Increased Intracellular Concentration of an Initiator Protein Markedly Reduces the Minimal Sequence Required for Initiation of DNA Synthesis

One of the most common sites used for cloning in the filamentous phages f1, fd, and M13 lies within the phage ``functional origin,'' a sequence of 140 nucleotides that is required for phage replication. Even small insertions (four nucleotides) at this location severely reduce origin functi...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1984-03, Vol.81 (5), p.1336-1340
Main Authors: Dotto, Gian Paolo, Zinder, Norton D.
Format: Article
Language:English
Subjects:
Bacteriophages
Base Sequence
Cloning, Molecular
Coliphages - genetics
DNA
DNA Replication
DNA Restriction Enzymes
Escherichia coli - genetics
Genetic mutation
Genetic vectors
Genomes
Infections
Molecules
Nucleotides
Peptide Initiation Factors - genetics
phage f1
phage fd
phage M13
Plasmids
Virus Replication
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Summary:One of the most common sites used for cloning in the filamentous phages f1, fd, and M13 lies within the phage ``functional origin,'' a sequence of 140 nucleotides that is required for phage replication. Even small insertions (four nucleotides) at this location severely reduce origin function. Secondary trans-acting mutations in the phage genome are necessary to restore efficient replication. One of these mutations, present in one of our cloning vectors, R218, has been fully characterized. It consists of a regulatory mutation within gene V that leads to a marked increase in the intracellular level of the phage gene II protein, the ``initiator'' of viral replication. Increased gene II protein production is sufficient to reduce the minimal sequence required for a functional origin to only 40 nucleotides, while the remaining 100 (containing the cloning site) become entirely dispensable. The general implications of these findings are discussed.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.81.5.1336