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W Fulford

Publications and source records attributed to W Fulford.

6 recordsLinked to original sources
PubMed1989

Phage Trojan horses: a conditional expression system for lethal genes.

The EcoRI restriction enzyme (ENase) cleaves DNA molecules within the sequence GAATTC. Cells expressing this lethal activity normally make a second enzyme, the M.EcoRI methyltransferase (MTase), which protects their chromosomal DNA by modifying the EcoRI recognition sites. To isolate mutants of the EcoRI ENase, its gene was cloned into a filamentous phage vector (M13mp18) under control of the lac promoter. Normally, filamentous phages (M13, f1 and their derivatives) form turbid plaques by impairing the growth of their host cell without killing it. In contrast, phages expressing the EcoRI ENase kill the host cell, but survive long enough to produce plaques which are very clear. Expression of the M.EcoRI MTase rescues the host and restores turbid plaque formation. EcoRI ENase mutants were isolated by screening for mutants that make turbid, instead of clear, plaques on an M- host. This conditional expression system may be useful for cloning and mutating genes for other toxic proteins.

Base Sequence↗
PubMed1988

Bacteriophage f1 DNA replication genes. II. The roles of gene V protein and gene II protein in complementary strand synthesis.

Filamentous phage gene V, which encodes a single-stranded DNA binding protein, has been cloned and placed under control of the lac promoter. Cells bearing the clone are refractory to filamentous phage infection if the expression of the gene is induced with isopropyl-1-thio-beta-D-galactoside. The inhibition of infection is shown to occur at an early stage, and can be reversed if the cells express gene II in addition to gene V protein. These observations support the hypothesis that gene II protein, in addition to its role in nicking and facilitating the synthesis of phage viral (+) strand DNA, functions to prevent the gene V-mediated inhibition of complementary (-) strand synthesis. We proposed a model in which the absolute and relative concentrations of the products of genes II, X and V determine whether a single strand is to be exported as phage or incorporated into double-stranded replicative form DNA.

Cloning, Molecular↗
PubMed1988

Regulation of bacteriophage f1 DNA replication. I. New functions for genes II and X.

Gene II protein is required for all phases of filamentous phage DNA synthesis other than the conversion of the infecting single strand to the parental double-stranded molecule. It introduces a specific nick into the double-stranded replicative form DNA, is required for the initiation of (+) strand synthesis and is responsible for termination and ring closure of the (+) strand product. Here we show that the gene II protein also promotes minus strand synthesis later in infection. Over-expression of gene II protein can induce the conversion of all nascent single-stranded phage DNA to the double-stranded form, even in the presence of the single-stranded DNA-binding gene V protein that would normally sequester the newly synthesized single strands. We also present evidence that the gene X protein (separately translated from an initiator codon within gene II, and identical to the C-terminal one-third of the gene II protein) is a powerful inhibitor of phage-specific DNA synthesis in vivo.

Base Sequence↗
PubMed1984

Gene X of bacteriophage f1 is required for phage DNA synthesis. Mutagenesis of in-frame overlapping genes.

The gene II protein of bacteriophage f1 is a site-specific endonuclease required for initiation of phage viral strand DNA synthesis. Within gene II is another gene, X, encoding a protein of unknown function identical to the C-terminal 27% of the gene II protein, and separately translated from codon 300 (AUG) of gene II. By oligonucleotide mutagenesis, we constructed phage mutants in which this codon has been changed to UAG (amber) or UUG (leucine), and propagated them on cells carrying a cloned copy of gene X on a plasmid. The amber mutant makes no gene X protein, and cannot grow in the absence of the complementing plasmid; the leucine-inserting mutant can make gene X protein, and grows normally without the plasmid. Without gene X protein, phage DNA synthesis (particularly viral strand synthesis) is impaired. We discuss this finding in the context of other known in-frame overlapping genes (particularly genes A and A* of phage phi X174), many of which are also involved in the specific initiation of DNA synthesis, and suggest applications for the mutagenic strategy we employed.

Coliphages↗
PubMed1984

Specificity of translational regulation by two DNA-binding proteins.

The gene V protein of the filamentous bacteriophages fl, fd and M13, and the gene 32 protein of bacteriophage T4 share the property of binding strongly and co-operatively to single-stranded nucleic acids, especially DNA. Moreover, both are capable of repressing the translation of specific mRNAs (gene 32 protein its own, and gene V protein that of the filamentous phage gene II), both in vivo and in vitro. If the mechanism of repression by either of these proteins were based solely on its ability to bind single strands co-operatively, then the other would be expected to mimic or interfere with its effect in vitro. We have found no such mimicry or interference, even at protein concentrations high enough to have substantial non-specific effects on translation. This suggests that the sites of repression on the mRNAs must offer something other than simple "unstructuredness" for binding and repression to occur.

Bacteriophages↗