The Restriction of Non-Glucosylated T-Even-Bacteriophage Dna by Escherichia Coli (original) (raw)
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European Journal of Biochemistry, 1969
The decrease in the activity of DNA-dependent RNA polymerase in Escherichia coli after infection with bacteriophage T4 is the consequence of a structural change of the polymerase and not of the appearance of an inhibitor. The kinetics of this change were followed using polymerases highly purified a t different times after infection (a) by measuring the specific enzymatic activity with T,and calf thymus DNA as templates and (b) by following the appearance of the modified polymerase-subunit in disc-electrophoresis.
Conversion of T4 gene 46 mutant deoxyribonucleic acid into nonviable bacteriophage particles
Journal of Virology
The T4 amber mutant Ni 30 in gene 46 produces, upon infection of a nonpermissive host, about 10 times more phage equivalent of deoxyribonucleic acid (DNA) than viable phage particles. Intracellular DNA is shorter than its mature phage counterpart and is converted into nonviable, light phage particles (287S) with about half of the normal DNA content, incapable either of adsorbing to or killing host bacteria. This DNA is highly unstable and breaks down, upon extraction, into subunits one-tenth the normal length of T4 DNA. Electron micrographs showed that the nonviable particles consist of normal-sized capsids of various degrees of fullness. Abnormal tail structures (naked cores with the sheaths missing) were also observed. Under conditions of arrested DNA synthesis, late messenger ribonucleic acid is produced, but some species are rare or missing, resulting in uneven transcription of the late genes. Our findings indicate that continuous DNA replication is necessary for normal transcription in T4.
Journal of Molecular Biology, 1975
We have studied the in ~vo effects ofT4 endonucleases II and IV on the cytosinecontaining T4 DNA made after infection ofEscherichia coli B with dGTPase amber mutants of bacteriophage T4. Both nucleases are specific for cytosine-containing DNA; endonuclease II is a niekase, and endonuelease IV is specific for singlestranded stretches of DNA. The small amount of DNA made by dCTPase amber mutants is rapi,dly degraded; in contrast, the (dCTPase, endonuclease II, endonuclease IV) mutant makes normal amounts of DNA. Although this cytosine-containing T4 DNA is actually larger than that made by T4D +, as determined by both neutral and alkaline density-gradient sedimentation, no viable phage particles are produced.
Journal of Bacteriology
The akc gene product (gpalc) of bacteriophage T4 inhibits the transcription of cytosine-containing DNA in vivo. We examined its effect on transcription in vitro by comparing RNA polymerase isolated from Escherichia coli infected with either wild-type T4D+ or alc mutants. A 50 to 60% decline in RNA polymerase activity, measured on phage T7 DNA, was observed by 1 min after infection with either T4D+ or alc mutants; this did not occur when the infecting phage lacked gpalt. In the case of the T4D+ strain but not alc mutants, this was followed by a further decrease. By 5 min after infection the activity of akc mutants was 1.5 to 2.5 times greater than that of the wild type on various cytosine-containing DNA templates, whereas there was little or no difference in activity on T4 HMdC-DNA, in agreement with the in vivo specificity. Effects on transcript initiation and elongation were distinguished by using a T7 phage DNA template. Rifampin challenge, end-labeling with [_y-32P]ATP, and selective initiation with a dinucleotide all indicate that the decreased in vitro activity of the wild-type polymerase relative to that of the alc mutants was due to inhibition of elongation, not to any difference in initiation rates. Wild-type (but not mutated) gpalc copurified with RNA polymerase on heparin agarose but not in subsequent steps. Immunoprecipitation of modified RNA polymerase also indicated that gpalc was not tightly bound to RNA polymerase intracellularly. It thus appears likely that gpalc inhibits transcript elongation on cytosine-containing DNA by interacting with actively transcribing core polymerase as a complex with the enzyme and cytosine-rich stretches of the template.
Host specificity of DNA produced by Escherichia coli
Molecular and General Genetics, 1972
Bacteria with A-specific restriction plate unmodified phage λ with an efficiency of 10-2. One mutational event can produce restriction insensitive (sAo) mutants of λ. These differ from the original sA form of λ by no other property than their response to A-host specificity. Two-parental phage crosses involving sA and sAo, respectively, as non-selective marker allowed to map sA between genes cII and O. These data indicate that sA is the only site on λDNA with affinity for A-specific restriction. λDNA is thus an interesting substrate in in vitro A-specific restriction and modification. Using an assay based on the infectivity of λDNA on helper-infected bacteria, A-specific modification activity was found in partially purified sonicates of bacteria with A-host specificity. In parallel to modification, 3H-methyl label from s-adenosylmethionine, the only cofactor required for modification, was transferred to unmodified λDNA. No association of radioactivity was observed in control experiments with DNA from either modified λ·A or from aλsAo mutant. These data suggest that A-specific modification is brought about by DNA methylation and that the sAo mutation not only abolished the affinity for A-specific restriction, but also for A-specific modification.