Summary
The recQ gene of Escherichia coli K12 was subcloned from plasmid pKO1 (Oeda et al. 1981) by monitoring the capacity of the resulting recombinant plasmids partially to reverse the increased ultraviolet (UV) sensitivity of a recF143 recQ1 double mutant. We were able to trace this complementation activity to a 3.4 kilobase (kb) SalI-PvuII fragment. Furthermore, analysis of the Tn3 insertion mutations that abolished the complementation revealed the exclusive localisation of such insertions in the same 3.4 kb segment. This segment was situated about 4 kb clockwise from corA on the chromosome, a result consistent with the transductional data previously reported. In addition, a comparison of our restriction endonuclease cleavage map with the published data has placed recQ between pldA and pldB. When relocated to the recQ site on the chromosome, the recQ::Tn3 mutations conferred partial resistance to thymineless death (TLD) or, in the case of a recBC sbcB background, recombination deficiency and increased UV sensitivity. This has provided the firm evidence that both the TLD resistance and the deficiency in the RecF recombination pathway result from loss of the functional recQ gene. We also identified the recQ gene product as a 74 kilodalton polypeptide by using the maxicell technique.
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Abbreviations
- TLD:
-
thymineless death
- UV:
-
ultraviolet light
- Ap:
-
ampicillin
- Km:
-
kanamycin
- Sm:
-
streptomycin
- Tc:
-
tetracycline
- r :
-
resistant
- s :
-
sensitive
- kb:
-
kilobase
- kdal:
-
kilodalton
References
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles II: a multipurpose cloning system. Gene 2:95–113
Cohen SS, Barner HD (1954) Studies on unbalanced growth in Escherichia coli. Proc Natl Acad Sci USA 40:885–893
Grindley NDF, Joyce CM (1980) Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903. Proc Natl Acad Sci USA 77:7176–7180
Guyer MS (1978) The γδ sequence of F is an insertion sequence. J Mol Biol 126:347–365
Homma H, Kobayashi T, Ito Y, Kuto I, Inoue K, Ikeda H, Sekiguchi M, Nojima S (1983) Identification and cloning of the gene coding for lysophospholipase L2 of E. coli K-12. J Biochem 94:2079–2081
Homma H, Kobayashi T, Chiba N, Karasawa K, Mizushima H, Kudo I, Inoue K, Ikeda H, Sekiguchi M, Nojima S (1984) The DNA sequence encoding pldA gene, the structural gene for detergent-resistant phospholipase A of E. coli. J Biochem 96:1655–1664
Horii Z, Clark AJ (1973) Genetic analysis of the RecF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J Mol Biol 80:327–344
Kato T, Rothman RH, Clark AJ (1977) Analysis of the role of recombination and repair in mutagenesis of Escherichia coli by UV irradiation. Genetics 87:1–18
Kretchmer PJ, Cohen SN (1977) Selected translocation of DNA segments containing antibiotic resistance genes. In: Bukhari AI, Shapiro JA, Adhya SL (eds) DNA insertion elements, plasmids and episomes. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 549–554
Kushner SR (1978) An improved method for transformation of Escherichia coli with ColE1-derived plasmid. In: Boyer HS, Nicosia S (eds) Genetic engineering. Elsevier, Amsterdam, pp 17–23
Maki S, Kuribayashi M, Miki T, Horiuchi T (1983) An amber replication mutant of F plasmid mapped in the minimal replication region. Mol Gen Genet 191:231–237
Manis JJ, Kline BC (1977) Restriction endonuclease mapping and mutagenesis of the F sex factor replication region. Mol Gen Genet 152:175–182
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Nakabeppu Y, Kondo H, Sekiguchi M (1984) Cloning and characterization of the alkA gene of Escherichia coli that encodes 3-methyladenine DNA glycosylase II. J Biol Chem 259:13723–13729
Nakayama H, Couch JL (1973) Thymineless death in Escherichia coli in various assay systems: viability determined in liquid medium. J Bacteriol 114:228–232
Nakayama H, Nakayama K, Nakayama R, Nakayama Y (1982) Recombination-deficient mutations and thymineless death in Escherichia coli K12: reciprocal effects of recBC and recF and indifference of recA mutations. Can J Microbiol 28:425–430
Nakayama H, Nakayama K, Nakayama R, Irino N, Nakayama Y, Hanawalt PC (1984) Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12; identification of a new mutation (recQ1) that blocks the RecF recombination pathway. Mol Gen Genet 195:474–480
Oeda K, Horiuchi T, Sekiguchi M (1981) Molecular cloning of the uvrD gene of Escherichia coli that controls ultraviolet sensitivity and spontaneous mutation frequency. Mol Gen Genet 184:191–199
Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693
Vapnek D, Alton NK, Bassett CL, Kushner SR (1976) Amplification in Escherichia coli of enzymes involved in genetic recombination: construction of hybrid ColE1 plasmids carrying the structural gene for exonuclease I. Proc Natl Acad Sci USA 73:3492–3496
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Communicated by P.T. Emmerson
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Nakayama, K., Irino, N. & Nakayama, H. The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants. Molec. Gen. Genet. 200, 266–271 (1985). https://doi.org/10.1007/BF00425434
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DOI: https://doi.org/10.1007/BF00425434