Bacteriophage N4-induced transcribing activities in Escherichia coli. I. Detection and characterization in cell extracts.
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Biomedical subjects
Publications and source records attributed to S C Falco.
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A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described. The principle of containment is sterility: the haploid host strains all contain a mating-type-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.
A DNA-dependent RNA polymerase has been purified from disrupted virions of the Escherichia coli bacteriophage N4. The RNA polymerase is phage-coded and is required for class I N4 RNA synthesis, which is defined as RNA synthesized in vivo in the absence of post-infection protein synthesis. A polypeptide of molecular weight 350,000 is detected when the purified enzyme is analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. N4 RNA polymerase requires denatured DNA as a template in vitro and shows a strong preference for denatured N4 DNA. With this template, transcription is asymmetric. The RNA product is complementary to only the H strand of N4 DNA. Furthermore, only class I N4 RNA is synthesized. In vivo transcription by the N4 virion RNA polymerase is inhibited by coumermycin. This result suggests that the activity of E. coli DNA gyrase, an enzyme that introduces negative supertwists into DNA, is required for N4 transcription.
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Escherichia coli phage N4 transcription is resistant to rifampicin, an inhibitor of the host RNA polymerase, even when the drug is added prior to infection. A rifampicin-resistant RNA polymerase has been detected in disrupted N4 virions. This enzyme shows a requirement for the four ribonucleoside 5'-triphosphates and exogenous denatured DNA. With denatured N4 DNA, the preferred template, transcription is asymmetric. The virion RNA polymerase apparently is necessary for phage development because a conditional lethal N4 mutant shows temperature-sensitive RNA synthesis in vivo as well as a temperature-sensitive RNA polymerase in disrupted virions.