Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.639367
Title: Transposition of a mycobacterial insertion sequence, IS6100, in Streptomyces avermitilis
Author: Weaden, J. A.
Awarding Body: University of Wales Swansea
Current Institution: Swansea University
Date of Award: 1998
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Abstract:
This project tested the suitability of insertion sequence IS6100, originally isolated as part of the compound transposon Tn610 from Mycobacterium fortuitum, for use as a molecular tool in streptomycetes. This project successfully demonstrated transposition of pUCS30 into both the chromosome and a giant linear plasmid, pSA1, of S. avermitilis. An optimized procedure was developed to efficiently generate and select recombinants in which cointegrate formation had taken place. Single colonies were incubated at 28°C for 3-4 days, then transferred to 40°C until outgrowing sectors developed. Subsequent Southern hybridization analysis of sector DNA, using an IS6100 probe, confirmed that plasmid integration had arisen as a result of transposition. PFGE and conventional agarose gel electrophoresis of DNA restricted with AflII and BamHI respectively, followed by Southern hybridization with IS6100, indicated that insertion took place at random sites throughout the genome. Prolonged growth of transposants at 28°C in the absence of antibiotics did not lead to loss or rearrangement of the cointegrate structure. Studies on eight auxotrophic mutants, generated by pUCS30 mutagenesis, revealed prototrophic revertants in three strains. Reversion frequencies were highly variable and resulted from precise excision of the cointegrate. Amplifications were observed in several transposants and three such strains involved IS6100 sequences.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.639367  DOI: Not available
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