AN OPERATOR ASSOCIATED WITH AUTOREGULATION OF THE REPRESSOR GENE IN ACTINOPHAGE-PHI-C31 IS FOUND IN HIGHLY CONSERVED COPIES IN INTERGENIC REGIONS IN THE PHAGE GENOME

C J INGHAM, C E OWEN, Stephen Wilson, Irene Hunter, Margaret Caroline MacHin Smith

Research output: Contribution to journalArticle

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Abstract

Previous reports have suggested that the repressor gene, c, of phiC31 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 - aphII fusion was repressed in a Streptomyces coelicolor A3(2) phiC31 lysogen and characterisation of an operator-constitutive (O(c)) 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 phiC31 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.

Original languageEnglish
Pages (from-to)821-827
Number of pages7
JournalNucleic Acids Research
Volume22
Issue number5
Publication statusPublished - 11 Mar 1994

Keywords

  • CLONING VECTORS
  • STREPTOMYCES
  • PHI-C31
  • TRANSCRIPTION
  • SITE
  • BACTERIOPHAGES
  • PHAGE-PHI-C31
  • CONSTRUCTION
  • SEQUENCE
  • PSAM2

Cite this

AN OPERATOR ASSOCIATED WITH AUTOREGULATION OF THE REPRESSOR GENE IN ACTINOPHAGE-PHI-C31 IS FOUND IN HIGHLY CONSERVED COPIES IN INTERGENIC REGIONS IN THE PHAGE GENOME. / INGHAM, C J ; OWEN, C E ; Wilson, Stephen; Hunter, Irene; Smith, Margaret Caroline MacHin.

In: Nucleic Acids Research, Vol. 22, No. 5, 11.03.1994, p. 821-827.

Research output: Contribution to journalArticle

INGHAM, C J ; OWEN, C E ; Wilson, Stephen ; Hunter, Irene ; Smith, Margaret Caroline MacHin. / AN OPERATOR ASSOCIATED WITH AUTOREGULATION OF THE REPRESSOR GENE IN ACTINOPHAGE-PHI-C31 IS FOUND IN HIGHLY CONSERVED COPIES IN INTERGENIC REGIONS IN THE PHAGE GENOME. In: Nucleic Acids Research. 1994 ; Vol. 22, No. 5. pp. 821-827.
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abstract = "Previous reports have suggested that the repressor gene, c, of phiC31 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 - aphII fusion was repressed in a Streptomyces coelicolor A3(2) phiC31 lysogen and characterisation of an operator-constitutive (O(c)) 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 phiC31 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.",
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N2 - Previous reports have suggested that the repressor gene, c, of phiC31 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 - aphII fusion was repressed in a Streptomyces coelicolor A3(2) phiC31 lysogen and characterisation of an operator-constitutive (O(c)) 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 phiC31 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.

AB - Previous reports have suggested that the repressor gene, c, of phiC31 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 - aphII fusion was repressed in a Streptomyces coelicolor A3(2) phiC31 lysogen and characterisation of an operator-constitutive (O(c)) 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 phiC31 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.

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KW - CONSTRUCTION

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