1 School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
2 University Hospital Lausanne (CHUV), Institute of Microbiology, CH-1011 Lausanne, Switzerland
Correspondence
Neil A.R. Gow
n.gow{at}abdn.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Several models have been proposed for the role of chitin synthesis and chitin lysis during hyphal growth. In the unitary model of cell wall growth (Bartnicki-Garcia, 1973), chitinase is suggested to play an active role in cell wall biosynthesis at the growing tips of buds of yeast and the hyphae of filamentous fungi. Accordingly, wall growth represents a delicate balance between biosynthetic and hydrolytic processes. It has been proposed that hydrolytic enzymes such as chitinase cause sufficient cell wall lysis to maintain the wall in a plastic, compliant condition that allows insertion of new chitin fibrils as well as turgor-driven expansion of the cell surface. Zymogenic, membrane-associated forms of chitinase, which seem to be regulated in a way compatible with this role, have been described (Humphreys & Gooday, 1984a
, b
; Dickinson et al., 1991
). However the chitin synthase and zymogenic chitinase activities of Candida albicans did not co-purify (Dickinson et al., 1991
). Fungi have also been shown to possess complex chitinase families, suggestive of a range of roles in addition to the hydrolysis of chitin for nutrition (Rast et al., 1991
; Gooday et al., 1992
). The regulation of spore germination, budding, hyphal growth, hyphal branching and septum formation may all involve the direct participation of cell wall hydrolysis as well as synthesis, and thus these enzymic functions may be coordinately regulated (Gooday et al., 1992
). Further evidence for the association of chitinase with chitin synthase was suggested by the parallel stimulation of the two activities during spore germination of Mucor mucedo, and exponential growth of yeast cells of Mucor rouxii (Rast et al., 1991
) and C. albicans (Barrett-Bee & Hamilton, 1984
).
An alternative steady-state model for fungal cell growth suggests that the plasticity of the hyphal apex and growing bud does not require the participation of hydrolytic enzymes such as chitinases (Wessels, 1984, 1986
, 1990
; Sietsma & Wessels, 1994
), but instead is an inherent property of the process of chitin and glucan synthesis. Nascent polysaccharides have been suggested to be plastic. They are poorly cross-linked (Wessels et al., 1983
) and are amorphous (Vermeulen & Wessels 1984
), because the polysaccharide chains that are synthesized de novo have not yet hydrogen-bonded and crystallized into structural microfibrils (Wessels, 1986
, 1990
). Nascent chitin is more susceptible to chitinase digestion (Vermeulen & Wessels, 1986
), which supports the view that chitinase could play a role in moulding and shaping the expanding cell wall. The most recent models of hyphal tip morphogenesis downplay the importance of the cell wall and emphasize instead the importance of the mechanism that deposits secretory vesicles from the vesicle supply centre to the cell surface (Bartnicki-Garcia, 2002
).
Most or all fungi have multiple genes encoding chitin synthase families (Munro & Gow, 1995, 2001
; Roncero, 2002
). These chitin synthases are not usually redundant, but instead perform distinct functions at specific stages of the cell cycle and are regulated by several gene products that influence chitin synthase activation and localization. C. albicans has four chitin synthase genes CHS1, CHS2, CHS3 and CHS8 (Munro & Gow, 2001
; Munro et al., 2003
) and at least four chitinases whose functions have yet to be fully explored. CaCHT1 is not expressed under any known conditions in vitro (McCreath et al., 1995
, 1996
). CHT2 and CHT3 are expressed preferentially in the yeast form of the fungus (McCreath et al., 1995
). CHT4 has been identified recently in the C. albicans genome, but has yet to be characterized. Saccharomyces cerevisiae has only three chitin synthase enzymes (CHS1, CHS2 and CHS3), and a single chitinase enzyme (CTS1) (Bulawa et al., 1986
; Bulawa, 1992
; Bulawa & Osmond, 1990
; Cabib et al., 1982
, 1989
; Kuranda & Robbins, 1991
; Shaw et al., 1991
; Valdivieso et al., 1991
).
In this study, we examine the hypothesis that chitin synthase and chitinase activity are coupled, by measuring the consequences of disruptions in specific chitin synthase and chitinase genes on both chitin synthesis and chitinase activity. We show that the total specific chitin synthase activity is not affected by mutations in chitinase genes, and reciprocally that the specific chitinase activity in cell extracts is not altered in any of the chs mutant backgrounds tested. The data do not provide support for the hypothesis that chitin synthesis and chitin hydrolysis are coordinately regulated.
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METHODS |
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Measurement of chitinase activity.
The fluorogenic microtitre plate assay developed by McCreath & Gooday (1992) was used to measure endochitinase activity, employing the substrate 4-methylumbelliferyl-
-D-N-tetraacetylchitotetraoside (4-MU-[GlcNAc]4). The substrate is sensitive to endochitinase activity and is relatively insensitive to action by N-acetylglucosaminidases (Jackson et al., 1996
). Chitinase activity was measured both in the extracellular supernatant and in washed whole cells. For hyphal cultures, bovine chitinase was removed from the serum used to induce the morphological transition by adding colloidal chitin, then removing the chitinase (which binds to this chitin) by centrifugation. Assays employing washed cells reflect periplasmic and cell-bound activity, since the substrate is not transported into the cell (Gooday et al., 1992
). Assays were performed in microtitre plates, with 5 µl substrate, 80 µl 0·1 M McIlvaine buffer, pH 5·0, and 20 µl sample per well. Samples were incubated at 40 °C for 30 min in an Ascent Labsystem fluorimeter. The reaction was stopped by adding 120 µl glycine/NaOH buffer, pH 10·6, and after a further 5 min incubation a final reading was obtained. Chitinase activity was expressed per 106 cells for yeast cells, and per mg of dry weight for hyphae.
Measurement of chitin synthase activity.
Mixed membrane fractions were prepared from exponential-phase yeast and hyphal cells, and chitin synthase activity was measured as described previously (Munro et al., 1998). Chitinase and chitin synthase activities were measured in cells grown under identical culture conditions. Differences in the activity of chitin synthase and chitinase were analysed using Student's t-test and ANOVA.
Measurement of cell wall chitin content.
Cell walls were prepared from 10 ml volumes of C. albicans exponential-phase yeast culture grown in YPD, or hyphal cultures grown in FCS. Hyphal growth was induced by inoculating 5x107 washed stationary phase yeast cells into 20 % (v/v) FCS in water, followed by incubation for 6 h at 37 °C, with shaking. Washed yeast and hyphal cells were broken with glass beads (Sigma, G9268), using a Fastprep cell breakage machine (Thermo Savant), until at least 95 % of the cells had been disrupted. Cell lysates were centrifuged at 5000 r.p.m. to pellet the cell walls, which were washed five times with 1 M NaCl. The washed cell walls were treated with 1 ml extraction buffer (50 mM Tris, 2 % (w/v) SDS, 0·3 M -mercaptoethanol, 1 mM EDTA, pH 8·0) at 100 °C for 10 min, then washed three times in distilled water. Cell wall pellets were resuspended with sterile distilled water, prior to lyophilization, and the dry weight of the cell walls was measured. Chitin content was measured by assaying the glucosamine released by acid hydrolysis of chitin (Kapteyn et al., 2000
). Cell wall material (15 mg) was resuspended in 6 M HCl and hydrolysed at 100 °C for 17 h, together with N-acetylglucosamine (GlcNAc) standards. Samples were then dried and resuspended in 300 µl distilled water. The quantity of glucosamine released by hydrolysis of 100 µl of this material was determined as follows. An equal volume of 4 % (v/v) acetylacetone in 1·5 M Na2CO3 was added, and the preparation heated at 100 °C for 20 min. Samples were then diluted with 700 µl 96 % ethanol, and 200 µl Ehrlich's reagent (26 mg p-dimethylaminobenzaldehyde ml1, 5·8 M HCl, 50 % ethanol) was added. Triplicate samples were incubated for 1 h at 20 °C before the absorbance was read at 520 nm.
Calcofluor White resistance.
Calcofluor White (CFW, Sigma) was incorporated into YPD agar plates at 50, 75 and 100 µg ml1. Yeast cells, grown to late exponential phase in YPD, were diluted to 5x105 cells ml1 in fresh YPD. Plates were inoculated with 5 µl drops of cell suspension and incubated for 24 h at 30 °C.
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RESULTS |
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Chitinase activities were also examined in four C. albicans chitin synthase mutant strains: MRP1 : CHS1/chs1, chs2
/chs2
, chs3
/chs3
and chs8
/chs8
. The conditional MRP1-CHS1 strain was grown under repressing conditions in the absence of maltose and the presence of glucose. In the yeast form, all four chs
mutants had a slight but statistically significant decrease in cell-associated chitinase activity (Fig. 1a
). Supernatant chitinase activity was only significantly lowered in chs1
and chs8
mutants grown in the yeast form. In the hyphal form, chs1
had significantly lower cell-associated chitinase activity, and significantly higher activity in the supernatant. Likewise, the chs1
, chs3
and chs8
hyphal forms had slightly but significantly higher supernatant chitinase activity (P<0·05). The chitinase activity in the other mutants remained unchanged (Fig. 1a
).
Chitinase activity was also examined in three S. cerevisiae chitin synthase mutant strains (chs1, chs2
, chs3
) and in the chitinase mutant cts1
. For the chitinase mutant, low residual chitinase activity was observed (Fig. 2
). No difference in chitinase activity against 4-MU-[GlcNAc]4 was observed for any of the three chitin synthase mutants. Therefore, although the level of total chitinase activity was in general higher in C. albicans than in S. cerevisiae, mutations in any of the three chitin synthase genes of S. cerevisiae or four chitin synthase genes of C. albicans had little or no effect on the total chitinase activity expressed by either fungus.
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DISCUSSION |
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Our investigations did reveal, however, that both chitin synthase and chitinase activity were regulated during yeasthypha morphogenesis in C. albicans and that CaCht2p and CaCht3p were regulated differentially in hyphal and yeast forms. Both chitinases were active in the yeast form, and loss of function of either of the two chitinase genes resulted in significantly decreased total endochitinase activity. However, during hyphal development, chitinase activity was inferred to be due mainly to the CHT3 gene product. These results would not be predicted by earlier reports of the transcriptional analysis of these genes, where both CHT2 and CHT3 were shown to be expressed preferentially in the yeast form of C. albicans (McCreath et al., 1995). However, in the latter study, hyphae were induced using pH and temperature to regulate morphogenesis, while our experiments used serum to induce hypha formation. The residual chitinolytic activity of single and double cht mutant strains may be due to the CHT4 gene product, recently identified in the C. albicans genome (GenBank AAG35112 and C. Specht, personal communication). Expression of CHT1 has not been detected in either yeast or hyphae and is therefore unlikely to contribute to measured chitinase activity in these experiments (McCreath et al., 1995
).
Chitinase activity was not greatly affected by deletion of chitin synthase genes in either growth form of C. albicans. During hyphal growth, most chitinase activity was measured in the supernatant, while in the yeast form, most chitinase activity fractionated with whole cells. These results are compatible with reports showing that CaCht2p is attached to the yeast cell wall (Iranzo et al., 2002) and that CaCht3p is a secreted protein (C. Specht, personal communication).
Similar findings were obtained for S. cerevisiae: mutations in the chitin synthase genes had little effect on chitinase activity. In C. albicans, deletion of CHT2 and CHT3 led to a slight increase in the overall cell chitin content and slightly increased sensitivity to Calcofluor White. This suggests that Cht2p and Cht3p may act on the cell wall of C. albicans and influence its chitin content.
The results obtained for the chitin synthase activities of the chs mutants in C. albicans and S. cerevisiae are consistent with previous findings. CaCHS2 and ScCHS1 encode the major in vitro chitin synthase activity for C. albicans and S. cerevisiae, respectively (Bulawa et al., 1986
; Munro et al., 1998
). In the yeast form, cht3
and cht2
cht3
mutant strains of C. albicans showed significant changes in chitin synthase activity. However, chitin synthase activity in the hyphal form of these mutants was unaffected. In addition, in S. cerevisiae, no change in chitin synthase activity was observed in the cts1
mutant. Chitin synthase genes are known to be regulated post-transcriptionally (Bulawa, 1992
; Choi et al., 1994
; Chuang & Schekman, 1996
). Little transcriptional regulation of CHS3 has been observed, although the gene product is activated by the Slt2-dependent salvage pathway that responds to cell wall damage (Popolo et al., 1997
, 2001
). The formal possibility remains that specific chitin synthase and chitinase enzymes may be coordinately regulated, but our in vitro enzyme assays were not sufficiently discriminating to evaluate how particular genes are regulated under different mutant backgrounds. It is possible that specific zymogenic forms are regulated through activation of proenzyme forms of the enzymes at specific cellular sites. To carry this study forward, we are now constructing reporter fusions to examine the transcriptional responses of CHS genes to specific genetic and environmental perturbations. However, taken at face value, the results presented here do not support the hypothesis that there is a strong interplay between total chitinase and chitin synthase activities in these fungi.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Barrett-Bee, K. & Hamilton, M. (1984). The detection and analysis of chitinase activity from the yeast form of Candida albicans. J Gen Microbiol 130, 18571861.[Medline]
Bartnicki-Garcia, S. (1973). Fundamental aspects of hyphal morphogenesis. In Microbial Differentiation (Society for General Microbiology Symposium no. 23), pp. 245267. Edited by J. M. Ashworth & J. E. Smith. Cambridge: Cambridge University Press.
Bartnicki-Garcia, S. (2002). Hyphal tip growth: outstanding questions. In Molecular Biology of Fungal Development, pp. 2959. Edited by H. D. Osiewacz. New York: Marcel Dekker.
Bulawa, C. E. (1992). CDS2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CDS2 gene product is related to chitin synthase and to developmentally regulated protein in Rhizobium species and Xenopus laevis. Mol Cell Biol 12, 17641776.[Abstract]
Bulawa, C. E. & Osmond, B. C. (1990). Chitin synthase I and chitin synthase II are not required for chitin synthesis in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 87, 74247428.[Abstract]
Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L. & Robbins, P. W. (1986). The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo. Cell 46, 213225.[Medline]
Bulawa, C. E., Miller, D. W., Henry, L. K. & Becker, J. M. (1995). Attenuated virulence of chitin-deficient mutants of Candida albicans. Proc Natl Acad Sci U S A 92, 1057010574.[Abstract]
Cabib, E. (1987). The synthesis and degradation of chitin. Adv Enzymol Relat Areas Mol Biol 59, 59101.[Medline]
Cabib, E., Roberts, R. & Bowers, B. (1982). Synthesis of the yeast cell wall and its regulation. Annu Rev Biochem 51, 763793.[CrossRef][Medline]
Cabib, E., Sburlati, A., Bowers, B. & Silverman, S. J. (1989). Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae. J Cell Biol 108, 16651672.[Abstract]
Cabib, E., Silverman, S. J. & Shaw, J. A. (1992). Chitinase and chitin synthase 1: counterbalancing activities in cell separation of Saccharomyces cerevisiae. J Gen Microbiol 138, 97102.[Medline]
Choi, W.-J., Santos, B., Durán, A. & Cabib, E. (1994). Are yeast chitin synthases regulated at the transcriptional or the posttranslational level? Mol Cell Biol 14, 76857694.[Abstract]
Chuang, J. S. & Schekman, R. W. (1996). Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p. J Cell Biol 135, 597610.[Abstract]
Dickinson, K., Keer, V., Hitchcock, C. A. & Adams, D. J. (1991). Microsomal chitinase activity from Candida albicans. Biochim Biophys Acta 1073, 177182.[Medline]
Fèvre, M. (1977). Subcellular localization of glucanase and cellulase in Saprolegnia monoica Pringsheim. J Gen Microbiol 103, 287295.
Fonzi, W. A. & Irwin, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717728.
Gooday, G. W. (1995). The dynamics of hyphal growth. Mycol Res 99, 385394.
Gooday, G. W., Zhu, W. Y. & O'Donnell, R. W. (1992). What are the roles of chitinases in the growing fungus? FEMS Microbiol Lett 100, 387392.[CrossRef]
Gow, N. A. R. & Gooday, G. W. (1982). Growth kinetics and morphology of colonies of the filamentous form of Candida albicans. J Gen Microbiol 128, 21872194.[Medline]
Humphreys, A. M. & Gooday, G. W. (1984a). Properties of chitinase activity from Mucor mucedo: evidence for a membrane-bound zymogenic form. J Gen Microbiol 130, 13591366.
Humphreys, A. M. & Gooday, G. W. (1984b). Phospholipid requirement of microsomal chitinase from M. mucedo. Curr Microbiol 11, 187190.
Iranzo, M., Aguado, C., Pallotti, C., Canizares, V. J. & Momeneo, S. (2002). The use of trypsin to solubilize wall protein from Candida albicans led to the identification of chitinase 2 as an enzyme covalently linked to the yeast wall structure. Res Microbiol 153, 227232.[CrossRef][Medline]
Jackson, D. J., Saunders, V. A., Gooday, G. W. & Humphreys, A. M. (1996). Chitinase activities from yeast and hyphal cells of Candida albicans. Mycol Res 100, 321327.
Kapteyn, J. C., Hoyer, L. L., Hecht, J. E., Muller, W. H., Andel, A., Verkleij, A. J., Makarow, M., Van den Ende, H. & Klis, F. M. (2000). The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants. Mol Microbiol 35, 601611.[CrossRef][Medline]
Klis, F. M., de Groot, P. & Hellingwerf, K. (2001). Molecular organization of the cell wall of Candida albicans. Med Mycol 39S, 18.
Kuranda, M. J. & Robbins, P. W. (1991). Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. J Biol Chem 266, 1975819767.
Martín-Cuadrado, A. B., Dueñas, E., Sipiczki, M., Vásquez de Aldana, C. R. & del Rey, F. (2003). The endo--1,3-glucanase eng1p is required for dissolution of the primary septum during cell separation in Schizosaccharomyces pombe. J Cell Sci 116, 16891698.
McCreath, K. J. & Gooday, G. W. (1992). A rapid and sensitive microassay for determination of chitinolytic activity. J Microbiol Methods 14, 229237.[CrossRef]
McCreath, K. J., Specht, C. A. & Robbins, P. W. (1995). Molecular cloning and characterization of chitinase genes from Candida albicans. Proc Natl Acad Sci U S A 92, 25442548.[Abstract]
McCreath, K. J., Specht, C. A., Liu, Y. & Robbins, P. W. (1996). Molecular cloning of a third chitinase gene (CHT1) from Candida albicans. Yeast 12, 501504.[CrossRef][Medline]
Mio, T., Yabe, T., Sudoh, M., Satoh, Y., Nakajima, T., Arisawa, M. & Yamada-Okabe, H. (1996). Role of three chitin synthase genes in the growth of Candida albicans. J Bacteriol 178, 24162419.[Abstract]
Mullins, T. J. (1973). Lateral branch formation and cellulase production in water molds. Mycologia 65, 10071014.[Medline]
Munro, C. A. & Gow, N. A. R. (1995). Chitin biosynthesis as target for antifungals. In Antifungal Agents: Discovery and Mode of Action, pp.161171. Edited by G. K. Dixon, L. G. Copping & D. W. Hollomon. Oxford: Bios.
Munro, C. A. & Gow, N. A. R. (2001). Chitin synthesis in human pathogenic fungi. Med Mycol 39S, 4153.
Munro, C. A., Schofield, D. A., Gooday, G. W. & Gow, N. A. (1998). Regulation of chitin synthesis during dimorphic growth of Candida albicans. Microbiology 144, 391401.[Abstract]
Munro, C. A., Winter, K., Buchan, A., Henry, K., Becker, J. M., Brown, A. J. P., Bulawa, C. E. & Gow, N. A. R. (2001). Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity. Mol Microbiol 39, 14141426.[CrossRef][Medline]
Munro, C. A., Whitton, R., Hughes, B., Reilla, M., Selvaggini, S. & Gow, N. A. R. (2003). CHS8 a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth. Fungal Genet Biol 40, 146158.[CrossRef][Medline]
Popolo, L., Gilardelli, D., Bonfante, P. & Vai, M. (1997). Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1 mutant of Saccharomyces cerevisiae. J Bacteriol 179, 463469.[Abstract]
Popolo, L., Gualtieri, T. & Ragni, E. (2001). The yeast cell-wall salvage pathway. Med Mycol 39S, 111121.
Rast, D. M., Horsch, M., Furter, R. & Gooday, G. W. (1991). A complex chitinolytic system in exponentially growing mycelium of Mucor rouxii: properties and function. J Gen Microbiol 137, 27972810.[Medline]
Roncero, C. (2002). The genetic complexity of chitin synthesis in fungi. Curr Genet 41, 367378.[CrossRef][Medline]
Shaw, A. J., Mol, P. C., Bowers, B., Silverman, S. J., Valdivieso, M. H., Durán, A. & Cabib, E. (1991). The function of chitin synthase 2 and 3 in the Saccharomyces cerevisiae cell cycle. J Cell Biol 114, 111123.[Abstract]
Sietsma, J. H. & Wessels, J. G. H. (1994). Apical wall biogenesis. In The Mycota I, pp.125141. Edited by J. G. H. Wessels & H. Meinhardt. Berlin: Springer.
Valdivieso, M. H., Mol, P. C., Shaw, J. A., Cabib, E. & Durán, A. (1991). CAL1, a gene required for activity of chitin synthase 3 in Saccharomyces cerevisiae. J Cell Biol 114, 11011109.
Vermeulen, C. A. & Wessels, J. G. H. (1984). Ultrastructural differences between wall apices of growing and non-growing hyphae of Schizophyllum commune. Protoplasma 120, 123131.
Vermeulen, C. A. & Wessels, J. G. H. (1986). Chitin biosynthesis by a fungal membrane preparation. Evidence for a transient non-crystalline state of chitin. Eur J Biochem 158, 411415.[Abstract]
Wessels, J. G. H. (1984). Apical hyphal wall extension. Do lytic enzymes play a role? In Microbial Cell Wall Synthesis and Autolysis, pp. 3142. Edited by C. Nombela. Berlin: Elsevier.
Wessels, J. G. H. (1986). Cell wall synthesis in apical hyphal growth. Int Rev Cytol 104, 3779.
Wessels, J. G. H. (1990). Role of cell wall architecture in fungal tip growth generation. In Tip Growth in Plant and Fungal Cells, pp. 128. Edited by I. B. Heath. San Diego: Academic Press.
Wessels, J. G. H., Sietsma, J. H. & Sonnenberg, S. M. (1983). Wall synthesis and assembly during hyphal morphogenesis in Schizophyllum commune. J Gen Microbiol 129, 16071616.
Received 22 July 2003;
revised 18 November 2003;
accepted 22 December 2003.
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