Browsing by Author "Kirby, Ralph"

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    Generic studies on the oxytetracycline producing organism, Streptomyces alboflavus
    (1981) Botha, Christine Leone; Kirby, Ralph
    The Actinomycete organism, Streptomyces alboflavus, produces the antibiotic oxytetracycline. Treatment with curing agents resulted in S. alboflavus losing the ability to produce oxytetracycline. However, the loss of oxytetracyc1ine production was reversible, and non-producing colonies regained the ability to produce oxytetracycline when the curing agent was removed. Therefore the loss of oxytetracyc1ine production was not due to the irreversible loss of genetic material specifying oxytetracycline production, but was possibly due to genetic instability. Two extrachromosomal DNA species were isolated from S. alboflavus, (SAP1 and SAP2). SAP1 was approximately 8 - 10 x 10â ¶ da1tons, and appeared to be linear and heterogenous. SAP2 was 20 - 25 x 10â ¶ daltons and appeared to be covalently closed circular. The functions of SAP1 and SAP2 are unknown, although transformation experiments with SAP1 suggested that it may play a role in the production of an antibiotic-like substance, possibly by acting as a promotor of the genes coding for the antibiotic-like substance.
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    Genetic studies of Streptomyces cattleya and Streptomyces olivaceus
    (1985) Coyne, Vernon Errol; Kirby, Ralph
    Actinophage VCll is able to virulently infect 11 of the 20 Streptomyces strains tested. Examination of VCll infection of Streptomyces cattleya, Streptomyces olivaceus and Streptomyces lividans TC10 indicated the absence of restriction-modification systems which affect VCll infectivity of these Streptomyces strains.
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    Investigation of Streptomyces promoters
    (1995) Bourn, William Richard; Thomson, Jennifer Ann; Kirby, Ralph
    [The work described here had multiple aims: to create a promoter probe that was suitable for the isolation of developmentally regulated Strepcomyces promoters, to isolate such promoters, to develop a computer assisted analysis system whereby potential promoter sequences could be determined and to use this in the analysis of the cloned promoters. Initially the suitability of the Streptomyces antibioticus me1C operon for use as a reporter system in Streptomyces was investigated. It was established that late-expressed promoters could be identified and that it was possible to use the me1C2 gene alone for this purpose. However, it was shown that the use of both me1C1 and me1C2 resulted in a more sensitive reporter system. High copy number promoter probe vectors were constructed and tested. A low copy number promoter probe (which used the Streptomyces penemefaciens pSPN1 origin of replication) was also constructed. The characteristics (copy number, stability and mobility) of the probe were established. The conditions in which sporulation was induced by phosphate limitation were identified. Under such conditions late expressing, phosphate dependent promoters were isolated, using the promoter probes previously developed. The expression of these promoters was tested in Streptomyces coelicolor bldA mutants, and the bldA dependent promoters identified. These were sequenced. Computer assisted analysis of DNA sequence bias was conducted, with the intention of using bias patterns to identify potential regulatory regions. The initial approach of using the sequence bias of protein coding regions (based on the premise that regulatory sites are likely to be under represented in these regions) was unsuccessful. Further analysis in which the positional preference of sequences that were over represented in regulatory regions was conducted. Based on this the known promoters of Streptomyces were partially classified. The sequence bias of protein coding DNA regions was used to develop a novel method to identify the protein coding regions of Streptomyces DNA. The computer programs were then used to identify protein coding and potential regulatory regions.
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    A Study of genetic instability and DNA repair in selected mutants of streptomyces cattleya
    (1987) Hromic, Alma; Kirby, Ralph
    Three mutants of Streptomyces cattleya NRRL 8057 with lesions in the DNA repair pathway were obtained by NTG mutagenesis. The mutants were selected on the basis of their resistance or sensitivity to Mitomycin c. Of the three mutants, two, R6 and Rl2, were selected as MMC resistant and one, S26, as MMC sensitive. They were also UVr and UVˢ, respectively. The mutants were subjected to UV irradiation in order to obtain UV kill curves. The curves generated for the two MMCʳ mutants showed induction or derepression of a second repair system. In instability studies performed on them, the two MMCʳ mutants showed increased instability compared to the wild type at low UV dosage. However, the instability dropped to below that of the wild type at high UV dosages. Both these mutants appeared to roughly mimic the plateau shown by the wild type as higher levels of UV irradiation were achieved. S26 in contrast showed a much higher initial incidence of instability, and no plateau was observed at high UV dosages. Single strand breaks were induced in the wild type and mutant strains by incorporation of P³² into their DNA. R6 showed a five-fold increase in instability after this treatment. Rl2 showed no significant change, and S26 showed a slight decrease, which may be due to the increased lethality of DNA damage sustained by this strain due to its relatively labile nature. The strains were further analysed using polyacrylamide gel electrophoresis of total cellular proteins. S26 showed a startlingly different protein profile. R6 was almost identical to the wild type, while Rl2 was extremely similar, showing only the loss of one major band and a significant lowering of the amount of detectable protein in the region below this band.