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PMID: 6316261 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

The bacteriophage lambda O replication protein: isolation and characterization of the amplified initiator.

Nucleic acids research ·Vol. 11 ·No. 21 ·1983-11-11 ·Pages 7435-52

Roberts JD, McMacken R

Abstract

The bacteriophage lambda O protein participates in the initiation of lambda DNA replication. The lambda O gene was cloned into plasmid pKC30 such that its expression was controlled by the lambda PL promoter. A lambda prophage-coded thermosensitive cI repressor was used to regulate transcription of the cloned O gene. Thermal inactivation of the lambda cI repressor resulted in overproduction of the O protein until it constituted approximately 20% of the total cellular protein of Escherichia coli. A simple three-step purification protocol was developed that yields several milligrams of homogeneous O protein per gram of cell paste. The precise position of the O gene in the known lambda DNA sequence was identified from the amino-terminal sequence of the isolated O protein. Purified O protein stimulated the replication of plasmid lambda dv DNA in vitro and specifically bound to duplex DNA fragments carrying the lambda replication origin.

MeSH Terms
Amino Acid Sequence Bacteriophage lambda/genetics Base Sequence DNA Replication DNA Restriction Enzymes Escherichia coli/genetics Gene Amplification Genes Genes, Viral Hot Temperature Plasmids Protein Biosynthesis Viral Proteins/genetics,isolation & purification Virus Replication
Chemicals
Viral Proteins DNA Restriction Enzymes
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Roberts J D
McMacken R
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32 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
Published
1983-11-11
Pages
7435-52
Language
English
Region
England
NLM ID
0411011
PMCID
PMC326494
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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