Effect of chloramphenicol and starvation for an essential amino acid on polypeptide and polyribonucleotide synthesis in Escherichia coli infected with bacteriophage T4

Witmer, H; Baros, Anna; Forbes, J.

doi:10.1016/0003-9861(75)90182-4

Cited by 9 publications

(2 citation statements)

References 27 publications

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“…When Gl was infected at a nonpermissive temperature, DHFR messenger metabolism was comparable to that observed when chloramphenicol-treated E. coli B/r was infected at the same temperature (H. Witmer, unpublished data). In this context, it is relevant to note that, at a nonpermissive temperature, no measurable protein synthesis occurs in Gl (20), whereas small polypeptides (10 to 70 amino acid residues) are synthesized in the presence of chloramphenicol (20,75 Two plausible hypotheses can be advanced concerning the apparently normal repression of DHFR messenger transcription in the absence of postinfection protein synthesis. (i) Several modifications of the host DNA-dependent RNA polymerase occur even when cells are adsorbed by DNA-less ghosts or are infected with intact phage in the presence of chloramphenicol (53,55,63,78).…”

Section: Discussionmentioning

confidence: 99%

Control of synthesis of mRNA's for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase

Witmer¹,

Baros²,

Ende³

et al. 1976

J Virol

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A 300C, functional messengers for dCMP hydroxymethylase first appeared 3 to 6 min postinfection and reached their maximum levels at 12 min. Chloramphenicol, added before the phage, reduced the rate of mRNA accumulation. When the antibiotic was added 6 min postinfection, mRNA levels increased at their normal rate but there was no obvious repression of messenger accumulation. Delaying the addition of drug until 8 or 12 min had progressively less effect on the pattern of hydroxymethylase mRNA metabolism. When chloramphenicol was present from preinfection times or from 6 min postinfection, all hydroxymethylase mRNA's synthesized were stable; at later times, however, the ability of the drug to stabilize mRNA decreased with its ability to delay the turnoff of mRNA production. An overaccumulation of hydroxymethylase mRNA was also seen when phage-specific DNA synthesis was inhibited either by mutational lesion in an essential viral gene or by 5-fluorodeoxyuridine. By min 20 of a DNA-negative program, hydroxymethylase mRNA synthesis was repressed to the point where it no longer compensated for decay. However, a finite level of hydroxymethylase mRNA synthesis was maintained at later times of a DNA-negative infection. Such results indicate that replication of the phage chromosome is necessary but not sufficient for a complete turnoff of hydroxymethylase mRNA production. Functions controlled by the maturation-defective proteins (the products of genes 55 and 33) played only a minor role in the regulation of hydroxymethylase mRNA metabolism. Thus, we favor the hypothesis that a complete turnoff of hydroxymethylase messenger production requires one or more new proteins as well as an interval of DNA replication. The absence of DNA synthesis had no particular effect upon dihydrofolate reductase messenger production. The preinfection addition of chloramphenicol likewise had little effect on dihydrofolate reductase messenger metabolism. These latter data imply that prior synthesis of a phage-coded protein synthesis may not be required for the turnoff of reductase messenger production.

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Section: Discussionmentioning

confidence: 99%

Control of synthesis of mRNA's for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase

Witmer¹,

Baros²,

Ende³

et al. 1976

J Virol

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“…In the absence of the N anti-terminator, p factor acts at specific sites in early transcription, and one finds, as a result, only promoter-proximal early RNA (21). In T4, the CAM effect has been more difficult to interpret, because the polarity-suppressing strains examined up to now do not reverse the CAM effect (22,23), because DE RNA expression seems not to depend on an N-like T4 gene (ref. 10 and our own unpublished results), and because a second mode of expressing many early genes is superimposed on the expression of polycistronic early transcription units (5).…”

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confidence: 99%

In vitro system for induction of delayed early RNA of bacteriophage T4.

Thermes¹,

Daegelen²,

Franciscis³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Recent experiments in vivo show that T4 infection can modify the cell to allow production of rIIA and rIB mRNA in the presence of CAM (11). When the rIIA and rIIB genes are introduced into a cell only after that cell has become insensitive to the CAM effect, these RNA species appear to be expressed as middle species (11). We have interpreted this to mean that overcoming the CAM effect and middle mode expression depend on a change in the state of the infecting DNA. We report here on an in vitro system which supports this conclusion.MATERIAL AND METHODS Bacteria, Bacteriophage, and T4-Infected Cells. E. coil BE is the standard host for T4 D infection. BE-rif 4 was selected as a spontaneous mutant resistant to 150.ug/ml of rifampicin in minimal medium agar plates. The strain grows normally in liquid culture with 200,gg/ml of rifampicin.T4 am 292 is a derivative of T4 D which contains a mutation in gene 55; it has a normal development during the early period. T4 NB 2226 is a derivative of T4 B; it is the largest known deletion in the rII region, covering all of nIIA, rIIB, D1, D2,, and D2b (see ref. 12 for full details). This phage and the other smaller deletions used to prepare competitor RNA for rIIA and rIIB messenger analysis are described in refs. 11 and 12.Bacteria were grown to 5 X 108 cells per ml at 36' in M9 medium with 1% casamino acids (13). When restricted lysates were to be prepared, CAM was added to 200,gg/ml 5 min before infection. L-Tryptophan was added to 20 ,ug/ml and then the cells were infected at a multiplicity of 5 phage per bacterium. Infections were usually stopped 5 min after infection by chilling rapidly to 1-3°. In preparation of "exhausted" unrestricted lysates, rifampicin was added to 200 .ug/ml 5 min after infection, and the infection was allowed to continue another 10 min, after which the cells were chilled. Infected cells were centrifuged for 10 min at 4000 X g, then resuspended in Yio the original volume of 10 mM Tris-HCI at pH 7.5, 10 mM MgCI2, 100 mM NaCl, and recentrifuged. The cell pellet was then resuspended in Aoo the original volume of 0.5% Brij 58, 10 mM Tris-HCl at pH 7.5, 10mM NaCl, after which cells were frozen at -20°. Frozen cells were stored at -200 until they were ready to be used for lysate preparation and in vitro synthesis.Lysate Preparation and In Vitro RNA Synthesis. Frozen infected cells at 5 X 1010/ml were thawed and mixed with 0.8 volumes of lysozyme at 1 mg/ml. They were then incubated 5 min at 370, after which the lysates were kept at 00 until they were used for RNA synthesis.Between 100 and 300 jul of these lysates (or combinations of various lysates) were then added per ml of incubation mixture for RNA synthesis. The standard incubation mixture contained 20 mM 3-(N-morpholino)propanesulfonic acid (Mops)

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Reproduction of Large Virulent Bacteriophages

Mathews¹

1977

Comprehensive Virology

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Effect of chloramphenicol and starvation for an essential amino acid on polypeptide and polyribonucleotide synthesis in Escherichia coli infected with bacteriophage T4

Cited by 9 publications

References 27 publications

Control of synthesis of mRNA's for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase

Control of synthesis of mRNA's for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase

In vitro system for induction of delayed early RNA of bacteriophage T4.

Reproduction of Large Virulent Bacteriophages

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