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Title: Isolation of developmental mutants using Tn 1792-based mutagenesis in Streptomyces avermitilis
Author: Pitman, A. B.
Awarding Body: University of Wales Swansea
Current Institution: Swansea University
Date of Award: 1997
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The use of transposon mutagenesis for the identification of genes involved in Streptomcyes development has been frustrated in part by the high target site specificity of many transposable elements. This investigation reports the low insertion site specificity of the IS6100-based mini-transposon Tn/792 from a temperature-sensitive delivery plasmid to the genome of the avermectin producer Streptomyces avermitilis. Sequencing of Tn/792 insertion sites in S. avermitilis transposants revealed no meaningful shared homology of the target sites or predilection of Tn/792 for GC or AT rich regions of the genome. Furthermore, the cointegrate structure normally formed as the end product of the IS6100 family of transposable elements was found to undergo resolution resulting in the precise excision of one of the duplicated copies of the transposon and the delivery vector. Several insertions revealed previously uncharacterized genes, of which one termed orf219 was implicated in morphological differentiation. Disruption of wild-type S. avermitilis with the cloned insertion site carrying Tn1792 generated colonies unable to sporulate (whi), suggesting orf219 was involved in development of the aerial hyphae. Complementation of the disruptants with a functional copy of the gene confirmed this involvement in development, producing reversion of the whi phenotype to that of wild-type. Microscopic analysis revealed that in strains in which orf219 was disrupted the aerial hyphae are grossly elongated and unable to develop spore compartments. This suggests that orf219 is involved in differentiation at or prior to septation of aerial hyphae.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available