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  • 1
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: A gene encoding a chitin synthase with a myosin motor-like domain (csm1) was isolated from Pyricularia oryzae using a PCR fragment amplified from a fungal chitin synthase conserved region. The deduced amino acid sequence of csm1 is homologous to that of CsmA of Aspergillus nidulans (65% identity). The putative gene product of csm1 is consisted of the myosin motor-like domain and a chitin synthase domain as in A. nidulans csmA. The chitin synthase domain of its C-terminus was also homologous to Aspergillus fumigatus ChsE (61.4% identity) and Ustilago maydis Chs6 (48.6% identity) that encode class V chitin synthases. Northern analysis demonstrated that the csm1 was expressed throughout the mycelial growth of P. oryzae. This is the first report on the isolation of the gene encoding a class V chitin synthase with the myosin motor-like domain from P. oryzae.
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 169 (1998), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: We found the presence of DNA sequence which shows sequence similarity to the class IV chitin synthase gene (CHS3) of Saccharomyces cerevisiae in the genome of 14 Rhizopus species which belong to zygomycetes. We cloned a gene (chs3), which might correspond to one of these homologous sequences, from Rhizopus oligosporus by low stringency plaque hybridization probed with CHS3. The deduced amino acid sequence of this gene showed highest similarity to the class IV chitin synthase of Neurospora crassa (46.7% identity over 1087 amino acids), showing that this gene encodes a class IV chitin synthase. Northern analysis revealed the differential expression pattern of this gene in the asexual life cycle with highest expression in the early stage of asexual spore formation. This is the first report of the isolation and analysis of a class IV chitin synthase gene from zygomycete fungi.
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  • 3
    ISSN: 1617-4623
    Keywords: Key words Chitin synthase ; Multigene family ; Cell wall ; Conidia ; Aspergillus nidulans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.
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  • 4
    ISSN: 1617-4623
    Keywords: Key words Chitin synthase ; Multigene family ; Cell wall ; Conidia ; Aspergillus nidulans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.
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  • 5
    ISSN: 1617-4623
    Keywords: Centromere ; Dicentric plasmid ; Overexpression ; Candida maltosa
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A centromeric activity was identified in the previously isolated 3.8 kb DNA fragment that carries an autonomously replicating sequence (ARS) from the yeast Candida maltosa. Plasmids bearing duplicated copies of the centromeric DNA (dicentric plasmids) were physically unstable and structural rearrangements of the dicentric plasmids occurred frequently in the transformed cells. The centromeric DNA activity was dissociated from the ARS, which is 0.2 kb in size, and was delimited to a fragment at least 325 by in length. The centromeric DNA region included the consensus sequences of CDEI (centromeric DNA element I) and an AT-rich CDEII-like region of Saccharomyces cerevisiae but had no homology to the functionally critical CDEIII consensus. A plasmid bearing the whole 3.8 kb fragment was present in 1–2 copies per cell and was maintained stably even under non-selective culture conditions, while a plasmid having only the 0.2 kb ARS was unstable and accumulated to high copy numbers. The high-copy-number plasmid allowed us to overexpress a gene to a high level, which had never been attained before, under the control of both constitutive and inducible promoters in C. maltosa.
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  • 6
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; endoplasmic reticulum ; chaperone ; unfolded protein response ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: RNAP-I, an aspartic proteinase from a filamentous fungus Rhizopus niveus, is secreted very efficiently in Saccharomyces cerevisiae. It is synthesized first as a precursor form with signal sequence and prosequence in its amino-terminus. Our previous study indicated that the prosequence of RNAP-I had important roles in its correct folding and secretion in yeast, and that a prosequence-deleted derivative of RNAP-I, Δpro, was not secreted but was retained and degraded in the yeast endoplasmic reticulum (ER). In the present study, we show that the accumulation of Δpro in the yeast ER caused elevated synthesis of ER resident chaperones, indicating that Δpro is recognized as an unfolded protein species in the ER. Our biochemical data demonstrated that Δpro formed aggregates which contained BiP, but not protein disulfide isomerase (PDI), in the ER. Immunoelectron microscopical analysis revealed that the Δpro aggregates were indeed visible as electron-dense regions in the ER and nuclear envelope. Such ‘chaperone-associated misfolded protein bodies’ were observed for the first time in yeast. Morphologies of the ER and nucleus were drastically altered by the accumulation of the Δpro aggregates. The ER lost its flat cisternal shape; the ER lumen extended aberrantly and the ER membrane irregularly proliferated. The misfolded Δpro proteins are probably sorted from the ordinary ER lumen to form the aggregates so that the ER function would not be grossly impaired, and the dilated ER may represent an ER subcompartment where the Δpro aggregates are degraded. © 1997 by John Wiley & Sons, Ltd.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 0749-503X
    Keywords: GAL genes ; expression vector ; cytochrome P-450 ; Candida maltosa ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The GAL1 and GAL10 gene cluster encoding the enzymes of galactose utilization was isolated from an asporogenic yeast, Candida maltosa. The structure of the gene cluster in which both genes were divergently transcribed from the central promoter region resembled those of some other yeasts. The expression of both genes was strongly induced by galactose and repressed by glucose in the medium. Galactose-inducible expression vectors in C. maltosa were constructed on low- and high-copy number plasmids using the promoter regions of both genes. With these vectors and the β-galactosidase gene from Kluyveromyces lactis as a reporter, galactose-inducible expression was confirmed. Homologous overexpression of members of the cytochrome P-450 gene family in C. maltosa was also successful by using a high-copy-number vector under the control of these promoters. © 1997 by John Wiley & Sons, Ltd.
    Additional Material: 5 Ill.
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  • 8
    ISSN: 0749-503X
    Keywords: Yarrowia lipolytica ; cytochrome P450 ; n-alkane metabolism ; n-alkane-inducible gene ; RT-PCR ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B. The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family. The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B. The nucleotide sequence of one of the positive clones was determined. An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified. It was of 523 amino acid residues, 60·2 kDa in molecular mass, and had 30-45% sequence identity with the other members of the CYP52 family of Candida species so far analysed. The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol. A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source. Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation. These results suggest that the YlALK1 gene product is the major P450Alk to metabolize short-chain n-alkanes such as decane and dodecane in Y. lipolytica. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 6 Ill.
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  • 9
    ISSN: 0749-503X
    Keywords: Candida maltosa ; codon usage ; heterologous gene expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa.In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNA SerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation.The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon.The nucleotide sequence datum reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the Accession Number D26074.
    Additional Material: 4 Ill.
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