Search tips
Search criteria 


Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. 1986 April; 166(1): 34–37.
PMCID: PMC214552

Analysis of forward mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine in the bacteriophage P22 mnt repressor gene.


We describe the isolation and genetic characterization of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutations in the phage P22 mnt repressor gene cloned in plasmid pBR322. Mutations in the mnt repressor gene or its operator on this plasmid, pPY98, confer a tetracycline resistance phenotype, whereas the wild-type plasmid confers tetracycline sensitivity. Cells carrying pPY98 were briefly exposed to MNNG to give 20 to 40% survival and a 50- to 100-fold increase in tetracycline-resistant cells. DNA sequence analysis showed that 29 of 30 MNNG-induced mutations were GC-to-AT transitions and one was an AT-to-GC transition. About 80% of the mutations are in three hotspots. This mutation spectrum is consistent with the proposed mechanism of mutagenic action of MNNG, which involves mispairing of an alkylated base, O6-methylguanine. The mnt gene may be a useful target for determining mutagenic specificity at the nucleotide level because forward mutations are easily isolated, the target size is small, and the DNA sequence changes of mutations can be determined rapidly.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (894K), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Clewell DB, Helinski DR. Effect of growth conditions on the formation of the relaxation complex of supercoiled ColE1 deoxyribonucleic acid and protein in Escherichia coli. J Bacteriol. 1972 Jun;110(3):1135–1146. [PMC free article] [PubMed]
  • Coulondre C, Miller JH. Genetic studies of the lac repressor. IV. Mutagenic specificity in the lacI gene of Escherichia coli. J Mol Biol. 1977 Dec 15;117(3):577–606. [PubMed]
  • DAVIS BD, MINGIOLI ES. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. [PMC free article] [PubMed]
  • Dente L, Cesareni G, Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. [PMC free article] [PubMed]
  • Guerola N, Ingraham JL, Cerdá-Olmedo E. Induction of closely linked multiple mutations by nitrosoguanidine. Nat New Biol. 1971 Mar 24;230(12):122–125. [PubMed]
  • Hawley DK, McClure WR. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. [PMC free article] [PubMed]
  • Hochschild A, Irwin N, Ptashne M. Repressor structure and the mechanism of positive control. Cell. 1983 Feb;32(2):319–325. [PubMed]
  • Koffel-Schwartz N, Verdier JM, Bichara M, Freund AM, Daune MP, Fuchs RP. Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli. Strain specificity and mutation-prone sequences. J Mol Biol. 1984 Jul 25;177(1):33–51. [PubMed]
  • Lawley PD, Orr DJ. Specific excision of methylation products from DNA of Escherichia coli treated with N-methyl-N'-nitro-N-nitrosoguanidine. Chem Biol Interact. 1970 Aug;2(2):154–157. [PubMed]
  • LeClerc JE, Istock NL, Saran BR, Allen R., Jr Sequence analysis of ultraviolet-induced mutations in M13lacZ hybrid phage DNA. J Mol Biol. 1984 Dec 5;180(2):217–237. [PubMed]
  • Lindahl T. DNA repair enzymes. Annu Rev Biochem. 1982;51:61–87. [PubMed]
  • Loechler EL, Green CL, Essigmann JM. In vivo mutagenesis by O6-methylguanine built into a unique site in a viral genome. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6271–6275. [PubMed]
  • Loveless A. Possible relevance of O-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides. Nature. 1969 Jul 12;223(5202):206–207. [PubMed]
  • Maxam AM, Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. [PubMed]
  • Olsson M, Lindahl T. Repair of alkylated DNA in Escherichia coli. Methyl group transfer from O6-methylguanine to a protein cysteine residue. J Biol Chem. 1980 Nov 25;255(22):10569–10571. [PubMed]
  • Samson L, Cairns J. A new pathway for DNA repair in Escherichia coli. Nature. 1977 May 19;267(5608):281–283. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Sauer RT, Krovatin W, DeAnda J, Youderian P, Susskind MM. Primary structure of the immI immunity region of bacteriophage P22. J Mol Biol. 1983 Aug 25;168(4):699–713. [PubMed]
  • Schendel PF, Robins PE. Repair of O6-methylguanine in adapted Escherichia coli. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6017–6020. [PubMed]
  • Skopek TR, Hutchinson F. DNA base sequence changes induced by bromouracil mutagenesis of lambda phage. J Mol Biol. 1982 Jul 25;159(1):19–33. [PubMed]
  • Walker GC. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. [PMC free article] [PubMed]
  • Wallace RB, Schold M, Johnson MJ, Dembek P, Itakura K. Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA. Nucleic Acids Res. 1981 Aug 11;9(15):3647–3656. [PMC free article] [PubMed]
  • Yamamoto N, Wohlhieter JA, Gemski P, Baron LS. lambdaimm P22dis: a hybrid of coliphage lambda with both immunity regions of Salmonella phage P22. Mol Gen Genet. 1978 Nov 9;166(3):233–243. [PubMed]
  • Youderian P, Vershon A, Bouvier S, Sauer RT, Susskind MM. Changing the DNA-binding specificity of a repressor. Cell. 1983 Dec;35(3 Pt 2):777–783. [PubMed]
  • Zagursky RJ, Berman ML. Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing. Gene. 1984 Feb;27(2):183–191. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)