Effects of nitrous acid on transduction by Salmonella phage P22

Adye, Jimmy

doi:10.1016/0042-6822(62)90065-x

Cited by 9 publications

(3 citation statements)

References 8 publications

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“…Several investigators had used a similar approach, but the method was not perfected or exploited. Adye (21) showed that histidinerequiring mutants could be obtained by transduction of a histidine deletion with phage mutagenized with nitrous acid. He pointed out that the method was useful for the isolation of leaky mutants that usually escaped a pencillin selection.…”

Section: Discussionmentioning

confidence: 99%

Localized Mutagenesis of Any Specific Small Region of the Bacterial Chromosome

Hong

Ames

1971

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

237

130

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A method, which we call localized mutagenesis, is described for the isolation of temperaturesensitive and other types of mutations in any specific small region (about 1%) of the bacterial chromosome. The principle of this method is to mutate the transducing DNA rather than the bacterial DNA. One can select for the introduction of this mutated DNA into any particular region of the bacterial chromosome by transducing an auxotrophic marker in that region to prototrophy, thereby introducing new mutations in the neighborhood. We have used this method to isolate many different temperaturesensitive mutations in genes of unknown function in particular regions of the chromosome. Since the method is very simple, it can be used to saturate any region of the map with mutations in essential genes, or for various types of genetic manipulations. Although we have used hydroxylamine-mutagenized phage P22 and Salmonella typhimurium, the method should be applicable to other mutagens and bacteria and transducing phage.Temperature-sensitive (ts) mutations in bacteria are extremely useful because they can be obtained in genes that code for proteins that are essential for the growth of the cell. Since the pioneering work of Horowitz and Leupold (1), temperature-sensitive mutants in bacteria have been used to study activating enzymes (2), ribosomes (3-5), DNA synthesis (6), and other components and aspects of macromolecular syntheses.A number of workers have isolated ts mutants by mutagenizing Escherichia coli or Salmonella typhimurium. Although the isolation of these mutants is easy, the mapping of them involves a considerable amount of work since they may map anywhere on the chromosome. We have developed a simple method, which we call localized mutagenesis, for the easy isolation of ts and other mutants in any specific small region of the chromosome. MATERIALS AND METHODSBacterial Strains. All strains were derived from S. typhimurium strain LT-2.Phage. Phage used for mutagenesis and transduction was P22-int-4, a nonintegrating mutant of P22 (7). The phage sensitivity test was done with P22-H5, a clear (c2) mutant of P22. Phage were stored in T2 buffer (8).Media. Nutrient broth (Difco) was used as maximally supplemented medium. E medium (9) (made 0.4% in glucose) Abbreviation: ts, (i.e., heat-sensitive).was used as minimal-salts medium. Solid medium contained 1.5% agar.Transducing Phage Mutagenesis. Phage (P22-int-4) was grown on wild-type strain LT-2 at 370C with shaking in a medium composed of 100 parts of nutrient broth, 3.5 parts of 40% glucose, and 1.3 parts of concentrated (50X) medium E. The phage, a mixture of phage and bacterial DNA in phage heads, usually had a titer of 1-3 X 1011/ml. Concentrated phage (1012/ml) in 0.9% saline was mutagenized at 370C with hydroxylamine (10). To 1 part of phage in saline were added 5 parts of 0.1 M sodium phosphate buffer (pH 6.0) containing 1 mM EDTA, and 4 parts of 1 M hydroxylamine * HCl (adjusted to pH 6.0 with NaOH) containing 1 mM EDTA. At the desired time the phage were collec...

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Section: Discussionmentioning

confidence: 99%

Localized Mutagenesis of Any Specific Small Region of the Bacterial Chromosome

Hong

Ames

1971

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

237

130

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“…subsequently explained as the result of conversion of abortive to complete transductants by increased recombination (3). A similar phenomenon caused by NA was explained in the same way (1).…”

Section: Time (Hr)mentioning

confidence: 82%

“…Thus, the procedure is of doubtful use as a general means for obtaining new mutations at specific loci, except possibly where the mutation induced is very close to the selected locus. Such a procedure has been used for mutagenizing transforming DNA and transducing phage, and later recovering new mutations closely linked to the donor locus selected in transformation or transduction (1,2,27). However, the frequency of recovery of such mutants was low (see 6) and many of the mutants obtained were leaky (1, 32).…”

Section: \ \mentioning

confidence: 99%

Recognition of Altered Deoxyribonucleic Acid in Recombination

Helling

1969

J Bacteriol

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Kinetics of inactivation of transduction by phage Plbt which had been treated with ultraviolet light (UV) or nitrous acid (NA) was examined. With Escherichia coli B/r (radiation-resistant), low doses of UV increased transduction frequency, but the frequency was exponentially inactivated by higher doses. Little initial stimulus was observed in strain B8.1 (radiation-sensitive). The final rate of decay was the same as in B/r. The initial stimulus of transduction in B/r was probably a consequence of increased recombination resulting from dark repair. It was estimated that another nucleotide within 1000 nucleotide pairs had to be damaged by UV to prevent a given nucleotide from successful transduction. The NA dose response was the same for the two strains. An initial stimulus of transduction was followed by exponential decline. The UV-repair enzymes missing in B.-, were not required for repair of NA-induced damage to transducing or lytic phage DNA. Low recovery of new mutations in the transductants showed that mutagen-induced damage to transducing DNA was excluded from recombinant chromosomes. The few recovered mutants may have resulted from "normal" error in recombination. After treatment of transforming deoxyribonucleic acid (DNA) or transducing phage with a mutagen, new mutations closely linked to donor genes selected in the transformants or transductants have been recovered (1, 2, 27). The procedure appeared to be a useful one for obtaining mutations in small selected regions of the chromosome. This paper shows that in transduction of 224 Vol. 100, No. I

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The Chemical Basis of Mutation

Le¹

1965

Advances in Enzymology - And Related Areas of Molecular Biology

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Effects of nitrous acid on transduction by Salmonella phage P22

Cited by 9 publications

References 8 publications

Localized Mutagenesis of Any Specific Small Region of the Bacterial Chromosome

Localized Mutagenesis of Any Specific Small Region of the Bacterial Chromosome

Recognition of Altered Deoxyribonucleic Acid in Recombination

The Chemical Basis of Mutation

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