Summary
The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the recA gene was inserted into the promoter region of the cloned kan gene to produce a fused operon, recA-kan. Plasmid pMCR685 carrying recA-kan expressed a low level of activity of the kan gene product (kanamycin phosphotransferase; KPT) in the wildtype cells of Escherichia coli, while the plasmid showed an increased level of the activity in the Spr- mutant cells which produce the inactive lexA protein. The KPT activity in the wildtype cells harboring the plasmid increased 6-to 11-fold upon treatment of the cells with mitomycin C or nalidixic acid, both of which are known to induce synthesis of recA protein.
Expression of the recA-kan operon fusion was remakably repressed by the lexA gene cloned into a plasmid carrying the operon fusion. Higher concentrations of mitomycin C were required for maximal induction of KPT activity in the cells harboring the resulting plasmid pMCR687. These results strongly suggest that the lexA gene product can by itself repress the recA gene, and that pMCR687 is a useful vector to clone genes whose expression is harmful to the host cell growth.
Similar content being viewed by others
References
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW (1977) Construction and characterization of new cloning vehicles: II. A multipurpose cloning system. Gene 2:95–113
Brent R, Ptashne M (1980) The lexA gene product represses its own promoter. Proc Natl Acad Sci USA 77:1932–1936
Clarke L, Carbon J (1976) A colony bank containing synthetic colE1 hybrid plasmids representative of the entire E. coli genome. Cell 9:91–99
Craig NL, Roberts JW (1980) E. coli recA protein-directed cleavage of phage λ repressor requires polynucleotide. Nature 283:26–30
Clowes RC, Hayes W (1968) Experiments in microbial genetics. Blackwell Scientific Publications, Oxford and Edinburgh
Cunningham RP, Shibata T, DasGupta C, Radding CM (1979) Homologous pairing in genetic recombination: Single strands induce recA protein to unwind duplex DNA. Nature 281:191–195
Haas MJ, Dowding JE (1975) Annoglycoside-modifying enzymes. In: Hash JH (ed) Methods in enzymology, vol 43. Academic Press, New York, pp 611–628
Horri T, Ogawa T, Ogawa H (1980) Organization of the recA gene of Escherichia coli. Proc Natl Acad Sci USA 77:313–317
Horii T, Ogawa T, Ogawa H (1981) Nucleotide sequence of the lexA gene of E. coll. Cell 23:689–697
Itakura K, Hirose T, Crea R, Riggs AD, Heyneker H, Bolivar F, Boyer HW (1977) Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198:1056–1063
Kanazawa H, Tamura F, Mabuchi K, Miki T, Futai M (1980) Organization of unc gene cluster of Escherichia coli coding for protontranslocating ATPase of oxidative phosphorylation. Proc Natl Acad Sci USA 77:7005–7009
Lennox ES (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190–206
Little JW (1979) Construction and characterization of a plasmid coding for a fragment of the Escherichia coli recA protein. Mol Gen Genet 177:13–22
Little JW, Harper J (1979) Identification of the lexA gene product of Escherichia coli K-12. Proc Natl Acad Sci USA 76:6147–6151
Little JW, Edmiston SH, Pacelli LZ, Mount DW (1980) Cleavage of the Escherichia coli lexA protein by the recA protease. Proc Natl Acad Sci USA 77:3225–3229
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
McEntee K (1978) Studies of the recA and lexA genes of Escherichia coli K-12. In: Hanawalt PC, Friedberg EC, Fox CF (eds) DNA repair mechanisms. Academic Press, New York, pp 349–360
McEntee K, Weinstock GM, Lehman IR (1979) Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli. Proc Natl Acad Sci USA 76:2615–2619
Miki T (1978) Genetic and physical analyses of the replication origin and dnaA gene of the Escherichia coli chromosome by molecular cloning and cleavage mapping with restriction endonucleases. Ann Rep Inst Virus Res, Kyoto Univ 21:1–26
Miki T, Hiraga S, Nagata T, Yura T (1978) Bacteriophage λ carrying the Escherichia coli chromosomal region of the replication origin. Proc Natl Acad Sci USA. 75:5099–5103
Miki T, Kimura M, Hiraga S, Nagata T, Yura T (1979) Cloning and physical mapping of the dnaA region of the Escherichia coli chromosome. J Bacteriol 140:817–824
Miki T, Ebina Y, Kishi F, Nakazawa A (1981) Organization of the lexA gene of Escherichia coli and nucleotide sequence of the regulatory region. Nucl Acids Res 9:529–543
Mount DW (1977) A mutant of Escherichia coli showing constitutive expression of the lysogenic induction and error-prone DNA repair pathways. Proc Natl Acad Sci USA 74:300–304
Nossal NG, Heppel LA (1966) The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 241:3055–3062
Ogawa T, Wabiko H, Tsujimoto T, Horii T, Masukata H, Ogawa H (1978) Characteristics of purified recA protein and the regulation of its synthesis in vivo. Cold Spring Harbor Symp Quant Biol 43:909–915
Ogura T, Miki T, Hiraga S (1980) Copy number mutants of the plasmid carrying the replication origin of the Escherichia coli chromosome: Evidence for a control region of replication. Proc Natl Acad Sci USA. 77:3993–3997
Rao NR, Rogers SG (1976) Plasmid pKC7: A vector containing ten restriction endonuclease sites suitable for cloning DNA segments. Gene 7:79–82
Rothstein SJ, Jorgensen RA, Postle K, Reznikoff WS (1980) The inverted repeats of Tn5 are functionally different. Cell 19:795–805
Roberts JW, Roberts CW, Mount DW (1977) Inactivation and proteolytic cleavage of phage λ repressor in vitro in an ATP-dependent reaction. Proc Natl Acad Sci USA 74:2283–2287
Ross GP, O'Callaghan CH (1975) β-Lactamase assays. In: Hash JH (ed) Methods in enzymology, vol 43. Academic Press, New York, pp 69–85
Sancar A, Rupp WD (1979) Physical map of the recA gene. Proc Natl Acad Sci USA 76:3144–3148
Sancar A, Stachelek C, Konigsberg W, Rupp WC (1980) Sequences of the recA gene and protein. Proc Natl Acad Sci USA 77:2611–2615
Shibata T, Cunningham RP, DasGupta C, Radding CM (1979) Homologous pairing in genetic recombination: Complexes of recA protein and DNA. Proc Natl Acad Sci USA 76:5100–5104
Tabak HF, Flavell RA (1978) A method for the recovery of DNA from agarose gels. Nucl Acids Res 5:2321–2332
Weinstock GM, McEntee K, Lehman IR (1980) ATP-dependent renaturation of DNA catalyzed by the recA protein of Escherichia coli. Proc Natl Acad Sci USA 76:126–130
Witkin EM (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev 40:869–907
Author information
Authors and Affiliations
Additional information
Communicated by T. Yura
Rights and permissions
About this article
Cite this article
Miki, T., Kumahara, H. & Nakazawa, A. Construction of a fused operon consisting of the recA and kan (Kanamycin resistance) genes and regulation of its expression by the lexA gene. Molec. Gen. Genet. 183, 25–31 (1981). https://doi.org/10.1007/BF00270133
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00270133