Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan1
Author for correspondence: Hiroyuki Horiuchi. Tel: +81 3 5841 5170. Fax: +81 3 5841 8015. e-mail: ahhoriu{at}mail.ecc.u-tokyo.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: cell wall, conidiation, filamentous fungi
Abbreviations: AP, alkaline phosphatase; RACE, rapid amplification of cDNA ends
The GenBank accession number for the the new version of the chsD nucleotide sequence is D83246.
a These authors contributed equally to this work.
b Present address: The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The functions of chitin synthases in filamentous fungi have been analysed by gene disruptions or gene deletions. The respective null mutants of class I (Motoyama et al., 1994a ), class II (Beth Din & Yarden, 1994
; Yanai et al., 1994
) and class IV (Beth Din et al., 1996
; Motoyama et al., 1996
; Specht et al., 1996
) chitin synthase genes of filamentous fungi exhibited no obvious phenotypic defect under standard conditions with the exception of that studied by Beth Din & Yarden (2000)
. Recently, it was reported that a class II chitin synthase gene (CHS1) of the dimorphic yeast Candida albicans was essential even for the growth of the yeast form (Sudoh et al., 2000
; Munro et al., 2001
). Disruptions or deletions of class III (Yarden & Yanofsky, 1991
; Yanai et al., 1994
; Borgia et al., 1996
; Mellado et al., 1996
) or class V (Specht et al., 1996
; Aufauvre-Brown et al., 1997
; Horiuchi et al., 1999
) chitin synthase genes, with the exception of two class III genes (Gold & Kronstad, 1994
; Mellado et al., 1996
), caused some phenotypic changes. These observations indicate that the respective chitin synthase genes (not all genes) alone do not bear essential functions for hyphal growth and conidiation, and that the class of chitin synthase is not necessarily related to the function and importance of these genes among different fungal species.
To reveal the function of individual chitin synthase genes, double and triple disruption mutants were constructed and analysed. We previously found that the class II and class IV chitin synthases of A. nidulans have some overlapping function(s) in conidiation (Motoyama et al., 1996 ), and that the class I and class II chitin synthases of A. nidulans share important roles in hyphal growth and conidiation (Fujiwara et al., 2000
). A partial redundancy of function of class II and class IV chitin synthases was also reported by another group (Culp et al., 2000
). From these results, we speculated that chitin synthases in class I, class II and class IV may play important roles in growth or differentiation, and that these synthases cooperate with each other. In the yeast Saccharomyces cerevisiae, the class IV chitin synthase is involved in most of the cellular chitin synthesis and serves redundant functions with class II chitin synthase in growth (Valdivieso et al., 1991
; Shaw et al., 1991
). These results suggest that fungal chitin is synthesized by the overlapping and cooperative function of multiple chitin synthase genes.
In our previous study, we showed that A. nidulans chsD, which encodes a class IV chitin synthase, is expressed in the growing hyphae (Motoyama et al., 1996 ), and chsB, which encodes a class III chitin synthase, plays essential roles in normal hyphal growth. These results suggest that ChsD and ChsB have some related functions in chitin synthesis and other activities of growing hyphae. In this study, we focused on the functional relationship between ChsB and ChsD in hyphal growth and in conidiation. The construction of double deletion mutants of chsB and chsD seemed to be difficult because null mutants of chsB are heavily damaged in hyphal growth (Yanai et al., 1994
; Borgia et al., 1996
). Therefore, we constructed conditional double mutants in which chsD was replaced with the argB gene of A. nidulans, and chsB was placed under the control of a repressible promoter (the alcA promoter). We then analysed the functional relationship between the two chitin synthases.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Total DNA isolation and Southern analysis.
Total DNA of filamentous fungi was extracted as described by Oakley et al. (1987) . Southern analysis was done with ECL direct nucleic acid labelling and detection systems (Amersham Pharmacia Biotech). DNA was separated by electrophoresis on agarose gels and further manipulation was done as described in the manufacturers instructions.
Total RNA isolation and Northern analysis.
Total RNA was prepared from mycelia by using the RNeasy Mini Kit (QIAGEN). Five micrograms of total RNA were separated by electrophoresis on a formaldehyde-agarose gel, and blotted onto Hybond-XL membrane (Amersham Pharmacia Biotech) according to the manufacturers instructions. Blots were hybridized to 32P-labelled probes prepared by using Random Primer DNA Labelling Kit Version 2 (TaKaRa). As a probe for chsB expression, the 1·6 kb NcoI fragment from pchsB was used.
PCR-amplification of DNA fragments.
PCR-amplification was done by using a thermal cycler (model 480, Perkin-Elmer/Cetus) and Taq DNA polymerase (Boehringer Mannheim) or the Expand High Fidelity PCR system (Boehringer Mannheim).
RT-PCR amplification.
Twenty micrograms of total RNA were treated with deoxyribonuclease (RT Grade) (NIPPON GENE), then reverse transcriptions were performed by using 3 µg DNA-free RNA and ReverTra Ace reverse transcriptase (TOYOBO) according to the manufacturers instructions. The primer used for reverse transcriptions was chsB.3072as (5'-GGCATCCAGTCTAGGTTGC-3'). For PCR amplifications, 5 µl samples from 20 µl of reverse transcription reactions were used. The primers used were chsB.2055s (5'-CATCATGGATCTGGTAGG-3') and chsB.3075as (5'-ATACATACAATTGCAAGGC-3'). Since we designed the primer set to amplify the region that contains an intron, the products of RT-PCR were expected to be 57 bp smaller than the PCR products amplified from genomic DNA by using the same primer set. PCR products and 100 bp DNA ladder (New England Biolabs) were electrophoresed on a 1·0% agarose gel.
Plasmid constructions.
Plasmids used in this paper are shown in Table 2. To make the strain in which the expression of chsB is under the control of the alcA promoter, the plasmid p5BP-ALCB was constructed as follows. The alcA promoter was amplified from the genomic DNA of strain AU1 by the PCR method using the two primers, M16-1 (5'-AAAATCGATGGCGGGGCGGAAATTGACA-3') and M16-2 (5'-AAAAAGCTTTGAGGCGAGGTGATAGGAT-3'), according to the sequence reported by Kulmburg et al. (1992)
. Using these two primers, 35 cycles were run consisting of a 94 °C 1 min melting step, a 56 °C 2 min annealing step, and a 72 °C 3 min extension step. The 5·5 kb HindIII fragment containing the chsB gene was prepared from the DNA purified from the phage clone that contains the chsB gene (Yanai et al., 1994
) and ligated with HindIII-digested and bacterial alkaline phosphatase (AP)-treated pUC118 (Takara Shuzo) to yield pchsB and pchsBR. The direction of chsB transcription was from the HindIII site to the EcoRI site on the multiple cloning site of pchsB, and the direction of the chsB insert of pchsBR was reverse compared to pchsB.
|
The plasmid pSS-chsB, for chsB expression, was constructed by ligating the 5·5 kb HindIII fragment, which contains the chsB gene derived from pchsB, and BamHI-digested, blunted, and AP-treated pSS1 (Motoyama et al., 1994a ).
Plasmids for the analysis of promoter activities in A. nidulans with lacZ of E. coli as a reporter gene were constructed as follows. The 3·0 kb BamHI fragment which contains lacZ of E. coli derived from pMC1871 (Casadaban et al., 1983 ) was ligated with BamHI-digested and AP-treated pSS1 to yield pSS-LZ. The blunted 0·9 kb SphI fragment, which is expected to contain the terminator region of chsB, derived from pchsB5.5H, was ligated with XbaI-digested, blunted, and AP-treated pSS-LZ to yield pSS-TB-LZ. The 1·8 kb HindIIINaeI fragment, which contains the chsB promoter (1·3 kb) and a part of the chsB ORF (0·5 kb), derived from pchsB, was blunted and ligated with SmaI-digested and AP-treated pSS-TB-LZ to yield pB-LAC. The 1·8 kb NspV fragment, which contains the chsD promoter (1·4 kb) and a part of the chsD ORF (0·4 kb), derived from pchsD, was blunted and ligated with SmaI-digested and AP-treated pSS-TB-LZ to yield pD-LAC. Linkage points were confirmed by sequencing with a primer, prm-LZ2 (5'-TTCTGGTGCCGGAAACCA-3'). We found some sequencing errors in the chsD and the new version of the chsD nucleotide sequence is registered with accession no. D83246. Consequently, total amino acid residues of ChsD were changed from 1086 to 1184 due to the extension of 96 aa residues in its N-terminus. From the results of 5'-RACE experiments, the transcriptional start point(s) were estimated approximately 530 bp upstream of the revised ATG initiation codon and an intron of 151 bp was identified from -6 to -157 when the first A of the revised initiation codon was designated +1.
Construction of A. nidulans strains by transformation.
All the strains originated from AU1 or ABPU1. The argB gene of A. nidulans and the pyr4 gene of N. crassa were used as selectable markers to complement arginine- and uridine-auxotrophy, respectively.
Conditional mutants of chsB were made as follows. The promoter of chsB in the genome of A. nidulans strain ABPU1 was interrupted with the alcA promoter by transformation with the 3·9 kb DraIClaI fragment from p5BP-ALCB. By Southern analysis of HindIII-digested or PstI-digested total DNA of ABPU1 transformants probed with the 1·3 kb HindIII fragment (Fig. 1a, probe) from pchsB5.5H, homologous recombination at the chsB locus was confirmed in strains named BM-3, BM-4 and BM-12 by a shift of the signal size from 5·5 kb to 1·9 kb and from 1·6 kb to 3·8 kb, respectively (Fig. 1b
, lanes 18). By the same strategy, the promoter of chsB in the genome of the chsD null mutant D3-2 (Table 1
, Motoyama et al., 1996
) was exchanged with the alcA promoter to yield strains DB-13 and DB-19. The exchange of these promoters was confirmed by Southern analysis (data not shown). Strain BB-3 was created by transformation of strain BM-3 with pSS-chsB. The strains ABPU/A and BM-3/A1 were constructed by transformation of strains ABPU1 and BM-3, respectively, with pSS1. chsD of strain BM-3 was replaced with the argB gene by using pD
A7 as described previously (Motoyama et al., 1996
), generating a strain designated as BD-2.
|
|
Carbon source exchange experiment.
Conidial suspensions of A. nidulans were inoculated into liquid MMFT (200 ml, fructose and threonine as carbon sources) in a 500 ml Erlenmeyer flask to a final concentration of 2x106 conidia ml-1. After agitating the flasks on a rotary shaker for 17 h at 37 °C, mycelia were collected by filtration through G3 glass filter, washed by liquid MM (glucose as a carbon source), and spread on MM plates (glucose as a carbon source). After 25 h incubation at 37 °C, conidia were collected from the plates and the numbers were counted using a haemocytometer. All the MMFT and MM used were supplemented with 0·20 mg arginine ml-1, 0·02 µg biotin ml-1, 0·50 µg pyridoxine ml-1 and 10 mM uridine. To examine the abundance of the chsB transcript, total RNA was prepared from BM-3/A1 (chsB mutant) mycelia taken at 0, 5, 15 and 25 h after transfer onto a MM plate.
Chitin content determination.
Chitin content was determined as described previously (Fujiwara et al., 2000 ), except that strains were grown in 100 ml YGU medium for 18 h at 37 °C, and that the cell-wall-containing pellets were sonicated in distilled water to a fine suspension before enzymic treatment. The protein levels, by which we normalized the amount of N-acetylglucosamine, were not very different between the cell extracts from different strains.
In situ staining of the ß-galactosidase activity.
ß-Galactosidase activity was stained in situ by a modification of a method reported previously (Adams & Timberlake, 1990 ; Aguirre et al., 1990
). In brief, 20 µl of a conidial suspension (108 conidia ml-1) was spread on a MM plate (supplemented with biotin, pyridoxine and uridine). After incubating for 13 or 36 h at 37 °C, hyphae on a piece of agar (approx. 15 mmx15 mm) were cut off, and treated with chloroform vapour for 20 min at room temperature. The piece was transferred into a staining solution (0·05 M sodium phosphate, pH 7·5, 0·02% X-Gal), incubated for 2 h at 37 °C, and cells were observed under a microscope.
Microscopy.
Samples were observed and photographed by using a light microscope (model BHS-RFK, Olympus) equipped with an automatic camera (model PM-10ADS, Olympus) attachment.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of chsB repression on growth and differentiation
We analysed the effect of the repression of chsB expression on the growth of A. nidulans by comparing the radial growth rate of the chsB-repressed strain with that of the wild-type strain. Since auxotrophic markers could affect the growth rate, BM-3/A1 was constructed by the transformation of BM-3 with pSS1, a plasmid containing the argB gene, and the growth rate of BM-3/A1 was compared with that of the wild-type strain ABPU/AU. Conidia of each strain were point-inoculated on solid medium and incubated for 72 h at 37 °C. The colony diameter was then determined (Table 3). On MM plates [alcA(p)-repressing condition], the colony diameter of BM-3/A1 (15·5±4·3 mm) was much smaller than that of ABPU/AU (34·5±2·9 mm). Under this condition, dark brown pigmentation of hyphae in the agar was observed in BM-3/A1. Borgia et al. (1996)
reported that the chsB disruptant colonies became brown. Significant reduction in the growth rate of BM-3/A1 was also observed on YGU plates [alcA(p)-repressing condition]. Since the growth defects of some cell-wall-deficient mutants can occasionally be remedied by the addition of osmotic stabilizers into the media, we examined the effects of osmotic stabilizers. The growth defect of strain BM-3/A1 on YGU plates was not suppressed by the addition of 0·6 M KCl. On MMFT plates [alcA(p)-inducing condition], the colony diameter of BM-3/A1 (31·8±0·8 mm) was slightly smaller than that of ABPU/AU (36·7±0·9 mm). Taken together, these results suggest that the growth defect of BM-3/A1 on MM and YGU plate was caused by the repression of chsB expression.
|
|
|
|
When the hyphal morphology of DB-13 on YGU plate was observed microscopically, the hyphae were seen to be even more disorganized than those of BM-3/A1 (Fig. 3g, h
). The number of conidiophores was much smaller than that of BM-3/A1 (data not shown). The number was not greatly increased by 24 h of additional incubation (data not shown).
In the carbon source exchange experiment, it was shown that DB-13 generated approximately 60% of the number of conidia generated by BM-3/A1 (Table 4). Taking into account that the chsD single mutants show no significant reduction in conidiation efficiency (Motoyama et al., 1996
; Culp et al., 2000
, in which chsD is referred to as chsE), it is suggested that the importance of the ChsD function in conidiation also increased under the chsB-repressing condition.
We measured the chitin content of BM-3/A1 and DB-13 (Table 5). The chitin contents of BM-3/A1 in YGU and in YGU containing 0·6 M KCl were both approximately 70% of those of the wild-type strain (ABPU/AU), whereas those of BB-3 were almost the same as those of the wild-type strain. The chitin contents of DB-13 were approximately half those of BM-3/A1. Since the chitin contents of D3-2 under these conditions were not very different from those of the relevant wild-type strain (ABPU/A), the effect of the chsD deletion on chitin content was considered to be larger in the chsB-conditional mutant than in the wild-type strain. Taking these results together, we concluded that the chsB repression increased the importance of the ChsD functions in hyphal growth, conidiation and chitin content maintenance.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our laboratory reported previously that a chsB disruptant could grow only as a heterokaryon containing both wild-type and disrupted chsB alleles (Yanai et al., 1994 ). The hyphae of a haploid chsB disruptant stopped growing immediately after the germination of conidia, with their hyphal tips swelling. Borgia et al. (1996)
obtained viable homokaryons carrying the disrupted chsB allele. Their mutants grew very slowly with a high degree of branching, and contained normal septa. The colonies did not form conidiophores and conidia, and became brown after about 4 days incubation. These phenotypes of the Borgia groups chsB disruptants are very similar to those of the repressed chsB conditional mutant generated in this study (Fig. 3
, and our unpublished result). These results strongly suggest that chsB is necessary not only for hyphal growth but also for conidiation. The lethality of the chsB disruptant reported by Yanai et al. (1994)
might be caused not only by the disruption of chsB, but also by the production of the C-terminally truncated ChsB, which could be expressed from the disrupted chsB locus. As shown in Fig. 4
and will also be mentioned below, the repression of alcA(p)::chsB is imperfect in nature, and the viability of the conditional chsB mutant BM-3/A1 may depend on this imperfection. While Borgia et al. (1996)
reported that the chitin content of their chsB disruptant was almost the same as that of the parental strain, the chitin contents of the mutant BM-3/A1 in YGU and in YGU containing 0·6 M KCl were both approximately 70% of those of the wild-type strain (Table 5
). It is possible that these differences were simply derived from the differences in the genetic backgrounds of the strains employed.
The results in Table 4 clearly show the defect of chsB mutants in conidiation, though the reduction in conidiation efficiency seems rather moderate. When we use repressible promoters for the analysis of the functions of gene products, we should pay attention to two points. First, when the repression of the transcription of the gene is leaky, effects milder than gene disruptions or deletions would be observed under the repressing condition. It has been reported that the expression from the alcA promoter is not completely repressed in minimal medium (Som & Kolaparthi, 1994
; McGoldrick et al., 1995
). The results in Fig. 4
show that the chsB transcripts are actually present even at 25 h after the shift from MMFT to MM plate. Second, when transcripts and/or products of the gene are stable after the shift to the repressive condition, the effects of transcriptional repression would be observed only after prolonged cultivation. As shown in Fig. 4
(5 h lane), this was also the case. Taking these precautions into account, it is possible that the contribution of ChsB to conidiation was underestimated in the carbon source exchange experiment (Table 4
).
The defects in the hyphal growth of BM-3/A1 after the shift to a chsB-repressing MM plate might have resulted in a reduction of the amount of conidiation-competent hyphae. However, in combination with the chsB expression in conidiophores and conidia (Fig. 5), we take our result to suggest that the function of ChsB is not limited to the generation of conidiation-competent hyphae but is directly involved in conidiation.
We noticed that the colony diameter of BM-3/A1 on MMFT plates is smaller than that of the wild-type strain. This finding might be caused by the dominant negative effect of the chsB overexpression. However, the chsB chsD double mutant DB-13 showed almost the same colony diameter as the wild-type strain. Therefore, it is likely that the chsB overexpression per se does not cause growth inhibition, but the simultaneous expression of chsD disturbs the fully wild-type growth.
A reduction in the growth rate of DB-13 compared to that of BM-3/A1 was observed on MM plates and YGU plates containing 0·6 M KCl, but occurred only slightly on YGU plates (Table 3). This result indicates that, when chsB expression is repressed, the importance of the ChsD function in hyphal growth increases under conditions of higher osmolarity. ChsD seems to be dispensable for hyphal growth under conditions of low osmolarity in the absence of chsB expression. It is possible that ChsD either does not function or its loss can be compensated for by other chitin synthases when the osmolarity of the culture media is low. However, the hyphal morphology of DB-13 was more disorganized than that of BM-3/A1 on YGU plates (Fig. 3g
, h
), suggesting that ChsD functions and contributes to hyphal morphology even under low-osmolarity conditions. To measure the growth rates on solid media, we inoculated many conidia at one point on a plate. It is possible that, as the colony diameter and hyphal density increase and glucose is consumed, leaky expression of chsB occurs around the centre of a colony. Thus, it is likely that we have underestimated the effect of the chsD deletion on the growth rate on YGU. Supplementation of 0·6 M KCl did not greatly affect the ratio of the chitin content between DB-13 and BM-3/A1 (Table 5
), suggesting that chitin content does not necessarily correlate with the degree of physiological disorder.
The conidiation efficiency of DB-13 was approximately 60% that of BM-3/A1 in the carbon source exchange experiment (Table 4). In this experiment, we used as media MMFT and MM, which have higher osmolarity than YGU. Since the deletion of chsD affected hyphal growth under these conditions (Table 3
), the defect in conidiation may have been derived from some defects in hyphal growth. Furthermore, since we have obtained evidence that ChsD is present in conidiophore structures of the wild-type strain (Fig. 5
), we propose that ChsD may also be directly involved in conidiation.
Our analyses of DB-13 revealed the importance of the chsD function under chsB-repressing conditions. Beth Din et al. (1996) suggested that Chs4, the class IV chitin synthase of N. crassa, may serve as an auxiliary enzyme that supplements chitin synthesis when additional chitin is necessary. ChsD, also a class IV chitin synthase, may contribute to chitin synthesis in response to various conditions, including the chsB-repressing condition. Since the chsD deletion in the chsA mutants reduced the conidiation efficiency of the mutants (Motoyama et al., 1996
; Culp et al., 2000
), it is possible that the chsA deletion may also be the condition that increases ChsD activity. Alternatively, ChsD may have some redundant functions with ChsA and ChsB.
The phenotypes of the conditional chsB mutants have some similarity to the phenotypes of the null mutants of the class III chitin synthase genes of other filamentous fungi, namely chs-1 of N. crassa and chsG of A. fumigatus. Further analysis of the chitin synthase functions using the conditional mutation in combination with mutations of other related genes will help to elucidate their coordination for fungal morphogenesis and differentiation, and to develop anti-fungal reagents effective against filamentous fungi.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aguirre, J., Adams, T. H. & Timberlake, W. E. (1990). Spacial control of developmental regulatory genes in Aspergillus nidulans. Exp Mycol 14, 290-293.
Aufauvre-Brown, A., Mellado, E., Gow, N. A. R. & Holden, D. W. (1997). Aspergillus fumigatus chsE: a gene related to CHS3 of Saccharomyces cerevisiae and important for hyphal growth and conidiophore development but not pathogenicity. Fungal Genet Biol 21, 141-152.[Medline]
Ballance, D. J. & Turner, G. (1985). Development of a high-frequency transforming vector for Aspergillus nidulans. Gene 36, 321-331.[Medline]
Beth Din, A. & Yarden, O. (1994). The Neurospora crassa chs-2 gene encodes a non-essential chitin synthase. Microbiology 140, 2189-2197.[Abstract]
Beth Din, A. & Yarden, O. (2000). The Neurospora crassa chs3 gene encodes an essential class I chitin synthase. Mycologia 92, 65-73.
Beth Din, A., Specht, C. A., Robbins, P. W. & Yarden, O. (1996). chs-4, a class IV chitin synthase gene from Neurospora crassa. Mol Gen Genet 250, 214-222.[Medline]
Borgia, P. T., Iartchouk, N., Riggle, P. J., Winter, K. R., Koltin, Y. & Bulawa, C. E. (1996). The chsB gene of Aspergillus nidulans is necessary for normal hyphal growth and development. Fungal Genet Biol 20, 193-203.[Medline]
Bowen, A. R., Chen-Wu, J. L., Momany, M., Young, R., Szaniszlo, P. J. & Robbins, P. W. (1992). Classification of fungal chitin synthases. Proc Natl Acad Sci USA 89, 519-523.[Abstract]
Casadaban, M. J., Martinez-Arias, A., Shapira, S. K. & Chou, J. (1983). Beta-galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast. Methods Enzymol 100, 293-308.[Medline]
Chen-Wu, J., Zwicher, J., Bowen, A. R. & Robbins, P. W. (1992). Expression of chitin synthase genes during yeast and hyphal growth phases of Candida albicans. Mol Microbiol 6, 497-502.[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, 7685-7694.[Abstract]
Culp, D. W., Dodge, C. L., Miao, Y., Li, L., Sag-Ozkal, D. & Borgia, P. T. (2000). The chsA gene from Aspergillus nidulans is necessary for maximal conidiation. FEMS Microbiol Lett 182, 349-353.[Medline]
Fujiwara, M., Horiuchi, H., Ohta, A. & Takagi, M. (1997). A novel fungal gene encoding chitin synthase with a myosin motor-like domain. Biochem Biophys Res Commun 236, 75-78.[Medline]
Fujiwara, M., Ichinomiya, M., Motoyama, T., Horiuchi, H., Ohta, A. & Takagi, M. (2000). Evidence that the Aspergillus nidulans class I and class II chitin synthase genes, chsC and chsA, share critical roles in hyphal wall integrity and conidiophore development. J Biochem 127, 359-366.[Abstract]
Gold, S. E. & Kronstad, J. W. (1994). Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis. Mol Microbiol 11, 897-902.[Medline]
Horiuchi, H., Fujiwara, M., Yamashita, S., Ohta, A. & Takagi, M. (1999). Proliferation of intrahyphal hyphae caused by disruption of csmA which encodes a class V-chitin synthase with a myosin motor-like domain in Aspergillus nidulans. J Bacteriol 181, 3721-3729.
Kulmburg, P., Judewicz, N., Mathieu, M., Lenouvel, F., Sequeval, D. & Felenbok, B. (1992). Specific binding sites for the activator protein, ALCR, in the alcA promoter of the ethanol regulon of Aspergillus nidulans. J Biol Chem 267, 21146-21153.
May, G. (1992). Fungal technology. In Applied Molecular Genetics of Filamentous Fungi, pp. 127. Edited by J. R. Kinghorn & G. Turner. London: Chapman & Hall.
McGoldrick, C. A., Gruver, C. & May, G. S. (1995). myoA of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth. J Cell Biol 128, 577-587.[Abstract]
Mellado, E., Aufauvre-Brown, A., Gow, N. A. R. & Holden, D. W. (1996). The Aspergillus fumigatus chsC and chsG genes encode Class III chitin synthases with different functions. Mol Microbiol 20, 667-679.[Medline]
Miller, K. Y., Toennis, T. M., Adams, T. H. & Miller, B. L. (1991). Isolation and transcriptional characterization of a morphological modifier: the Aspergillus nidulans Stunted (stuA) gene. Mol Gen Genet 227, 285-292.[Medline]
Motoyama, T., Kojima, N., Horiuchi, H., Ohta, A. & Takagi, M. (1994a). Isolation of a chitin synthase gene (chsC) of Aspergillus nidulans. Biosci Biotechnol Biochem 58, 2254-2257.[Medline]
Motoyama, T., Sudoh, M., Horiuchi, H., Ohta, A. & Takagi, M. (1994b). Isolation and characterization of two chitin synthase genes of Rhizopus oligosporus. Biosci Biotechnol Biochem 58, 1685-1693.[Medline]
Motoyama, T., Fujiwara, M., Kojima, N., Horiuchi, H., Ohta, A. & Takagi, M. (1996). The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation. Mol Gen Genet 251, 442450. (Corrigendum in Mol Gen Genet 253, 520528.).
Munro, C. A., Schofield, D. A., Gooday, G. W. & Gow, N. A. R. (1998). Regulation of chitin synthesis during dimorphic growth of Candida albicans. Microbiology 144, 391-401.[Abstract]
Munro, C. A., Winter, K., Buchan, A., Henry, K., Becker, J. M., Brown, A. J., 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, 1414-1426.[Medline]
Oakley, C. E., Weil, C. F., Kretz, P. L. & Oakley, B. R. (1987). Cloning of the riboB locus of Aspergillus nidulans. Gene 53, 293-298.[Medline]
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]
Rowlands, R. T. & Turner, G. (1973). Nuclear and extranuclear inheritance of oligomycin resistance in Aspergillus nidulans. Mol Gen Genet 126, 201-216.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shaw, J. A., 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, 111-123.[Abstract]
Som, T. & Kolaparthi, V. S. R. (1994). Developmental decisions in Aspergillus nidulans are modulated by Ras activity. Mol Cell Biol 14, 5333-5348.[Abstract]
Specht, C. A., Liu, Y., Robbins, P. W. & 8 other authors (1996). The chsD and chsE genes of Aspergillus nidulans and their roles in chitin synthesis. Fungal Genet Biol 20, 153167.[Medline]
Sudoh, M., Nagahashi, S., Doi, M., Ohta, A., Takagi, M. & Arisawa, M. (1993). Cloning of the chitin synthase 3 gene from Candida albicans and its expression during yeasthyphal transition. Mol Gen Genet 241, 351-358.[Medline]
Sudoh, M., Yamazaki, T., Masubuchi, K., Taniguchi, M., Shimma, N., Arisawa, M. & Yamada-Okabe, H. (2000). Identification of a novel inhibitor specific to the fungal chitin synthase. Inhibition of chitin synthase 1 arrests the cell growth, but inhibition of chitin synthase 1 and 2 is lethal in the pathogenic fungus Candida albicans. J Biol Chem 275, 32901-32905.
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, 101-109.[Abstract]
Wang, Z. & Szaniszlo, P. J. (2000). WdCHS3, a gene that encodes a class III chitin synthase in Wangiella (Exophiala) dermatitidis, is expressed differentially under stress conditions. J Bacteriol 182, 874-881.
Xoconostle-Cázares, B., León-Ramirez, C. & Ruiz-Herrera, J. (1996). Two chitin synthase genes from Ustilago maydis. Microbiology 142, 377-387.[Abstract]
Xoconostle-Cázares, B., Specht, C. A., Robbins, P. W., Liu, Y., León, C. & Ruiz-Herrera, J. (1997). Umchs5, a gene coding for a class IV chitin synthase in Ustilago maydis. Fungal Genet Biol 22, 199-208.[Medline]
Yanai, K., Kojima, N., Takaya, N., Horiuchi, H., Ohta, A. & Takagi, M. (1994). Isolation and characterization of two chitin synthase genes from Aspergillus nidulans. Biosci Biotechnol Biochem 58, 1828-1835.[Medline]
Yarden, O. & Yanofsky, C. (1991). Chitin synthase 1 plays a major role in cell wall biogenesis in Neurospora crassa. Genes Dev 5, 2420-2430.[Abstract]
Received 8 October 2001;
revised 7 January 2002;
accepted 10 January 2002.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |