3 IN-FRAME N-TERMINALLY DIFFERENT PROTEINS ARE PRODUCED FROM THE REPRESSOR LOCUS OF THE STREPTOMYCES BACTERIOPHAGE-PHI-C31

Margaret Caroline MacHin Smith, C E OWEN

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

The sequence of the repressor locus, c, of the Streptomyces temperate phage, phi-C31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in-frame N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N-terminal sequencing, and used to raise antibody. The antibody cross-reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolor phi-C31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the c(ts) locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super-infection immunity. Data described here and current knowledge of the nature of other 'dual start' genes suggest a model for the regulation of lysis versus lysogeny in phi-C31.

Original languageEnglish
Pages (from-to)2833-2844
Number of pages12
JournalMolecular Microbiology
Volume5
Issue number11
Publication statusPublished - Nov 1991

Keywords

  • TRANSCRIPTIONAL ANALYSIS
  • NUCLEOTIDE-SEQUENCE
  • PHAGE PHI-C31
  • GENE
  • INITIATION
  • CLONING
  • IDENTIFICATION
  • QUANTITIES
  • EXPRESSION
  • PROMOTERS

Cite this

3 IN-FRAME N-TERMINALLY DIFFERENT PROTEINS ARE PRODUCED FROM THE REPRESSOR LOCUS OF THE STREPTOMYCES BACTERIOPHAGE-PHI-C31. / Smith, Margaret Caroline MacHin; OWEN, C E .

In: Molecular Microbiology, Vol. 5, No. 11, 11.1991, p. 2833-2844.

Research output: Contribution to journalArticle

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abstract = "The sequence of the repressor locus, c, of the Streptomyces temperate phage, phi-C31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in-frame N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N-terminal sequencing, and used to raise antibody. The antibody cross-reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolor phi-C31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the c(ts) locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super-infection immunity. Data described here and current knowledge of the nature of other 'dual start' genes suggest a model for the regulation of lysis versus lysogeny in phi-C31.",
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N2 - The sequence of the repressor locus, c, of the Streptomyces temperate phage, phi-C31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in-frame N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N-terminal sequencing, and used to raise antibody. The antibody cross-reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolor phi-C31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the c(ts) locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super-infection immunity. Data described here and current knowledge of the nature of other 'dual start' genes suggest a model for the regulation of lysis versus lysogeny in phi-C31.

AB - The sequence of the repressor locus, c, of the Streptomyces temperate phage, phi-C31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in-frame N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N-terminal sequencing, and used to raise antibody. The antibody cross-reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolor phi-C31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the c(ts) locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super-infection immunity. Data described here and current knowledge of the nature of other 'dual start' genes suggest a model for the regulation of lysis versus lysogeny in phi-C31.

KW - TRANSCRIPTIONAL ANALYSIS

KW - NUCLEOTIDE-SEQUENCE

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KW - INITIATION

KW - CLONING

KW - IDENTIFICATION

KW - QUANTITIES

KW - EXPRESSION

KW - PROMOTERS

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SP - 2833

EP - 2844

JO - Molecular Microbiology

JF - Molecular Microbiology

SN - 0950-382X

IS - 11

ER -