Summary
Mutants of a specialized λdmet transducing phage bearing the metJBLF gene cluster of Escherichia coli K12 were constructed using transposon Tn5. Two of these mutants, λdmet128::Tn5, MW77 and λdmet128::Tn5, 3–1, were used to locate precisely as well as confirm the existence of the metF transcription unit (approximately 1,000 base pairs in size). The introduction of new restriction sites within the metJBLF gene cluster due to the Tn5 insertion events allowed the metF transcription unit to be cloned into the high copy number plasmid pBR322. Analyses of the structures of two of these recombinant plasmids, pTJ77H and pTJ3-1H, are presented. Expression of the plasmid borne metF allele in cells grown in the absence, or presence, of exogenous L methionine (0.2 mM) demonstrates that the amplification of the metF copy number does not abolish met regulon mediated control of the gene's activity.
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References
Bachmann BJ (1983) Linkage map of Escherichia coli K12, edition 7. Micro Biol Rev 47:180–230
Berg DE, Davies J, Allet B, Rochaix JD (1975) Transposition of R factor genes to bacteriophage lambda. Proc Natl Acad Sci USA 72:3628–3632
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
Daubner SC, Matthews RG (1982) Purification and properties of methylenetetrahydrofolate reductase from pig liver. J Biol Chem 257:140–145
Dugaiczyk A, Boyer HW, Goodman HM (1975) Ligation of EcoRI endonuclease generated fragments into linear and circular structures. J Mol Biol 96:171–184
Flavin M (1975) Methionine biosynthesis. In: Greenberg DM (ed) Metabolic pathways, vol 7. Academic Press, New York. p 457–503
Greene RC, Krueger JH, Johnson JR (1982) of the metJBLF gene cluster of Escherichia coli in lambda met transducing phage. Mol Gen Genet 187:401–404
Greene RC, Smith AA (1982) Transcription organization of the metJBLF gene cluster in E. coli K12. Fed Proc Amer Soc Exp Biol 41:752
Ferguson J, Davis RW (1978) Quantitative electron microscopy of nucleic acids. In: Koehler JK (ed) Advanced techniques in biological electron microscopy. Springer Verlag, Berlin. p 123–171
Hellig RB, Goodman HM, Boyer H (1974) Analysis of endonuclease R. EcoRI fragments from lambdoid bacteriophage and other viruses by agarose gel electrophoresis. J Virol 14:1235–1244
Holmes DS, Quigley M (1981) A rapid boiling method for preparation of bacterial plasmids. Anal Biochem 114:193–197
Humphreys GO, Wilshaw GW, Anderson ES (1975) A simple method for the preparation of bacterial plasmids. Biochim Biophys Acta 383:457–463
Johnson JR, Greene RC, Krueger JH (1977) Isolation and characterization of specialized lambda transducing phage carrying the metBJLMF methionine gene cluster. J Bacteriol 131:795–800
Jørgensen RA, Rothstein SJ, Reznikoff WS (1979) A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet 177:6047–6051
Katzen HM, Buchanan JM (1965) Enzymatic biosynthesis of the methyl group of methionine. J Biol Chem 240:825–835
Kraus J, Soll D, Low KB (1979) Glutamyl-gamma-ethyl ester acts as a methionine analogue in Escherichia coli. Analogue resistant mutants map at the metJ and metK loci. Genet Res Cambridge 33:49–55
Krueger JH, Johnson JR, Greene RC (1978) In vitro synthesis of cystathionine-gamma-synthetase in Escherichia coli K12. J Bacteriol 133:1351–1357
Krueger JH, Johnson JR, Greene RC, Dresser M (1981) Structural studies of lambda transducing bacteriophage carrying bacterial deoxyribonucleic acid from the metBJLF region of the Escherichia coli chromosome. J Bacteriol 147:612–621
Kushner SA (1978) An improved method for transformation of Escherichia coli with Col E1 derived plasmids. In: Boyer HW and Nicosia S (eds) Genetic engineering. Elsevier North Holland, London. p 17–23
McDonell MW, Simon NM, Studier FW (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol 110:119–146
Patte JC, Le Bras G, Cohen GN (1967) Regulation by methionine of the synthesis of a third aspartokinase and of a second homoserine dehydrogenase in Escherichia coli K12. Biochim Biophys Acta 136:245–257
Reznikoff WS (1982) Tn5 transposition and its regulation. Cell 31:307–308
Suitcliffe JG (1979) Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp on Quant Biol 48, vol 1: 77–90
Zakin MM, Duchange N, Ferrara P, Cohen GN (1983) Nucleotide sequence of the metL gene of Escherichia coli. J Biol Chem 258:3028–3031
Zakin MM, Greene RC, Dautry-Varsat A, duchange N, Ferrara P, Py MC, Margarita D, Cohen GN (1982) Construction and physical mapping of plasmids containing the metJBLF gene cluster of E. coli K12. Mol Gen Genet 187:101–106
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Communicated by G.A. O'Donovan
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Treat, M.L., Weaver, M.L., Emmett, M.R. et al. Mutagenesis of the metJBLF gene cluster with transposon Tn5: Localization of the metF transcription unit. Molec Gen Genet 193, 370–375 (1984). https://doi.org/10.1007/BF00330695
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DOI: https://doi.org/10.1007/BF00330695