of mannan and 1,3-{beta}-glucan and are similar to the vegetative cell wall (Kreger-Van Rij, 1978). The two outer layers of the spore wall are unique to the spore and confer on the spore much of its resistance to environmental damage (Smits et al., 2001). Outside of the {beta}-glucan layer is a layer of chitosan, a 1,4-{beta}-glucosamine polymer (Briza et al., 1988). This layer is formed by the combined action of the CHS3-encoded chitin synthase and the chitin deacetylases encoded by CDA1 and CDA2 (Christodoulidou et al., 1996; Mishra et al., 1997; Pammer et al., 1992). Outside of the chitosan is a thin layer that consists predominantly of cross-linked molecules of dityrosine (Briza et al., 1986, 1990, 1996). The dityrosine monomers are produced in the spore cytosol by the combined action of the Dit1p and Dit2p enzymes and then exported for assembly into the wall (Briza et al., 1994; Felder et al., 2002). After spores are fully formed, the remains of the mother cell then collapse around the four completed spores to form the ascus. As a result, the four spores of a tetrad are enclosed together inside an ascal membrane and ascal wall, which are derived from the mother cell plasma membrane and cell wall, respectively.

In an effort to develop new assays of spore wall assembly, we examined wild-type and mutant spores by scanning electron microscopy (SEM). This analysis revealed that the wild-type spore wall has a distinctive surface texture that is altered as the dityrosine and chitosan layers are removed. Moreover, this analysis has led to the identification of a previously undescribed feature of the spore wall, the interspore bridge. These bridges connect the outer spore wall layers of adjacent spores and serve to maintain the physical association of spores upon release from the ascus.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and media.
Unless otherwise noted, standard media and methods were used (Rose & Fink, 1990). Strains used in this study are listed in Table 1. Strains were constructed as follows: DIT1 and CHS3 were individually deleted in the haploid strains AN117-4B and AN117-16D (Neiman et al., 2000) by PCR-mediated knockout (Longtine et al., 1998); the resulting haploids were mated to generate AN264 and AN262, respectively.


View this table:
[in this window]
[in a new window]
 
Table 1. Strains

 
Sporulation.
Intact cells were prepared for sporulation in 2 % potassium acetate (KOAc) as described previously (Neiman, 1998). For sporulation of spheroplasts, cells were first grown to mid-exponential phase in YPAcetate medium (1 % yeast extract, 2 % Bacto-peptone, 2 % KOAc). Cells were then pelleted, washed once in distilled water (dH2O) and then resuspended in 0·7 M sorbitol. Zymolyase 100T (US Biological) was added to a final concentration of 0·1 mg ml–1 and cells were incubated for 1 h at 30 °C. Cells were then washed once in SSM (0·7 M sorbitol, 1·4 % KOAc), resuspended in SSM at a concentration of 2x107 cells ml–1 and incubated at 30 °C.

Scanning electron microscopy.
For SEM analysis of spores, spheroplasts were first sporulated, washed once in 0·5 % SDS to remove the ascal membrane, washed once in dH2O, and then adhered to polylysine coated glass cover slips. The cells on the cover slips were fixed in 3 % glutaraldehyde in cacodylate buffer (0·1 M sodium cacodylate pH 7·4, 5 mM CaCl2) for 1 h at 23 °C, then stained for 1 h at 23 °C in 1 % osmium tetroxide and 1 % potassium ferricyanide in cacodylate buffer, washed with dH2O and then dehydrated by 10 min incubations in a graded acetone series: two incubations each in 30 %, 50 %, 70 % and 95 % acetone, and four incubations in 100 % acetone. The cover slips were then critical-point dried and sputter-coated with 4 nm of gold particles. Germinating cells, vegetative cells and asci were adhered to cover slips and prepared in the same way. Images were collected in a LEO1550 scanning electron microscope at 2·5 kV using an in-lens detector.

Transmission electron microscopy.
Cells were prepared for transmission electron microscopy (TEM) using an osmium-thiocarbohydrazide staining protocol (Rieder et al., 1996). Spores were prepared as for SEM, then fixed for 1 h in 3 % glutaraldehyde in cacodylate buffer, washed once in cacodylate buffer, resuspended in 1 % osmium tetroxide and 1 % potassium ferricyanide in cacodylate buffer, and incubated for 30 min at 23 °C. Cells were then washed four times in dH2O, resuspended in 1 % thiocarbohydrazide in water, and incubated for 5 min at 23 °C. Cells were again washed in dH2O, incubated in 1 % osmium tetroxide and 1 % potassium ferricyanide in cacodylate buffer for an additional 5 min, and washed again in dH2O. The cells were then incubated in saturated uranyl acetate for 2 h and dehydrated through a graded series of acetone washes as for the SEM samples. The dehydrated samples were embedded in Epon 812, sectioned, and images were collected on a JEOL 1200EX microscope at 80 kV.

Spore dispersal assay.
Wild-type, dit1 and chs3 mutant cells were sporulated. Zymolyase was then added to a final concentration of 0·1 mg ml–1 and the spores were incubated for 15 min at 37 °C. Spores were washed once in dH2O, resuspended in dH2O, and mounted on slides for examination by phase-contrast microscopy.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
SEM reveals a distinct surface to the spore wall
The outermost layer of the S. cerevisiae spore wall is composed primarily of dityrosine (Briza et al., 1994, 1986). By contrast, the exterior of the vegetative cell wall is primarily mannoproteins (Orlean, 1997). To determine if these two distinctive polymers could be distinguished by their surface appearance, vegetative cells, asci and isolated spores were examined by SEM. Vegetatively growing yeast cells had a smooth, velvety appearance (Fig. 1a). Intact asci had a similar surface texture to vegetative cells (Fig. 1b), consistent with the fact that the ascal wall is derived from the vegetative wall of the mother cell. By contrast, spore surfaces, visualized after removal of the ascus, had a distinctive ridged or scalloped appearance (Fig. 1c).



View larger version (153K):
[in this window]
[in a new window]
 
Fig. 1. The surface morphology of spores is distinct from that of vegetative cells. Vegetatively growing yeast cells (a), asci (b), isolated spores (c), and germinating spores (d) were examined by SEM. Representative cells from each type are shown. Over 100 cells of each class were examined. Arrowheads in (c) indicate interspore bridges. Arrows in (d) indicate germinating cells. Scale bars, 1 µm.

 
It was possible that the different surface features of the spore wall and the vegetative wall were in some way an artefact of the spore preparation process. To control for this possibility, isolated spores were transferred to rich medium for 3 h before fixation and preparation for SEM. Under these conditions, the germinating cells were seen to have buds, with the typical vegetative wall texture emerging from the scalloped spore coat (Fig. 1d). Thus, the spore wall and cell wall have distinct appearances even when present on different areas of the same cell.

Finally, it was possible that the appearance of the spores would be altered by the treatment necessary for removal of the ascus. In particular, removal of spores from the ascus requires digestion of the ascal wall with glucanases to release the spores. Although spores are resistant to glucanase digestion, it is nonetheless possible that the enzymes could cause changes in the spore wall. To avoid this problem, vegetative cells were spheroplasted before transfer to osmotically stabilized sporulation medium. After sporulation, the spores could then be removed from the ascus without enzymic digestion. When prepared in this way, spores appeared indistinguishable by SEM from spores isolated by direct digestion of asci with glucanases (data not shown). Thus, although we cannot exclude the possibility that the surface of the spore wall is affected by preparation for electron microscopy, the appearance of the wall is not an artefact of exposure to lytic enzymes.

Spores are connected to each other by bridges
The most striking finding of the SEM analysis was the presence of connections between spores (arrowheads, Fig. 1c). To explore the nature of these interspore bridges, spores were released from asci as for the SEM analysis and examined by TEM. Interspore bridges were readily visible in these preparations as darkly staining material connecting the outer layers of the walls of adjacent spores. In these preparations, the dityrosine and chitosan layers of the spore wall stain as a single dark layer (arrowheads, Fig. 2). This layer branches at the position of the bridges so that it both surrounds individual spores and connects adjacent spores. Thus, the bridges themselves appear to consist of chitosan and/or dityrosine.



View larger version (158K):
[in this window]
[in a new window]
 
Fig. 2. Individual spores are connected by interspore bridges. (a) An interspore bridge as seen by SEM. (b) Higher magnification of spores in (a). (c, e) TEM images of interspore bridges. (d, f) Higher magnification of bridges in (c) and (e), respectively. Representative cells from over 200 cells examined are shown. Arrowheads in (d) and (f) mark the position of branches in the outer spore wall layers. Scale bars: (a) 1 µm; (b–f) 200 nm.

 
Chitin synthase is required for construction of the bridges
To test the idea that the interspore bridges are constructed of outer spore wall materials, we examined the spore surfaces of dit1 and chs3 mutants, which lack the dityrosine and chitosan layers of the spore wall, respectively (Briza et al., 1994; Pammer et al., 1992). Because both dit1 and chs3 spores are sensitive to Zymolyase digestion, cells were prepared for analysis by sporulation of spheroplasts, as described above. The surfaces of dit1 mutant spores, which should consist primarily of chitosan without the overlying dityrosine, differed from that of wild-type spores in having a blistered, rather than scalloped appearance (Fig. 3a). Nonetheless, interspore bridges were readily visible in the dit1 mutant (Fig. 3b). When the bridges in dit1 cells were examined by TEM, the outer layer of the spore wall again appeared to branch and run between the spores (Fig. 3d). Because there is no dityrosine present in the dit1 mutant, the remaining bridge material is probably chitosan.



View larger version (161K):
[in this window]
[in a new window]
 
Fig. 3. Interspore bridges are still formed in dit1 mutant cells. (a) SEM image of dit1 spores. (b) Higher magnification of spores in (a). (c) TEM image of dit1 spores. (d) Higher magnification of spores in (c). Representative cells from over 200 cells examined are shown. Arrowheads in (d) mark the position of branches in the chitosan layer. Scale bars: (a) 1 µm; (b) 200 nm; (c) 500 nm; (d) 100 nm.

 
To confirm that the bridges are indeed constructed of chitosan, spores were examined in a chs3 mutant. CHS3 encodes the chitin synthase required for synthesis of spore wall chitosan; in the chs3 mutant both the chitosan and dityrosine layers of the spore wall are absent (Pammer et al., 1992). In chs3 spores, the surface of the underlying {beta}-glucan layer is exposed and has a very smooth appearance by SEM (Fig. 4a, b). It was very difficult to identify chs3 tetrads by SEM, as upon release from the ascal membrane the chs3 spores dispersed. Figs 4(a) and 4(b) show two tetrads of chs3 spores still held together by the remnants of the ascal membrane (arrowheads). These tetrads lack any structures characteristic of interspore bridges. Similar results were obtained by TEM analysis. Again, chs3 spores were visualized within the ascus to prevent spores from dispersing. No connections between spores were visible at positions where the {beta}-glucan layers of adjacent spores came into contact (arrow, Fig. 4d). Thus, CHS3 is required for the construction of interspore bridges, consistent with the inference that the bridges are themselves extensions of the chitosan layer of the spore wall.



View larger version (141K):
[in this window]
[in a new window]
 
Fig. 4. Interspore bridges are absent in chs3 mutant cells. (a, b) SEM images of chs3 asci. Examples of intact tetrads of chs3 were rare due to dispersal of the spores (see text). These spores are held together by remnants of the ascal membrane (arrowheads). (c) TEM image of chs3 spores within an ascus. (d) Higher magnification of spores in (c); the {beta}-glucan layers are adjacent but not connected (arrow). Scale bars: (a, b) 1 µm; (c) 500 nm; (d) 200 nm.

 
Interspore bridges provide a physical connection between spores
The electron microscopic analysis of the chs3 mutant suggests that the interspore bridges might hold spores together upon release from the ascus. To test this directly, wild-type, dit1 and chs3 mutant strains were sporulated and the spores were released from the ascus by digestion with Zymolyase. The released spores were then examined by light microscopy and scored as to whether they were in clumps of four, three, two, or isolated spores (Fig. 5). Before Zymolyase treatment, more than 90 % of the spores in all three cultures were grouped in tetrads. After digestion with Zymolyase, more than 80 % of the wild-type spores remained in groups of four. Consistent with the presence of bridges in dit1 mutants, the dit1 spores remained clustered together nearly as efficiently as wild-type. By contrast, less than 10 % of the chs3 mutant spores maintained intact tetrads after digestion and nearly 80 % of the of the chs3 mutant spores were dispersed as single, isolated spores. These results demonstrate that interspore bridges can maintain a physical connection between spores after they are released from the ascus.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5. Spore dispersal assay. Spores of the wild-type (a), a dit1 mutant strain (b), and a chs3 mutant strain (c) were examined by light microscopy and scored as clustered in groups of 1, 2, 3 or 4, both before (black bars) and after (grey bars) Zymolyase digestion. Over 500 spore clusters were scored in each culture.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Our SEM studies of the S. cerevisiae spore wall have revealed that the surface of the spore wall is distinct from that of the vegetative cell wall. It is interesting in this regard that SEM images of spores from the fission yeast, Schizosaccharomyces pombe, have a similar surface appearance to S. cerevisiae spores, though the Schiz. pombe spore wall has not been reported to contain dityrosine (Nakamura et al., 2004). The scalloped surface of the wall may therefore not be an inherent property of the constituents of the spore surface, but an architectural feature of the wall. When dit1 and chs3 mutations were used to expose different layers of the spore wall on the surface, these layers displayed distinct morphologies. Thus, SEM will be a useful technique in the characterization of other mutants with spore wall defects.

More strikingly, this analysis revealed that S. cerevisiae spores isolated from the ascus are connected by structures we have termed interspore bridges. TEM analysis of isolated spores suggested that the bridges are formed by a branching of the outer layers of the spore wall so that these layers both surround individual spores and connect adjacent spores. This inference was confirmed by analysis of chs3 mutant cells. In chs3 mutants, the interspore bridges were absent, indicating that chitin or chitosan is required for bridge construction. These results demonstrate that chitin or chitosan is essential for construction of the bridges. Whether there are additional constituents of the bridges is unclear. For instance, the bridges appear somewhat less robust in a dit1 mutants (Fig. 3) suggesting that, although bridges are still functional in dit1 mutants (Fig. 5), dityrosine may normally be present in the bridges. Additionally, the composition of the grey-staining material in the central region of the bridges is unknown.

The existence of these bridges raises several interesting issues. For example, how are the bridges constructed? One possibility is that bridges may be formed at points of contact during spore wall construction. Perhaps contact triggers branching and interconnection of the chitosan layers of adjacent spores. Understanding bridge assembly will require the identification of the enzymes responsible for this process. It may be possible to use the spore dispersal assay (Fig. 5) to identify genes involved in bridge synthesis.

Another important question raised by the presence of these bridges is what functional role these structures play in the yeast life cycle. One possibility is suggested by the homothallic nature of S. cerevisiae. Outside of the laboratory, S. cerevisiae is found primarily in a diploid (or higher ploidy) state (Mortimer & Hawthorne, 1969). Starvation induces sporulation and the generation of haploids, but HO-mediated mating-type switching guarantees that isolated haploid spores will be able to mate and return to diploidy upon germination (Herskowitz & Jensen, 1991). However, if haploid spores self-mate to restore diploidy, then any heterozygosities that may have been present in the parental population will be lost. A large body of literature suggests that maintenance of heterozygosity can be selectively advantageous to a population (Milton, 1997). Therefore, one possible role for bridges is to promote, by physically connecting spores, the mating of sister spores to produce diploids and thereby restore the heterozygous state present in the parental cell. The fact that mating-type switching does not begin until the second division after germination (Strathern & Herskowitz, 1979) might, in combination with the bridges, help promote mating between sister spores.

While this is a speculative hypothesis, some indirect support for it can be found by comparison to Schiz. pombe. Schiz. pombe is also a homothallic yeast, but unlike S. cerevisiae it exists primarily as a haploid, only mating to form diploids immediately prior to sporulation (Mortimer, 1969). Because Schiz. pombe haploids do not mate upon germination, our model would predict no role for interspore bridges in this yeast. Indeed, in Schiz. pombe, no bridges are evident by SEM and wild-type spores disperse upon release from the ascus (Gutz et al., 1974; Nakamura et al., 2004). It will be of interest to determine whether, in other homothallic yeasts, the presence of bridges between ascospores correlates with the tendency to mate immediately upon germination.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Greg Rudomen and Jim Quinn for assistance with electron microscopy. This work was supported by National Institutes of Health Grant GM62184 to A. M. N.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Briza, P., Winkler, G., Kalchhauser, H. & Breitenbach, M. (1986). Dityrosine is a prominent component of the yeast ascospore wall. A proof of its structure. J Biol Chem 261, 4288–4294.[Abstract/Free Full Text]

Briza, P., Ellinger, A., Winkler, G. & Breitenbach, M. (1988). Chemical composition of the yeast ascospore wall. The second outer layer consists of chitosan. J Biol Chem 263, 11569–11574.[Abstract/Free Full Text]

Briza, P., Ellinger, A., Winkler, G. & Breitenbach, M. (1990). Characterization of a DL-dityrosine-containing macromolecule from yeast ascospore walls. J Biol Chem 265, 15118–15123.[Abstract/Free Full Text]

Briza, P., Eckerstorfer, M. & Breitenbach, M. (1994). The sporulation-specific enzymes encoded by the DIT1 and DIT2 genes catalyze a two-step reaction leading to a soluble LL-dityrosine-containing precursor of the yeast spore wall. Proc Natl Acad Sci U S A 91, 4524–4528.[Abstract]

Briza, P., Kalchhauser, H., Pittenauer, E., Allmaier, G. & Breitenbach, M. (1996). N,N'-Bisformyl dityrosine is an in vivo precursor of the yeast ascospore wall. Eur J Biochem 239, 124–131.[Abstract]

Christodoulidou, A., Bouriotis, V. & Thireos, G. (1996). Two sporulation-specific chitin deacetylase-encoding genes are required for the ascospore wall rigidity of Saccharomyces cerevisiae. J Biol Chem 271, 31420–31425.[Abstract/Free Full Text]

Felder, T., Bogengruber, E., Tenreiro, S., Ellinger, A., Sa-Correia, I. & Briza, P. (2002). Dtrlp, a multidrug resistance transporter of the major facilitator superfamily, plays an essential role in spore wall maturation in Saccharomyces cerevisiae. Eukaryot Cell 1, 799–810.[Abstract/Free Full Text]

Gutz, H., Heslot, H., Leupold, U. & Loprieno, N. (1974). Schizosaccharomyces pombe. In Handbook of Genetics, pp. 365–446. Edited by R. C. King. New York: Plenum.

Herskowitz, I. & Jensen, R. E. (1991). Putting the HO gene to work: practical uses for mating-type switching. Methods Enzymol 194, 132–146.[Medline]

Kreger-Van Rij, N. J. (1978). Electron microscopy of germinating ascospores of Saccharomyces cerevisiae. Arch Microbiol 117, 73–77.[Medline]

Kupiec, M., Byers, B., Esposito, R. E. & Mitchell, A. P. (1997). Meiosis and sporulation in Saccharomyces cerevisiae. In The Molecular Biology of the Yeast Saccharomyces. Cell Cycle and Biology, pp. 889–1036. Edited by J. R. Pringle, J. R. Broach & E. W. Jones. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Longtine, M. S., McKenzie, A. 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P. & Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]

Lynn, R. R. & Magee, P. T. (1970). Development of the spore wall during ascospore formation in Saccharomyces cerevisiae. J Cell Biol 44, 688–692.[Free Full Text]

Milton, J. B. (1997). Selection in Natural Populations. Oxford: Oxford University Press.

Mishra, C., Semino, C. E., McCreath, K. J., de la Vega, H., Jones, B. J., Specht, C. A. & Robbins, P. W. (1997). Cloning and expression of two chitin deacetylase genes of Saccharomyces cerevisiae. Yeast 13, 327–336.[CrossRef][Medline]

Moens, P. B. (1971). Fine structure of ascospore development in the yeast Saccharomyces cerevisiae. Can J Microbiol 17, 507–510.[Medline]

Mortimer, R. K. & Hawthorne, D. C. (1969). Yeast genetics. In The Yeasts, pp. 386–453. Edited by A. H. Rose & J. S. Harrison. London: Academic Press.

Nakamura, T., Abe, H., Hirata, A. & Shimoda, C. (2004). ADAM family protein Mde10 is essential for development of spore envelopes in the fission yeast Schizosaccharomyces pombe. Eukaryot Cell 3, 27–39.[Abstract/Free Full Text]

Neiman, A. M. (1998). Prospore membrane formation defines a developmentally regulated branch of the secretory pathway in yeast. J Cell Biol 140, 29–37.[Abstract/Free Full Text]

Neiman, A. M., Katz, L. & Brennwald, P. J. (2000). Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae. Genetics 155, 1643–1655.[Abstract/Free Full Text]

Orlean, P. (1997). Biogenesis of yeast wall and surface components. In The Molecular and Cellular Biology of the Yeast Saccharomyces. Cell Cycle and Biology, pp. 229–262. Edited by J. R. Pringle, J. R. Broach & E. W. Jones. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Pammer, M., Briza, P., Ellinger, A., Schuster, T., Stucka, R., Feldmann, H. & Breitenbach, M. (1992). DIT101 (CSD2, CAL1), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls. Yeast 8, 1089–1099.[Medline]

Rieder, S. E., Banta, L. M., Kohrer, K., McCaffery, J. M. & Emr, S. D. (1996). Multilamellar endosome-like compartments accumulate in the yeast vps28 vacuolar protein sorting mutant. Mol Biol Cell 7, 985–999.[Abstract]

Rose, M. D. & Fink, G. R. (1990). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Smits, G. J., van den Ende, H. & Klis, F. M. (2001). Differential regulation of cell wall biogenesis during growth and development in yeast. Microbiology 147, 781–794.[Medline]

Strathern, J. N. & Herskowitz, I. (1979). Asymmetry and directionality in production of new cell types during clonal growth: the switching pattern of homothallic yeast. Cell 17, 371–381.[Medline]

Tachikawa, H., Bloecher, A., Tatchell, K. & Neiman, A. M. (2001). A Gip1p-Glc7p phosphatase complex regulates septin organization and spore wall formation. J Cell Biol 155, 797–808.[Abstract/Free Full Text]

Received 16 April 2004; revised 21 May 2004; accepted 25 May 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Coluccio, A.
Articles by Neiman, A. M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Coluccio, A.
Articles by Neiman, A. M.
Agricola
Articles by Coluccio, A.
Articles by Neiman, A. M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.