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  • Title: Dependence on reporter gene of apparent activity in gene fusions of a Streptomyces griseus streptomycin biosynthesis promoter.
    Author: Lindley HK, Deeble VJ, Peschke U, O'Neill M, Baumberg S, Cove J.
    Journal: Can J Microbiol; 1995; 41(4-5):407-17. PubMed ID: 8590416.
    Abstract:
    The adjacent genes strR-strA-strB1 lie within the large cluster of genes of streptomycin biosynthesis and resistance in Streptomyces griseus. strR encodes a pathway-specific activator StrR, suggested by previous work to be either an antiterminator or a conventional activator, binding to its DNA target via a helix-turn-helix motif. strB1 is transcribed in an StrR-dependent fashion from a promoter (PstrB1) that lies downstream from strA; between PstrB1 and strB1 there is a 300-bp leader region containing numerous inverted repeats that could represent modulatable transcription termination sites. Hybrid plasmids were constructed in vitro with transcriptional fusions in which fragments containing PstrB1 and either the entire leader region ("long" fragments) or a small part of it (the "short" fragment) were cloned upstream of (i) aph as reporter gene, in a high copy number plasmid background, or (ii) xylE as reporter gene, in a low copy number plasmid background. The short fragment directed high levels of APH (aminoglycoside 3'-phosphotransferase) whether StrR was present or not, while the long fragments did not do so in the absence of StrR; one long fragment directed high levels in wild-type S. griseus, in which StrR would be present. Insertion of an extraneous fragment into PstrB1 in the short fragment construct led to loss of APH activity, demonstrating that no adventitious promoter had been formed in the short construct. In vitro deletion of part of the leader region in a long fragment construct led to high APH expression with or without StrR present. Although these results are consistent with the target of StrR being within the leader region, and thus with an antiterminator role, it was found that both long and short fragments in the low copy number background failed to direct high expression of catechol oxygenase (the product of xylE) unless strR was also present on a compatible plasmid. Transfer of PstrB1-xylE fragments to the high copy number vector did not increase catechol oxygenase expression. We interpret these results in terms of an effect, in the hybrid constructs, of one of the reporter genes on promoter function, possibly by affecting local DNA topology.
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