The synthesis of deoxycytidylate deaminase and dihydrofolate reductase and its control in Escherichia coli infected with bacteriophage T4 and T4 amber mutants

Warner, Huber R.; Lewis, Nathan M.

doi:10.1016/0042-6822(66)90208-x

Cited by 55 publications

(18 citation statements)

References 8 publications

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“…1 and 3) that is not in sequence with their placement in the T4 genome (frd, td, cd). Furthermore, FH2 reductase activity is not enhanced in DO amber mutants (32), in contrast to other phage early enzymes. Similar conclusions were derived by Mathews (28).…”

Section: Discussionmentioning

confidence: 99%

“…From the results in Table 1, it appears that aside from promoting the synthesis of three internal capsid proteins (37), the immediate-early RNA may direct the synthesis of FH2 reductase and at least one-half of the dCMP hydroxymethylase. The refractoriness of the FH2 reductase (32) to the usual hyperactivation of delayed-early enzymes induced by amber T4 mutants (3) might now be explained by the location of the frd gene in a region distinct from that occupied by the delayed-early genes. Similar conclusions have been arrived at (20) with phage-infected spheroplasts treated with 5-methyltryptophan and chloramphenicol.…”

Section: Discussionmentioning

confidence: 99%

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The Temporal Expression of T2r ⁺ Bacteriophage Genes In Vivo and In Vitro

Trimble

Galivan

Maley

1972

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

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The kinetic order of synthesis of deoxycytidylate deaminase (EC 3.5.4.12), deooxycytidylate hydroxymethylase (EC 2.1.2.b), dihydrofolate reductase (EC 1.5.1.3), 5-hydroxymethyldeoxycytidylate kinase (EC 2.7.4.4), and thymidylate synthetase (EC 2.1.1.b) after infection of Escherichia coli with T2r+ bacteriophage was found not to correlate with their order of synthesis in an in vitro protein-synthesizing preparation. The in vivo and in vitro synthesis of enzyme-specific messenger RNA measured in the protein-synthesizing preparation preceded each enzyme by about 1 min. Through the use of sheared DNA, it was shown that the thymidylate synthetase gene was most susceptible to a loss in template activity, which suggests that tbis gene is further removed from its promoter than the other genes are from theirs. With a DNA segment of 2.5 X 10 daltons, the synthesis of dihydrofolate reductase alone was obtained, but at a much reduced rate. Translation of the RNA from phage-infected cells treated with chloramphenicol yielded amounts of dihydrofolate reductase and deoxycytidylate hydroxymethylase activities similar to those obtained with RNA from untreated infected cells. These results suggest that the chloramphenicol RNA, which consists primarily of immediate-early RNA, may contain most, if not all, of the information required for the synthesis of phage dihydrofolate reductase and deoxycytidylate hydroxymethylase.The prereplicative events in T-even bacteriophage have been divided into immediate-early, delayed-early, and late stages, each responsible for the elaboration of specific proteins that contribute to the formation of phage DNA and the completion of the virion. Immediate-early synthetic events are apparently host-mediated and are prescribed by a specific phage mRNA formed in the presence of chloramphenicol (1, 2). The impairment of DNA synthesis as a consequence of the deletion or restriction of a crucial enzyme prevents the transition from delayed-early to late transcription, an effect that promotes the enhanced production of delayed-early enzymes (3). It appears that the orderly transition from one event to the next in phage development is controlled by specific regulatory agents (4-8).DNA-RNA hybridization has been useful for clarification of the order of events after phage infection (4, 9). However, hybridization does not distinguish between the existence of an RNA and its capacity to act as a functional unit. Recent studies (10, 11) suggest that an in vitro protein-synthesizing preparation that uses DNA and RNA templates might be useful in determining the relative location of genes on an isolated transcriptional unit, since the order of appearance of immediate-early proteins, a single delayed-early enzyme, and a late enzyme was identical to that in vivo. To determine if the in vitro preparation could be used to reconstruct the events that occur in tivo, we asked whether the genes for delayed-early enzymes are expressed with the same fidelity in vitro as they are in uivo. The answer, as will be shown, is ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The Temporal Expression of T2r ⁺ Bacteriophage Genes In Vivo and In Vitro

Trimble

Galivan

Maley

1972

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

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“…Material from a 700-ml bacterial culture was chromatographed on a column (0.9 by 15 cm) and eluted at a rate of 12 mlI/h by a linear gradient of 0.05 to 0.50 M KCl in 0.05 M Tris-chloride buffer (pH 7.2) containing 1 mM dithiothreitol. Enzyme activity was determined as described by Warner and Lewis (26), and tests for inhibition by trimethoprim were performed by adding the drug to the indicated final concentration 2 min before the addition of dihydrofolate, which started the reaction. Enzyme assays on crude extracts were performed several times and agreed to within 10%o.…”

Section: Methodsmentioning

confidence: 99%

Plasmid-borne or chromosomally mediated resistance by Tn7 is the most common response to ubiquitous use of trimethoprim

Steen¹,

Sköld²

1985

Antimicrob Agents Chemother

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The folic acid analog trimethoprim has been in clinical use for more than 10 years. The use of it in Sweden has doubled in the last 6 to 7 years, and from the distribution statistics it can be calculated that during 1 year 4 to 5% of the population in Sweden are given this drug. The The folic acid analog trimethoprim has been used as an antimicrobial agent for more than a decade. A few years after the introduction of trimethoprim for clinical use, R plasmid-mediated resistance to this drug was reported (13). This plasmid-borne resistance also demonstrated a new principle regarding bacterial insensitivity, in that a drug-resistant variation of the target enzyme, dihydrofolate reductase, was found to be expressed from the plasmid (1, 22). Several types of plasmid-borne, drug-resistant dihydrofolate reductases have been subsequently discerned, and at least one of them was shown to be located on a transposon, Tn7 (3, 4). In later years, trimethoprim has been used extensively for clinical purposes, and resistance to it seems to have increased in frequency (7,9,25).The use of trimethoprim in Sweden can be defined quantitatively by data from the computerized Swedish Drug Information System (Department of Drugs, National Board of Health and Welfare). From these data it could thus be seen that more than 0.15% of the population receive trimethoprim each day or that during 1 year 4 to 5% of the population are given this drug. It was interesting to investigate the types of bacterial resistance mechanisms that could be found in response to such a selection pressure in a relatively isolated population in northern Sweden (the county of Jamtland), in which one centrally located bacteriological laboratory serves the area. During an 8-month period, trimethoprim-resistant strains were collected from consecutive specimens of bacteria from the urinary tracts of patients. Among highly resistant bacteria, transposon Tn7 was found to be common, either located chromosomally or borne on a conjugative Incl plasmid, 50 kilobases (kb) of size, which seemed to be endemic to the area. Two cases of marked overproduction of chromosomal dihydrofolate reductase which caused high resistance to trimethoprim were also found. * Corresponding author. MATERIALS AND METHODSBacterial strains and plasmids. Laboratory strains of bacteria and plasmids are listed in Table 1. Clinically isolated enterobacterial strains are listed in Table 2, and their collection is described in the text. Testing for antibiotic resistance was performed by the agar diffusion method with antibiotics-containing paper disks from AB Biodisk, Solna, Sweden.The serotyping of Escherichia strains was kindly performed by I. and F. Orskov, International Escherichia and Klebsiella Centre, Copenhagen.Media. The rich medium LB or the mineral salts medium M9, supplemented with Casamino Acids (0.05% [wt/vol]), was used throughout (19).Transfer of R plasmids. The transfer of R plasmids was performed as described earlier (21) and also on agar plates, and recombinants were selected on M9 agar ...

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“…DHFR activity was determined spectrophotometrically, as described earlier (35). A Zeiss PM6 spectrophotometer, thermostatically regulated at 30°C, connected to a graph recorder was used.…”

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confidence: 99%