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  • 1
    ISSN: 1432-072X
    Keywords: Multidrug resistance ; Mating ; P-glycoprotein ; Fission yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The pmd1 +, a multidrug resistance gene of the fission yeast Schizosaccharomyces pombe, encodes a protein similar to the budding yeast Saccharomyces cerevisiae STE6 gene product and mammalian P-glycoproteins. The STE6 protein is a membrane transporter of a-factor, a mating pheromone of a-type S. cerevisiae, which is structurally related to M-factor of the fission yeast. However, heterothallic or homothallic pmd1 null mutant cells of S. pombe, which were constructed by means of gene disruption, showed no significant decrease in the mating abilities. On the other hand, the multidrug resistance conferred by the pmd1 + was overcome by the treatment with verapamil, a typical inhibitor of mammalian P-glycoproteins. These results indicate that the pmd1 + gene product is functionally similar to mammalian P-glycoproteins, rather than to the budding yeast STE6.
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  • 2
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Symbiobacterium thermophilum is an obligately symbiotic thermophile that can grow only in coculture with a specific Bacillus strain. The amino acid sequences of fragments obtained by cyanogen bromide decomposition of the thermostable β-tyrosinase (tyrosine phenol-lyase, E.C. 4.1.99.2) from this organism resembled that of the tryptophanase produced by the same organism. DNA-probing with the tryptophanase gene as the hybridization probe led to cloning in Escherichia coli of the β-tyrosinase (tpl) gene. The nucleotide sequence revealed that the β-tyrosinase of 458 amino acids (relative molecular mass, 52269) showed significant similarity in amino acid sequence to the tryptophanase over the entire sequence. DNA manipulation of the cloned tpl gene in E. coli led to production of 375 times as much β-tyrosinase as that produced by the original S. thermophilum strain.
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  • 3
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Membrane-bound aldehyde dehydrogenase (ALDH) was purified from the membrane fraction of an industrial-vinegar-producing strain, Acetobacter polyoxogenes sp. nov. NBI1028 by solubilization with Triton X-100 and sodium N-lauroyl sarcosinate and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneos on polyacrylamide disc gel electrophoresis. Upon sodium dodecyl sulphate-polyacrylamide gelelectrophoresis, the enzyme showed the presence of two subunits with a molecular mass of 75 000 daltons and 19 000 daltons, respectively. From the absorption and fluorescence spectra, the absence of cytochrome c and the presence of pyrroloquinoline quinone in the purified enzyme were demonstrated. The ALDH preferentially oxidized aliphatic aldehyde with a straight carbon chain except for formaldehyde. The apparent K m for acetaldehyde was 12 mM. The optimum pH and temperature were 7.0 and 50°–60°C, respectively. The enzyme remained active after storage at 4°C for 20 days. p-Chloromercuribenzoic acid and heavy metal salts such as CuSO4 were inhibitory to the enzyme. Ferricyanide was effective as an electron acceptor.
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  • 4
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Membrane-bound alcohol dehydrogenase (ADH) was purified from the membrane fraction of an industrial-vinegar-producing strain, A. polyoxogenes sp. nov. NBI1028 by solubilization using Triton X-100 and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneous on polyacrylamide disc gel electrophoresis (PAGE). Upon sodium dodecyl sulphate-PAGE, the enzyme showed the presence of two subunits with a molecular mass of 72 000 daltons and 44 000 daltons, respectively. The small subunit was identified as cytochrome c. In addition, absorption and fluorescence spectra showed the the presence of pyrroloquinoline quinone in the purified ADH. The ADH preferentially oxidized aliphatic alcohols with a straight carbon chain except for methanol. Formaldehyde and acetaldehyde were also oxidizable substrates. The apparent K m for ethanol was 1.2 mM. The optimum pH and temperature were 5.0–6.0 and 40°C, respectively. p-Chloromercuribenzoic acid and heavy metals such as CuSO4 were inhibitory to the enzyme activity. Ferricyanide was effective as an electron acceptor.
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  • 5
    ISSN: 1432-041X
    Keywords: Trichostatin A ; n-Butyric acid ; Histone acetylation ; Embryos ; Echinodermata
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary External application of 10 rig/ml (R)-trichostatin A (TSA), a potent and specific inhibitor of mammalian histone deacetylase, to the embryo of the starfish Asterina pectinifera inhibited development during the early gastrula stage before formation of mesenchyme cells. The TSA-sensitive period was limited to the mid-blastula stage before hatching. The pulse-chase experiment clearly demonstrated that TSA induced an accumulation of acetylated histone species in blastulae through inhibition of historic deacetylation. Similar blockage of development at the early gastrula stage was observed with n-butyrate, which has been known as a weak inhibitor of historic deacetylase. These results suggest an intimate role for historic acetylation-deacetylation equilibria in starfish development.
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  • 6
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Host specific restriction was detected in 13 Bacillus strains, when 63 strains of Bacillus subtilis and 15 other Bacillus strains were tested with phage Φ105C. These 13 strains were classified into 8 groups (M,H,C,N,E,F,G,P) by the type of restriction. M-type strains (B. subtilis Marburg 168, its derivatives, and two other strains) showed relatively weak restriction, restricting Φ105C from other groups of Bacillus by ratios of 10-1 to 10-3. Strains of groups H,C,N,E,F,G, and P restricted Φ105C from other groups by ratios of 10-2 to 10-8. It was confirmed with some of the strains that type-specific modification was endowed only by the last host. Furthermore, we isolated one restriction deficient mutant of B. subtilis marburg 168-YS11, which had also lost its modification phenotype.
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  • 7
    ISSN: 1617-4623
    Keywords: Nitrogen fixation ; nifL ; nifA ; Klebsiella oxytoca ; DNA sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The complete nucleotide sequence of the regulatory operon nifLA of a nitrogen fixer Klebsiella oxytoca NG13 was determined, and the transcriptional start point was assigned by S1 mapping. The nifL protein (a repressor) was coded by an open reading frame of 1,485 bases, corresponding to a protein of 495 amino acids with a calculated molecular weight of 55,242. The open reading frame (1,572 bases) of the nifA protein (an activator), corresponding to a molecular weight of 58,649, was confirmed by in vitro transcription-translation experiments, using the wild type and artificially deleted nifA genes. The initiation codon (ATG) of nifA overlapped the termination condon(TGA) of nifL, sharing the two bases T and G. A conserved DNA contact point [Gln-(X)3-Ala-(X)3-Gly-(X)5-Val] common in many DNA binding proteins was found in the C-terminal region of the nifA sequence. The promoter sequences of nifLA, nifB and nifF in K. oxytoca coincided exactly with those of K. pneumoniae in the consensus regions at-12 and-26, although the overall homology in the promoter regions was 96%. Changes of four amino acids were found between the nifA coding sequences of K. oxytoca and K. pneumoniae.
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  • 8
    ISSN: 1617-4623
    Keywords: Streptomyces ; Secondary metabolism ; Transcriptional stimulation ; afsB ; Pleiotropic regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The pleiotropic regulatory gene, afsB, from Streptomyces coelicolor A3(2), possibly encoding a DNA-binding protein, is required for actinorhodin production in this organism. Northern blot hybridization using a DNA fragment covering part of the set of cloned actinorhodin biosynthetic gene cluster (act) as the probe showed lack of the act transcripts in an afsB-negative mutant of S. coelicolor A3(2); the transcripts were restored on introduction of a cloned afsB gene. Introduction of the cloned afsB gene into Streptomyces lividans stimulated transcription of the act genes under conditions in which they are normally silent in this strain, leading to production of actinorhodin in large quantity. These data show that afsB exerts its positive regulatory effect by means of transcriptional stimulation of its target genes.
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  • 9
    ISSN: 1617-4623
    Keywords: Streptomyces griseus ; Protein phosphorylation ; Protein kinase inhibitors ; Secondary metabolism ; Cellular differentiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and γ-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.
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  • 10
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A composite plasmid (pAT2010) has been constructed in vitro from RSF2124 and Bacillus subtilis IFO3022 plasmid (pAT1060) by covalent joining of the two DNA molecules by means of Escherichia coli DNA ligase through the cohesive ends generated by restriction endonuclease RI (EcoRI) cleavage. The composite plasmid was selected by transformation of E. coli C600 r −m− with the ligated mixture affer enrichment for composite plasmid by preparative agarose gel electrophoresis, and plating of the transformants on a medium containing ampicillin and colicin El. Treatment of the composite plasmid with EcoRI yielded two fragments corresponding to the linear forms of the parental plasmids. The composite plasmids replicated as biologically functionally units in E. coli, and expressed genetic information carried by RSF2124. In the presence of chloramphenicol, the composite plasmids continued to replicate and the copy number gradually increased. Such nature of replication in the presence of chloramphenicol is characteristic to RSF2124 derived from colicin El factor, and so it is suggested that the replicator of RSF2124 is functional in the composite plasmid. The composite plasmid was found to synthesize mRNA of B. subtilis plasmid in cell-free extracts of E. coli, by hybridization of the mRNA to the original plasmid DNA of pAT1060.
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