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Nucleic Acids Research, 1994, Vol. 22, No. 5 821-827
© 1994

MOLECULAR BIOLOGY

An operator associated with autoregulation of the repressor gene in actinophage øC31 is found in highly conserved copies in intergenic regions in the phage genome

Colin J. lngham, Charles E. Owen+, Stuart E. Wilson1,§, lain S. Hunter and Margaret C.M. Smith1,*,§

Robertson Institute of Biotechnology, Church Street, University of Glasgow Glasgow G11 5JS 1Department of Biological and Molecular Science, University of Stirling Stirling FK9 4LA, UK

*To whom correspondence should be addressed

Received November 15, 1993. Revised February 17, 1994. Accepted February 17, 1994.

Previous reports have suggested that the repressor gene, c, of øC31 is autoregulated and that likely operators are conserved Inverted repeat sequences (CIRs1&2) located just upstream of the promoters, cp1 and cp2. Evidence is now presented that the CIRs 1&2 are indeed binding sites for one of the three Inframe, N-terminally different protein isoforms of 42, 54 and 74 kDa produced by the c gene. A cp1 - aphll fusion was repressed in a Streptomyces coelicolor A3(2) øC31 lysogen and characterisation of an operator-constitutive (0c) mutant showed a single mutation In CIR-1. CIR-1 containing fragments were retarded in electrophoresis gels by the 42 kDa repressor protein isoform and this retardation was inhibited by the addition of competing DNA fragments containing either CIR-1 or CIR-2. Using a combination of Southern blotting and analysis of available DNA sequence we also show that at least 18 copies of the CIRs are present throughout the øC31 genome. Alignment of 9 CIR sequences showed that 8 contained a perfectly conserved 17 bp core whilst the exception had a single mismatch. The core includes a 16 bp inverted repeat (IR), and is usually part of a more extensive and less highly conserved palindrome. When superimposed on a previously derived transcription map of the early region, the CIRs lie in intergenic regions associated with transcription initiation and/or termination.

+Present addresses: ICRF, 44 Lincoln's Inn Fields, London WC2A 3PX

§Present addresses: Department of Genetics, Queens Medical Centre. University Park. Nottingham NG7 2UH, UK


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