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  • 1
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary A cyclcodextrin glucanotransferase (CGTase) gene of Bacillus ohbensis was cloned in Escherichia coli and the nucleotide sequence was determined. A single open reading frame (2112 bp) with a TTG codon as an initiator was identified that encodes a typical signal peptide of 29 amino acids followed by the mature enzyme (675 amino acids), of which the partial amino acid sequences of the N-terminal region and some lysyl-endopeptidase fragments were determined by Edman degradation. The CGTase gene was expressed in E. coli under control of the lac promoter only when the upstream region containing a long inverted repeat structure (located at −108 to −67 bp from the initiation codon) was deleted. Substitution of an ATG codon for the initiation TTG triplet doubled the expression of the CGTase gene in E. coli. Enzyme preparations purified from the culture supernatant of B. ohbensis and from the periplasmic fraction of the E. coli transformant exhibited the same molecular weight (M r) and enzymatic properties as follows: M r, 80 000; optimum pH for activity, 5.0 (and a suboptimum at 10.0); stability between pH 6.5 and 10.0; optimum temperature for activity, 55°C; and stability below 45°C. The yields of the products from starch as the substrate were 25% for β-and 5% for γ-cyclodextrin.
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  • 2
    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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  • 3
    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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  • 4
    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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  • 5
    ISSN: 1617-4623
    Keywords: Microbial protease ; Proenzyme ; Secretion ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH2-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH2-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.
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  • 6
    ISSN: 1617-4623
    Keywords: C-P bond forming enzyme ; Carboxyphosphonoenolpyruvate phosphomutase ; Bialaphos ; Streptomyces hygroscopicus ; Gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The carboxyphosphonoenolpyruvate (CPEP) phosphonomutase gene of bialaphos-producing Streptomyces hygroscopicus, which encodes a C-P bond forming enzyme was cloned into Streptomyces lividans and sequenced. The amino acid composition of the protein coded in an open reading frame of 295 codons and its calculated molecular mass, 32,800 Da, coincided well with those of the purified enzyme. Introduction of the CPEP phosphonomutase gene, the expression of which is controlled by the promoter of the aph gene, into S. lividans resulted in the production of this enzyme at a level almost equivalent to that in the parent strain.
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  • 7
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Group
    Nature 286 (1980), S. 754-754 
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] SIR-Dr Zeki's red filter described in your article (Nature, 31 July, p.435) is really excellent for use in photographing fluorescent UNA bands on agarose gels. One of the most important factors for a contrast filter is the absence of fluorescence of the filter itself. Conventional red filters ...
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  • 8
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract A 1.5-kb XbaI-SacII fragment containing the upstream region of the Trichoderma reesei cellobiohydrolase I gene (cbh1) has been sequenced. The 1.5-kb fragment contains eight 6-bp sites having an identical or similar sequence to the consensus sequence for binding a catabolite repressor, Aspergillus nidulans CreA. Results of binding assays with the maltose-binding protein: :Cre1(10–131) fusion protein (Cre1 is a catabolite repressor of T. reesei) and the cbhI upstream region revealed that a 504-bp XbaI-NspV fragment (nucleotide position − 1496 to − 993) bearing three 6-bp sites, Al, A2, and A3, and a 356-bp NspV-MunI fragment (nucleotide position −994 to −639) bearing three 6-bp sites, B1, B2, and B3, were shifted in the electrophoretic mobility shift assay. DNase I footprinting experiments showed that the 6-bp sites A2, B1, B2, and B3 were protected from DNase I digestion.
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  • 9
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The production of cyclodextrin glucanotransferase (CGTase) by Bacillus ohbensis is dependent on the presence of starch and inhibited by glucose in the medium. Northern blot analysis revealed that the CGTase gene (cgt) was transcribed to almost the same level irrespective of the presence or absence of starch, but glucose completely repressed the transcription. Furthermore, a relatively high amount of CGTase protein was detected on Western blotting only in the medium with starch, showing the lack of posttranslational control of the CGTase activity. These findings suggest some starch induction mechanism for the cgt gene, possibly at the posttranscriptional level, besides negative transcriptional regulation by glucose.
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  • 10
    ISSN: 1432-203X
    Keywords: Key words: Ion beam irradiation – Somatic embryogenesis – Somatic hybridization –Hibiscus rosa-sinensis–Lavatera thuringiaca
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary. The biological effects of irradiation with 12C+5 ion beam on plant cells have been analyzed. Protoplasts and cell suspensions of Lavatera thuringiaca, and a somatic hybrid callus (Hibiscus rosa-sinensis+Lavatera thuringiaca), were irradiated with doses from 0.05 to 50 Gy, and the effects on cell growth, cell division, cell viability and embryogenesis rates were analyzed. Irradiation with 12C+5 ion beam at relatively very low doses (5.0 Gy) significantly inhibited cell division, yet the survival rate and regeneration capability of the cells through somatic embryogenesis were conserved in more than 70 and 50%, respectively. These results indicate that cell division is the most sensitive parameter to irradiation, accounting for the inhibition of colony formation and callus growth. The potential use of the 12C+5 ion beam in asymmetric protoplast fusion experiments is discussed.
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