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  • 1
    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.
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  • 2
    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.
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  • 3
    ISSN: 0749-503X
    Keywords: Chromosome length ; chromosome VI ; electrophoretic karyotype ; RFLP ; Saccharomyces ; saké yeast ; taxonomy ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Previous studies have revealed that chromosome VI of saké yeasts is much larger than that of the other strains of Saccharomyces cerevisiae. Southern analysis using segments of chromosome VI of a laboratory strain as probes suggested that the nucleotide sequence of a major portion of this chromosome is conserved, but considerable diversity was found in the distal parts in the other strains. Physical maps also indicated that differences in length of chromosome VI were mainly due to differences in its ends. NotI was found to generate 9 kb and/or 16 kb fragments from the left telomere of chromosome VI in most saké yeasts, but no fragment in the case of AB972. SfiI produced one or two 30-50 kb fragments from the right end of this chromosome in all saké yeasts tested, but produced a 20 kb fragment in the case of AB972. All S. cerevisiae strains not employed in saké brewing were the same as AB972 in these respects. S. paradoxus had one NotI site in chromosome VI, while S. bayanus had two, one of which is possibly common to both species. The SfiI site mentioned above was present in chromosome VI of all species, while that of S. bayanus and S. paradoxus each had a second site distinct from the other. Chromosome VI of S. pastorianus was not distinguishable from that of S. bayanus. © 1998 John Wiley & Sons, Ltd.
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  • 4
    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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  • 5
    ISSN: 1432-0983
    Keywords: Candida maltosa ; C-HISS ; Host-vector system ; Nucleotide sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The host-vector system of an n-lkaneassimilating-yeast, Candida maltosa, which we previously constructed using an autonomously replicating sequence (ARS) region isolated from the genome of this yeast, utilizes C. maltosa J288 (leu2 −) as a host. As this host had a serious growth defect on n-alkane, we developed an improved host-vector system using C. maltosa CHI (his) as host. The vectors were constructed with the Candida ARS region and a DNA fragment isolated from the genome of C. maltosa. Since this DNA fragment could complement histidine auxotrophy of both C. maltosa CH1 and S. cerevisiae (hiss −), we termed the gene contained in this DNA fragment C-HIS5. The vectors were characterized in terms of transformation frequency and stability, and the nucleotide sequence of C-HISS was determined. The deduced amino acid sequence (389 residues) shared 51% homology with that of HISS of S. cerevisiae (384 residues; Nishiwaki et al. 1987).
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  • 6
    ISSN: 1399-0047
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: Meso-2,3-butanediol dehydrogenase (meso-BDH) has been crystallized and preliminary X-ray crystallographic characterization of meso-BDH crystals has been performed. Single crystals of meso-BDH were prepared in two forms by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. Form I crystals belong to space group C2, with unit-cell parameters a = 215.5, b = 79.4, c = 134.8 Å, β = 98.22°, and form II crystals belong to space group P21, with unit-cell parameters a = 69.16, b = 109.78, c = 127.28 Å, β = 102.29°. The crystals diffracted to 2.0 and 1.7 Å resolutions, respectively, using synchrotron radiation.
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  • 7
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The bacterial community colonizing the gut wall of the termite Reticulitermes speratus was characterized without cultivation. Analysis of 16S rRNA genes after fractionation of the gut revealed that the bacterial composition on the gut wall was diverse and significantly different from that able to move unconfined in the gut fluid or physically associated with the gut protists. Actinobacteria, Firmicutes and Bacteroidetes were dominant on the gut wall, but Spirochaetes and the Termite group 1 phylum, abundant in the gut lumen, were relatively rare. A sequence-specific probe enabled the in situ detection of a rod-shaped Actinobacteria member, abundantly colonizing the gut paunch epithelium.
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  • 8
    ISSN: 1432-0983
    Keywords: Candida maltosa ; PGK ; Expression vector
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A gene encoding phosphoglycerate kinase (PGK) was isolated from the genomic library of C. maltosa to construct an expression vector for this yeast. The PGK gene had an open reading frame of 1251 base pairs encoding approximately 47-kDA polypeptide of 417 amino-acid residues. Expression of this gene assayed by Northern-blot analysis was significantly induced in cells grown on glucose but not in cells grown on n-tetradecane, n-tetradecanol, or oleic acid. By using the promoter region of this gene, an expression vector (termed pMEA1) for C. maltosa was constructed and expression of an endogenous gene (P450alkl encoding one of cytochrome P450s for n-alkane hydroxylation in C. maltosa) and a heterologous gene (LAC4 encoding Kluyveromyces lactis β-galactosidase) was tested. Expression of P450alkl gene was confirmed at both mRNA and protein levels. LAC4 gene expression was confirmed by determining β-galactosidase activity. The activity in cells grown on various carbon sources correlated very well with the expression levels of PGK mRNA in these cells.
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  • 9
    ISSN: 1432-0983
    Keywords: Candida maltosa ; C-URA3 ; Triple auxotroph ; Gene disruption
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The C-URA3 gene of the n-alkane assimilating-yeast Candida maltosa was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae. The nucleotide sequence of C-URA3 and its deduced amino-acid sequence showed significant homology to those of the orotidine 5′-phosphate decarboxylases of other fungal species. To construct a useful host for genetic engineering of C. maltosa using C-URA3 as a marker, one allele of C-URA3 in a double auxotroph (his5, ade1) was disrupted by C-ADE1, and subsequently two kinds of ura3 mutants were isolated by selecting for spontaneous 5-fluoro-orotic acid (5FOA) resistance. One of the mutants was homozygous for the disruption (ura3::C-ADE1/ura3::C-ADE1); the other was heterozygous (ura3::C-ADE1/ura3). The ura3::C-ADE1 allele in the latter strain was re-substituted by C-URA3 to rescue the adenine auxotroph (his5, ade1, C-URA3/ura3). Finally, by selecting a 5FOA-resistant mutant, a triple auxotroph (his5, ade1, ura3/ura3) was isolated.
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  • 10
    ISSN: 1433-4909
    Keywords: Key words Minimum size of anoxic habitat ; Termite hindgut ; Molecular phylogeny
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A termite maintains an anaerobic microbial community in its hindgut, which seems to be the minimum size of an anaerobic habitat. This microbial community consists of bacteria and various anaerobic flagellates, and it is established that termites are totally dependent on the microbes for the utilization of their food. The molecular phylogene-tic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy that does not rely on cultivation of the resident microorganisms. Small subunit ribosomal RNA (ssrRNA) genes were directly amplified from the mixed-population DNA of the termite gut by polymerase chain reaction (PCR) and clonally isolated. Most sequenced clones were phylogenetically affiliated with the four major groups of the domain Bacteria: the Proteobacteria group, the Spirochete group, the Bacteroides group, and the Low G + C gram-positive bacteria. The 16S rRNA sequence data show that the majority of the intestinal microflora of the termite consists of new species that are yet to be cultured. The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite (R. speratus) was analyzed without cultivation. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order. The diversity of nitrogen-fixing organ-isms was also investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the mixed-population DNA of the termite gut and were clonally isolated. The phylogenetic analysis of the nifH amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. The molecular phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of R. speratus was also examined by the same strategy. The whole-cell hybridization experiments indicated that the sequence originated from a large hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that the hypermastigote and trichomonads shared a recent common ancestry.
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