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Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli

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Summary

The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E. coli genes cloned in the bacteriophage λD69 vector. A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed by lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells and the subunit molecular mass established as 33,000 daltons. Analysis, by the minicell procedure, of plasmids with deletions extending into the prs gene established the direction of transcription as counterclockwise. A putative leader sequence of approximately 400 bp preceded the coding sequence. By deletion analysis and by cloning fragments of this leader sequence in a galK expression vector it was found to contain the prs promoter as well as a potential transcription termination site.

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References

  • Bachmann BJ (1983) Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev 47:180–230

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    Google Scholar 

  • Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heynecker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113

    Google Scholar 

  • Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207

    Google Scholar 

  • Davis RW, Botstein D, Roth JR (1980) A manual for genetic engineering. Advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Degnen GE, Cox EC (1974) Conditional mutator gene in Escherichia coli: isolation, mapping, and effector studies. J Bacteriol 117:477–487

    Google Scholar 

  • Enquist LW, Weisberg RA (1976) A genetic analysis of the att-intxis region of coliphage lambda. J Mol Biol 111:97–120

    Google Scholar 

  • Hong JS, Ames BN (1971) Localized mutagenesis of any specific small region of the bacterial chromosome. Proc Natl Acad Sci USA 68:3158–3162

    Google Scholar 

  • Hove-Jensen B (1983) Chromosomal location of the gene encoding phosphoribosylpyrophosphate synthetase in Escherichia coli. J Bacteriol 154:177–184

    Google Scholar 

  • Hove-Jensen B, Nygaard P (1982) Phosphoribosylpyrophosphate synthetase of Escherichia coli. Identification of a mutant enzyme. Eur J Biochem 126:327–332

    Google Scholar 

  • Inselburg J (1970) Segregation into and replication of plasmid deoxyribonucleic acid in chromosomeless segregants of Escherichia coli. J Bacteriol 102:642–647

    Google Scholar 

  • Jensen KF (1983) Metabolism of 5-phosphoribosyl 1-pyrophosphate (PRPP) in Escherichia coli and Salmonella typhimurium. In: Munch-Petersen A (ed) Metabolism of nucleotides, nucleosides and nucleobases in microorganisms. Academic Press, London, pp 1–25

    Google Scholar 

  • Jensen KF, Houlberg U, Nygaard P (1979) Thin-layer chromatographic methods to isolate 32P-labeled 5-phosphoribosyl-α-1-pyrophosphate (PRPP): determination of cellular PRPP pools and assay of PRPP synthetase activity. Anal Biochem 98:254–263

    Google Scholar 

  • Jensen KF, Larsen JN, Schack L, Sivertsen A (1984) Studies on the structure and expression of Escherichia coli pyrC, pyrD and pyrF using the cloned genes. Eur J Biochem 140:343–352

    Google Scholar 

  • Jensen KF, Neuhard J, Schack L (1982) RNA polymerase involvement in the regulation of expression of Salmonella typhimurium pyr genes. Isolation and characterization of a fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to a mutation in rpoBC. EMBO J 1:69–74

    Google Scholar 

  • Jochimsen BU, Hove-Jensen B, Garber BB, Gots JS (1985) Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase. J Gen Microbiol 131:245–252

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 193:265–275

    Google Scholar 

  • Mandel M, Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53:159–162

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560

    Google Scholar 

  • McKenney K, Shimatake H, Court D, Schmeissner U, Brady C, Rosenberg M (1981) A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. In: Chirigiian JC, Papas TS (eds) Gene amplification and analysis, vol 2. Elsevier/North-Holland, New York, pp 383–415

    Google Scholar 

  • Mizusawa S, Ward DF (1982) A bacteriophage lambda vector for cloning with BamHI and Sau3A. Gene 20:317–322

    Google Scholar 

  • Neuhard J (1983) Utilization of preformed pyrimidine bases and nucleosides. In: Munch-Petersen A (ed) Metabolism of nucleotides, nucleosides and nucleobases in microorganisms. Academic Press, London, pp 95–148

    Google Scholar 

  • O'Callaghan CH, Morris A, Kirby SM, Shingler AH (1972) Novel method for detection of β-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother 1:283–288

    Google Scholar 

  • Oeschger MP, Oeschger NS, Wiprud GT, Woods SL (1980) High efficiency temperature-sensitive amber suppressor strains of Escherichia coli K12: isolation of strains with suppressor-enhancing mutations. Mol Gen Genet 177:545–552

    Google Scholar 

  • Olszowy J, Switzer RL (1972) Specific repression of phosphoribosylpyrophosphate synthetase by uridine compounds in Salmonella typhimurium. J Bacteriol 110:450–451

    Google Scholar 

  • Pandey NK, Switzer RL (1982) Mutant strains of Salmonella typhimurium with defective phosphoribosylpyrophosphate synthetase activity. J Gen Microbiol 128:1863–1871

    Google Scholar 

  • Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693

    Google Scholar 

  • Schubert KR, Switzer RL, Shelton E (1975) Studies of the quaternary structure and the chemical properties of phosphoribosyl-pyrophosphate synthetase from Salmonella typhimurium. J Biol Chem 250:7492–7500

    Google Scholar 

  • Schwartz M, Neuhard J (1975) Control of expression of the pyr genes in Salmonella typhimurium: effects of variations in uridine and cytidine nucleotide pools. J Bacteriol 121:814–822

    Google Scholar 

  • Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Sternberg N, Tiemeier D, Enquist L (1977) In vitro packaging of a λDam vector containing EcoRI DNA fragments of Escherichia coli and phage P1. Gene 1:255–280

    Google Scholar 

  • Sutcliffe JG (1978) Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77–90

    Google Scholar 

  • Switzer RL, Sogin DC (1973) Regulation and mechanism of phosphoribosylpyrophosphate synthetase. V. Inhibition by end products and regulation by adenosine diphosphate. J Biol Chem 248:1063–1073

    Google Scholar 

  • Vande Woude GF, Oskarson M, Enquist LW, Nomura S, Sullivan M, Fischinger PJ (1979) Cloning of integrated Moloney sarcoma proviral DNA sequences in bacteriophage λ. Proc Natl Acad Sci USA 76:4464–4468

    Google Scholar 

  • White MN, Olszowy J, Switzer RL (1971) Regulation and mechanism of phosphoribosylpyrophosphate synthetase: repression by end products. J Bacteriol 108:122–131

    Google Scholar 

  • Wilson DB, Hogness DS (1966) Galactokinase and uridine diphosphogalactose 4-epimerase from Escherichia coli. Methods Enzymol 8:229–240

    Google Scholar 

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Authors and Affiliations

  1. Enzyme Division, University Institute of Biological Chemistry B, Sølvgade 83, DK-1307, Copenhagen K, Denmark

    Biarne Hove-Jensen

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  1. Biarne Hove-Jensen
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Communicated by P.T. Emmerson

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Hove-Jensen, B. Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli . Molec. Gen. Genet. 201, 269–276 (1985). https://doi.org/10.1007/BF00425670

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  • DOI: https://doi.org/10.1007/BF00425670

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