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  • 11
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 5 (1990), S. 295-301 
    ISSN: 1476-5535
    Keywords: Alpha-amylase assay ; Turbidity ; Kinetic determination
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary A simple, reliable and sensitive assay for alpha-amylase activity is reported, together with its theoretical derivation, that overcomes many of the problems encountered with other assays, especially when attempting to assay alpha-amylase activity in crude cell extracts or culture supernatants. The method relies on the reduction in turbidity that occurs upon digestion of a starch suspension with alpha-amylase. The initial rate of decrease in turbidity is shown to be proportional to a wide range of enzyme concentrations, permitting a rapid spectrophotometric and kinetic determination of alpha-amylase activity.
    Type of Medium: Electronic Resource
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  • 12
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary When strains of Streptomyces coelicolor A3(2) lacking the previously identified autonomous plasmids SCP1 and SCP2 are crossed with Streptomyces lividans 66, some of the S. lividans progeny are able to elicit zones of growth inhibition (lethal zygosis), previously associated with the transfer of conjugative Streptomyces plasmids, when grown in contact with S. lividans 66. Some such progeny yield covalently closed circular (CCC) plasmid DNA, the size and restriction endonuclease cleavage pattern of which is constant for a particular isolate, but varies among isolates. These plasmid, which have been named SLP1.1, SLP1.2, ect., all confer resistance to lethal zygosis elicited by the others. Genetic and molecular characterization of the plasmids reveals that they are derived from the strA region of the chromosome of S. coelicolor. It is proposed that, before or during mating with S. lividans, the SLP1 sequences are excised from the chromosome, bringing varying regions of the surrounding chromosome with them, and can circularise to yield the SLP1 family of plasmids. Autonomous SLP1 plasmids can also be generated by cleaving total DNA of S. coelicolor with certain restriction enzymes, ligating it, and transforming the DNA into S. lividans. The autonomous SLP1 plasmids exist within S. lividans in a few copies per chromosome, and act as fertility factors. They provide suitable vectors for DNA cloning since the segments of chromosomal DNA carried by the larger members of the family are dispensable.
    Type of Medium: Electronic Resource
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 154 (1977), S. 155-166 
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Covalently closed circular (ccc) DNA of uniform monomer size (c. 18×106 daltons) and restriction endonuclease cleavage pattern was isolated from strains of S. coelicolor A3(2) of differing constitution in respect of the SCP1 sex factor: SCP1+, SCP1′, SCP1- and NF (integrated SCP1). No such ccc DNA was found in strains of S. lividans 66 or S. parvulus ATCC 12434 whether or not they contained SCP1. These results confirmed that the 18×106 dalton plasmid is not, and does not include, SCP1, which has not so far been isolated by any of a variety of methods. Genetic data served to identify a second sex factor, SCP2, postulated to be present in SCP2+ state in the starting strains and to be capable of mutation to a variant form, SCP2*, with enhanced sex factor activity. From SCP2* strains, SCP2- cultures were isolated, at an average spontaneous frequency of about 0.8%. Crosses of pairs of SCP1- SCP2- strains were almost, but not completely, sterile; thus SCP1 and SCP2 probably contribute nearly all the fertility naturally occurring in the A3(2) strain. The two sex factors share the property of exerting an effect that may be comparable with “lethal zygosis” caused by F in E. coli: it is shown by SCP1-carrying strains against SCP1-, or SCP2* (but not SCP2+) strains against SCP2- and is revealed as a narrow zone of growth inhibition surrounding the plasmid-carrying culture on a background of the appropriate plasmid-negative strain. Genetically defined SCP2- strains lacked the ccc DNA found in SCP2+ and SCP2* strains. Thus this DNA apparently represents the SCP2 sex factor. A preliminary restriction endonuclease cleavage map of SCP2 was constructed, with single sites for EcoRI and HindIII, four sites for SalPI (=PstI) and more than 20 sites for SalGI (SalI).
    Type of Medium: Electronic Resource
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  • 14
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 187 (1982), S. 265-277 
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Promoter-probe plasmid vectors were constructed for Streptomyces lividans using expression of the Escherichia coli chloramphenicol acetyltransferase gene as an indicator of promoter activity. These vectors have been used to isolate and to study the activity of DNA sequences that contain transcriptional control signals from Streptomyces, Bacillus licheniformis, E. coli, and Serratia marcescens. Studies of these promoter regions in heterospecific hosts indicate that genus or species-specific factors may present barriers to the expression of bacterial genetic material in certain heterologous cellular environments. While promoter regions isolated from E. coli, S. marcescens and B. licheniformis all appear to be recognized by the RNA polymerase of S. lividans, the Streptomyces transcriptional control signals isolated do not appear to function normally in E. coli.
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  • 15
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 213 (1988), S. 269-277 
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Helix-turn-helix motifs ; In vitro transcription-translation ; Phage immunity ; Exonuclease III deletions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The nucleotide sequence of the 3.4 kb SphI-G fragment that contained the repressor gene (c) of the temperate Streptomyces phage ϕc31 was determined. Analysis of this sequence revealed a large open reading frame with protein coding character and sequence changes in c gene point and deletion mutants identified this as the coding region of the repressor. Two of the mutants studied had undergone deletions of 1.1 kb and 1.4 kb that had occurred across short direct repeats of 6 bp and 11 bp, respectively. Coupled in vitro transcription-translation experiments using the cloned SphI-G fragment and Streptomyces lividans cell free extracts identified a protein product of approximately 72 kDa, in close agreement with that predicted from the nucleotide sequence. A strongly predicted helix-turn-helix motif that may be involved in DNA binding occurred towards the carboxy-terminus of the amino acid sequence. Initial attempts to clone the SphI-G fragment in Streptomyces failed; using information gained from the sequence analysis a smaller segment of this DNA fragment was cloned in S. lividans and conferred immunity to a clear plaque mutant (c1) of ϕc31.
    Type of Medium: Electronic Resource
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  • 16
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 211 (1988), S. 191-196 
    ISSN: 1617-4623
    Keywords: Streptomyces coelicolor ; Ribosomal RNA genes ; Streptomyces lividans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Using Southern hybridisation of radiolabelled purified ribosomal RNAs to genomic DNA the ribosomal RNA genes of Streptomyces coelicolor A3(2) were shown to be present in six gene sets. Each gene set contains at least one copy of the 5 S, 16 S and 23 S sequences and in at least two cases these are arranged in the order 16 S-23S-5S. Three gene sets, rrnB, rrnD and rrnF, were isolated by screening a λ library of S. coelicolor A3(2) DNA. The restriction map of one of these, rrnD, was determined and the nucleotide sequences corresponding to the three rRNAs were localised by Southern hybridisation. The gene order in rrnD is 16S-23S-5S.
    Type of Medium: Electronic Resource
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  • 17
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 209 (1987), S. 101-109 
    ISSN: 1617-4623
    Keywords: Promoters ; S1 mapping ; In vitro transcription ; Protein secretion ; Signal peptide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The DNA sequence of a 1.77 kb region of the Streptomyces coelicolor chromosome containing the coding and regulatory regions of the extracellular agarase (dagA) gene was determined. The sequence predicts a primary translation product of 309 amino acids and Mr 35132. Comparison of the N-terminal sequence determined for the mature extracellular protein with that of the primary translation product deduced from the DNA sequence predicts the presence of a 30 amino acid signal peptide. Analysis of the transcription of the dagA gene using high resolution S1 mapping, in vitro transcription, dinucleotide-primed in vitro transcription and in vivo promoter probing identified four promoters, initiating transcription approximately 32, 77, 125 and 220 nucleotides upstream of the coding sequence.
    Type of Medium: Electronic Resource
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  • 18
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 244 (1994), S. 135-143 
    ISSN: 1617-4623
    Keywords: Streptomyces coelicolor A3(2) ; Glucose kinase ; Glucose repression ; Agarase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The glucose kinase gene (glkA-ORF3) of Streptomyces coelicolor A3(2) plays an essential role in glucose utilisation and in glucose repression of a variety of genes involved in the utilisation of alternative carbon sources. These genes include dagA, which encodes an extracellular agarase that permits agar utilisation. Suppressor mutants of glkA-ORF3 deletion strains capable of utilising glucose (Glc+) arise at a frequency of about 10−5 on prolonged incubation. The Glc+ phenotype of the mutants is reversible (at a frequency of about 10−3) and reflects either the activation of a normally silent glucose kinase gene or the modification of an existing sugar kinase. Although the level of glucose kinase activity in the Glc+ supressor mutants is similar to that in the glkA + parental strain, glucose repression of dagA remains defective. Expression of the glucose kinase gene of Zymomonas mobilis in glkA-ORF3 mutants restored glucose utilisation, but not glucose repression of dagA. Over-expression of glkA-ORF3 on a high-copy-number plasmid failed to restore glucose repression of dagA in glkA-ORF3 mutants and led to loss of glucose repression of dagA in a glkA + strain. These results suggest that glucose phosphorylation itself is not sufficient for glucose repression and that glkA-ORF3 plays a specific regulatory role in triggering glucose repression in S. coelicolor A3(2).
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  • 19
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Promoter-probe plasmid vectors were used to isolate putative promoter-containing DNA fragments of three Streptomyces antibiotic resistance genes, the rRNA methylase (tsr) gene of S. azureus, the aminoglycoside phosphotransferase (aph) gene of S. fradiae, and the viomycin phosphotransferase (vph) gene of S. vinaceus. DNA sequence analysis was carried out for all three of the fragments and for the protein-coding regions of the tsr and vph genes. No sequences resembling typical E. coli promoters or Bacillus vegetatively-expressed promoters were identified. Furthermore, none of the three DNA fragments found to be transcriptionally active in Streptomyces could initiate transcription when introduced into E. coli. An extremely biased codon usage pattern that reflects the high G+C composition of Streptomyces DNA was observed for the protein-coding regions of the tsr and vph genes, and of the previously sequenced aph gene. This pattern enabled delineation of the protein-coding region and identification of the coding strand of the genes.
    Type of Medium: Electronic Resource
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  • 20
    ISSN: 1617-4623
    Keywords: Streptomyces ; Promoter-probes ; Transcription ; Gene expression ; fd terminator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans. An efficient transcriptional terminator from E. coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters. A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions. Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised. The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts. Some derivatives contain a poly-linker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors.
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