Isolation and characterization of mutants of bacteriophage T4 resistant to folate analogs

Johnson, James R.; Hall, Dwight H.

doi:10.1016/0042-6822(73)90221-3

Virology

1973

DOI: 10.1016/0042-6822(73)90221-3

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Isolation and characterization of mutants of bacteriophage T4 resistant to folate analogs

James R. Johnson

Dwight H. Hall

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1976

1996

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Cited by 24 publications

(30 citation statements)

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“…In a previous paper, a group of far mutants designated class IIB was tentatively characterized as being RDP reductase deficient (32). In the case of the class IIB far mutants and in the case of other independently isolated nrd mutants, loss of the phage-induced RDP reductase has now been shown to constitute a mechanism of resistance to folate analogue inhibition.…”

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“…Plating techniques that eliminate T4 plaque formation on E. coli by folate analogue inhibition of FH, reductase allow the isolation of folate analogue-resistant (far) mutants of T4 (32). These mutants can be divided into classes based on their proposed mechanism of re-VOL.…”

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“…The nrdA18-td8, nrdB10-frdl, and nrdB67-frdl double mutants were obtained from I. Tessman (56,57), as were mutants td8 and tdlO (47). Mutants farC31, farC61, farP22, farP82, farP85, and farP95 were previously isolated and characterized by this laboratory (32). Mutant cdNl 6 was isolated in this laboratory from the double mutant tdN16-cdN16 described by Hall (27).…”

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confidence: 99%

“…sistance (32,33). In a previous paper, a group of far mutants designated class IIB was tentatively characterized as being RDP reductase deficient (32).…”

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Novel Mechanism of Resistance to Folate Analogues: Ribonucleoside Diphosphate Reductase Deficiency in Bacteriophage T4

Johnson

Collins

Rementer

et al. 1976

Antimicrob Agents Chemother

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Some spontaneously occurring bacteriophage T4 mutants (far mutants) were able to form plaques in the presence of concentrations of folate analogues that completely inhibit plaque formation by wild-type phage T4. Some of these far mutants were shown to be ribonucleoside diphosphate (RDP) reductase (EC 1.17.4.1) deficient, and some independently isolated RDP reductase-deficient mutants (nrd mutants) were shown to be folate analogue resistant. The map positions of the RDP reductase-deficient far mutants were shown to be within the genes controlling the phage-induced RDP reductase activity.Escherichia coli infected by bacteriophage T4 offers several advantages as a model system in which to study mechanisms of resistance to antimetabolites. Since many of the T4-induced enzymes duplicate functions already present in the host cell, this system is similar to most other host-parasite relationships but is much easier to study in the laboratory because T4 and E. coli multiply rapidly in simple media. T4 is a genetically complex virus, and the results obtained with it may be comparable to higher biological systems. To date, about 100 T4 genes have been identified out of a maximum of approximately 200 potential genes. The products of over 30 of these genes have been identified as phage-induced enzymatic activities (review by Mathews [40]). Many types of T4 mutants are available for study, comparison, and confirmation of phenotype.Among the proteins encoded in the genome of phage T4 and synthesized early after infection is a six-enzyme system that controls the biosynthesis of thymidylic acid and the interconversion of pyrimidine nucleotides. The six enzymes are ribonucleoside diphosphate (RDP) reductase, thymidylate synthetase, dihydrofolate (FH2) reductase, deoxycytidine triphosphatase, deoxycytidylate (deoxycytidine monophosphate [dCMP]) deaminase, and thymidine kinase (5, 17, 27-29, 35, 42, 47, 56). The reactions catalyzed by these enzymes are diagramed in Fig. 1 dine at a low but detectable rate (34), is grown on agar plates containing synthetic medium and cytidine as the only pyrimidine (S + Cyd plates), T4 mutants deficient in the production of FH2 reductase activity, designated frd mutants (27), or thymidylate synthetase activity, designated td mutants (47), produce plaques that exhibit the white-halo phenotype (28). The white halo surrounding the plaques of these mutants is the result of an increase in bacterial cell growth at the periphery of the plaque. It is believed that a deficiency in either FH2 reductase or thymidylate synthetase activity allows deoxyuridine monophosphate (dUMP) or a related metabolite to accumulate within the mutant-infected cells (Fig. 1); lysis of these cells during the normal cycle of phage proliferation releases high levels of uracil or a related metabolite into the medium. On S + Cyd plates with E. coli OK305 as the host cell, this metabolite could either directly or indirectly stimulate cell growth by relieving the requirement for uracil. Mutations in the phage cd gene, which codes for...

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confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

“…sistance (32,33). In a previous paper, a group of far mutants designated class IIB was tentatively characterized as being RDP reductase deficient (32).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Novel Mechanism of Resistance to Folate Analogues: Ribonucleoside Diphosphate Reductase Deficiency in Bacteriophage T4

Johnson

Collins

Rementer

et al. 1976

Antimicrob Agents Chemother

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“…Although several T4 motA mutants have been isolated and characterized (7,17,18,20,25,26,39,47), analyses of the sequence changes present in these mutations reveal that all retain at least the first 59 amino acids of the wild type-MotA sequence (33,45,47). We describe the isolation and characterization of an N-terminal mutation of motA, mot21, in which the first 8 codons of the wild-type motA gene are replaced with 11 different codons.…”

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An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription

Gerber

Hinton

1996

J Bacteriol

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The bacteriophage T4 MotA protein is a transcriptional activator of T4-modified host RNA polymerase and is required for activation of the middle class of T4 promoters. MotA alone binds to the ؊30 region of T4 middle promoters, a region that contains the MotA box consensus sequence [(t/a)(t/a)TGCTT(t/c)A]. We report the isolation and characterization of a protein designated Mot21, in which the first 8 codons of the wild-type motA sequence have been replaced with 11 different codons. In gel retardation assays, Mot21 and MotA bind DNA containing the T4 middle promoter P uvsX similarly, and the proteins yield similar footprints on P uvsX . However, Mot21 is severely defective in the activation of transcription. On native protein gels, a new protein species is seen after incubation of the 70 subunit of RNA polymerase and wild-type MotA protein, suggesting a direct protein-protein contact between MotA and 70 . Mot21 fails to form this complex, suggesting that this interaction is necessary for transcriptional activation and that the Mot21 defect arises because Mot21 cannot form this contact like the wild-type activator.Transcription of the middle class of bacteriophage T4 genes requires T4-modified host RNA polymerase and a transcriptional activator, the product of the T4 motA gene (reviewed in reference 43). The modification to the polymerase required for MotA-dependent transcription has been identified as the T4 AsiA protein (13, 32), a factor that binds tightly to 70 (41, 42). A mutation in the motA gene was discovered because the mutant phage fails to give normal patterns of T4 prereplicative protein synthesis (25). Subsequent work demonstrated that this abnormal protein pattern observed during a T4 motA mutant infection occurs because the middle (MotA-dependent) class of promoters does not become active (6). Middle promoters are characterized by an excellent match to the Ϫ10 consensus sequence for 70 , the specificity subunit of RNA polymerase, but they lack a recognizable 70 Ϫ35 sequence. Instead they share a 9-bp sequence, a MotA box, centered 30 bp upstream of the start of transcription (2, 6). The MotA protein binds to this sequence and is required for transcription initiating at T4 middle promoters by T4-modified polymerase in vitro (10,24,37). Recent results indicate that while T4-modified polymerase can bind a middle promoter in the absence of the activator, MotA protein is needed to form the open complex (12).The motA gene encodes a protein of 211 amino acids (45). Although several T4 motA mutants have been isolated and characterized (7,17,18,20,25,26,39,47), analyses of the sequence changes present in these mutations reveal that all retain at least the first 59 amino acids of the wild type-MotA sequence (33,45,47). We describe the isolation and characterization of an N-terminal mutation of motA, mot21, in which the first 8 codons of the wild-type motA gene are replaced with 11 different codons. The wild-type MotA and Mot21 proteins bind similarly to the T4 middle promoter P uvsX , but Mot21 fails to activ...

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Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

1993

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The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of the td gene by recombinational marker rescue using plasmid or M13 subclones of the td gene. Phage crosses using deletion mutants with known breakpoints in the 3' end of the td intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5' and 3' ends of the intron sequence, in accordance with the hypothesis that the 5' and 3' domains of the T4 td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mapped td mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within the td gene.

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Isolation and characterization of mutants of bacteriophage T4 resistant to folate analogs

Cited by 24 publications

References 17 publications

Novel Mechanism of Resistance to Folate Analogues: Ribonucleoside Diphosphate Reductase Deficiency in Bacteriophage T4

Novel Mechanism of Resistance to Folate Analogues: Ribonucleoside Diphosphate Reductase Deficiency in Bacteriophage T4

An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription

Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

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