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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 81; no. 5; pp. 1336 - 1340
• Main Authors: Dotto, Gian Paolo, Zinder, Norton D.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.03.1984; National Acad Sciences
• 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
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.81.5.1336

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.