A simple procedure for localized mutagenesis using nitrosoguanidine

Oeschger, Max P.; Berlyn, Mary K. B.

doi:10.1007/bf00332814

Cited by 44 publications

(14 citation statements)

References 15 publications

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“…8). Such transitions have often been found after mutagenesis with N-methyl-N'-nitro-Nnitrosoguanidine (11,31). The observed point mutation results in a threonine-to-isoleucine substitution in the Cterminal part of the chorismate mutase.…”

Section: Resultsmentioning

confidence: 99%

A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae

et al. 1989

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The Saccharomyces cerevisiae ARO7 gene product chorismate mutase, a single-branch-point enzyme in the aromatic amino acid biosynthetic pathway, is activated by tryptophan and subject to feedback inhibition by tyrosine. The ARO7 gene was cloned on a 2.05-kilobase EcoRI fragment. Northern (RNA) analysis revealed a 0.95-kilobase poly(A)+ RNA, and DNA sequencing determined a 771-base-pair open reading frame capable of encoding a protein of 256 amino acids. In addition, three mutant alleles of ARO7 were cloned and sequenced. These encoded chorismate mutases which were unresponsive to tyrosine and tryptophan and were locked in the on state, exhibiting a 10-fold-increased basal enzyme activity. A single base pair exchange resulting in a threonine-to-isoleucine amino acid substitution in the C-terminal part of the chorismate mutase was found in all mutant strains. In contrast to other enzymes in this pathway, no significant homology between the monofunctional yeast chorismate mutase and the corresponding domains of the two bifunctional Escherichia coli enzymes was found.In the yeast Saccharomyces cerevisiae, the biosynthesis of aromatic amino acids is regulated either at the transcriptional or at the enzyme level. At the transcriptional level, the general control system is known to regulate at least 30 structural amino acid genes in various pathways, among them most of the ARO and TRP genes. This transcriptional control responds to amino acid starvation and results in an increased transcription rate of these genes through binding of the activator protein GCN4 (36). In contrast to many bacteria, no aromatic amino acid-specific regulation is known at the transcriptional level.At least four ARO and TRP gene products are also or exclusively regulated at the enzyme level (Fig. 1). The two isoenzymes 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 4.1.2.15) encoded by the genes AR03 and AR04 control the entrance of the shikimate pathway and are subject to feedback inhibition by the pathway end products phenylalanine and tyrosine, respectively (37). The TRP2 gene product anthranilate synthase (EC 4.1.3.27) and the AR07 gene product chorismate mutase (EC 5.4.99.5)

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Section: Resultsmentioning

confidence: 99%

A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae

et al. 1989

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“…Since nitA maps close to ilv (81% cotransduction, using phage P1) (4), we could use ilv as the linked selective marker in the localized mutagenesis technique (13). The basic procedure employed was the same as that used before (4); mutants resistant to XsusN7nin5 were selected in an E. coli K-12 strain that was lysogenic for phage P2.…”

Section: Resultsmentioning

confidence: 99%

“…1); PR-promoted transcription in vio is apparently' prevented at these sites unless the N product acts as an anti-rho factor. To investigate the effects of the nitA Ts mutations on termination of rightward transcription, expression of early indispensable genes, such as 0, P (13), and Q (14), was -examined in terms of growth of phages XsusN7N53byp, XsusN7, and XsusN7N53 at 30' in nitA Ts cells nonlysogenic for P2. The byp mutation close to tR2 does not allow sufficient' N-independent synthesis of the Q product for normal growth of N defective phage (17).…”

Section: Resultsmentioning

confidence: 99%

Isolation and characterization of conditional-lethal rho mutants of Escherichia coli.

Inoko¹,

Shigesada²,

Imai³

1977

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

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Temperature-sensitive nitA (rho) mutants of E. coli were isolated; one of them was characterized as an amber mutant. These strains show the Nit phenotype (transcription of phage X DNA independent of the Ngene) at low temperatures and are inviable at high temperatures. The mutated sites appear to be between cya and metE on the chromosome. Temperature-sensitive nitA bacteria not only permit leftward transcription of the A genome at a hi'h rate in the absence of the A Nprotein, but also allow X gro at low temperatures. At high temperatures, phages A and T4 are incapable of normal development in these cells, while growth of 17 is not affected. The production of thermally unstable rho by the nitA temperaturesensitive mutant suggests that nitA is the structural geie for rho.Studies of host mutants that specifically affect early gene expression of phage A have provided useful information on the mechanism of transcription (1-4). We have taken advantage of the Spi phenomenon (5, 6)-the fact that bacteria lysogenic for phage P2 will not permit growth of wild-type X but will allow AsusN7nin5 to multiply-to develop a method for the isolation of bacterial mutants (nit) which permit N-independent transcription of the X genome.

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“…Although As2 can tolerate only 5 mM arsenate, the complemented mutants (with pXL10c) can tolerate up to 20 mM. It has been shown that MNNG can introduce mutations at multiple sites (22). However, the results of the complementation experiment provide strong evidence that the specific mutation in the arsC1 region of As2 is responsible for loss of arsenate resistance.…”

Section: Vol 189 2007 Regulation Of D Desulfuricans G20 Arsenate Rmentioning

confidence: 99%

Regulation of Arsenate Resistance in Desulfovibrio desulfuricans G20 by an arsRBCC Operon and an arsC Gene

Li¹,

Krumholz

2007

J Bacteriol

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Desulfovibrio desulfuricans G20 grows and reduces 20 mM arsenate to arsenite in lactate-sulfate media. Sequence analysis and experimental data show that D. desulfuricans G20 has one copy of arsC and a complete arsRBCC operon in different locations within the genome. Two mutants of strain G20 with defects in arsenate resistance were generated by nitrosoguanidine mutagenesis. The arsRBCC operons were intact in both mutant strains, but each mutant had one point mutation in the single arsC gene. Mutants transformed with either the arsC1 gene or the arsRBCC operon displayed wild-type arsenate resistance, indicating that the two arsC genes were equivalently functional in the sulfate reducer. The arsC1 gene and arsRBCC operon were also cloned into Escherichia coli DH5␣ independently, with either DNA fragment conferring increased arsenate resistance. The recombinant arsRBCC operon allowed growth at up to 50 mM arsenate in LB broth. Quantitative PCR analysis of mRNA products showed that the single arsC1 was constitutively expressed, whereas the operon was under the control of the arsR repressor protein. We suggest a model for arsenate detoxification in which the product of the single arsC1 is first used to reduce arsenate. The arsenite formed is then available to induce the arsRBCC operon for more rapid arsenate detoxification.

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A simple procedure for localized mutagenesis using nitrosoguanidine

Cited by 44 publications

References 15 publications

A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae

A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae

Isolation and characterization of conditional-lethal rho mutants of Escherichia coli.

Regulation of Arsenate Resistance in Desulfovibrio desulfuricans G20 by an arsRBCC Operon and an arsC Gene

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