Summary
The pepN gene, that encodes aminopeptidase N in Escherichia coli, has been cloned into the multicopy plasmid pBR322. Expression of the cloned pepN gene results in overproduction of the enzyme. The restriction map of the 6.7 Kb insert was established and the gene was further localized by analysis of the in vitro constructed deletion plasmid and mutant plasmids generated by Tn5 insertions. Chromosome mobilization experiments, using pepN-lac fusion strains allowed us to infer a clockwise direction of transcription for the pepN gene.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bally M, Murgier M, Tommassen J, Lazdunski A (1983a) Physical mapping of the gene for Aminopeptidase N in E. coli K12. Mol Gen Genet (in press)
Bally M, Murgier M, Lazdunski A (1983b) Molecular cloning and amplification of the gene for aminopeptidase of E. coli. FEMS Microbiol Lett 19:261–265
Bankaitis VA, Bassford PJ (1982) Regulation of adenylate cyclase synthesis in E. coli: studies with cya-lac operon and protein fusion strains. J Bacteriol 151:1346–1357
Berg, DE (1977) Insertion and excision of the transposable kanamycin resistance determinant Tn5. In: Bukhari AI, Shapiro JA, Adhya SL (eds) DNA insertion elements, plasmids and episomes. Cold Spring Harbor Press, New York pp 205–212
Chappelet-Tordo, D, Lazdunski C, Murgier M, Lazdunski A (1977) Aminopeptidase N from E. coli: Ionizable active center groups and substrate specificity. Eur J Biochem 81:299–305
Chumley FG, Menzel R, Roth JR (1979) Hfr formation directed by Tn10. Genetics 91:639–655
Clarke L, Carbon J (1976) A colony bank containing synthetic ColE1 hybrid plasmids representative of the entire E. coli genome. Cell 9:91–99
Humphreys GO, Willshaw GA, Anderson ES (1975) A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta 383:457–463
Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucl. Acids Res 9:529–543
Laskey RA, Mills AD (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56:335–341
Latil M, Murgier M, Lazdunski A, Lazdunski C (1976) Isolation and genetic mapping of E. coli aminopeptidase mutants. Molec Gen Genet 148:43–47
Lazdunski A, Pellissier C, Lazdunski C (1975a) Regulation of E. coli K10 aminoendopeptidase synthesis. Effects of mutations involved in the regulation of alkaline phosphatase. Eur J Biochem 60:357–362
Lazdunski A, Murgier M, Lazdunski C (1975b) Evidence for an aminopeptidase localized near the cell surface of E. coli. Eur J Biochem 60:349–355
Lederberg EM, Cohen S (1974) Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol 119:1072–1074
Lugtenberg B, Meijers J, Peters R, Van der Hoek P, Van Alphen L (1975) Electrophoretic resolution of the “Major outer membrane protein” of E. coli K12 into four bands. FEBS Lett 58:254–258
McCaman MT, Villarejo MR (1982) Structural and catalytic properties of peptidase N from Escherichia coli K12. Arch Biochem Bioph 213:384–394
Michaels G, Kelln RA (1983) Construction and use of pyr::lac fusion strains to study regulation of pyrimidine biosynthesis in Salmonella typhimurium. Mol Gen Genet 189:463–470
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Miller CG, Mackinnon K (1974) Peptidase mutants of Salmonella typhimurium. J Bacteriol 120:355–363
Miller CG, Schwartz G (1978) Peptidase deficient mutants of Escherichia coli. J Bacteriol 135:603–611
Murgier M, Gharbi S (1982) Fusion of the lac genes to the promoter for the aminopeptidase N gene of E. coli. Mol Gen Genet 187:316–319
Pages C, Lazdunski C, Lazdunski A (1982) The receptor of bacteriophage Lambda: evidence for a biosynthesis dependent on lipid synthesis. Eur J Biochem 122:381–386
Rothstein SJ, Reznikoff WS (1981) The functional differences in the inverted repeats of Tn5 are caused by a single base pair non homology. Cell, 23:191–199
Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693
Tommassen J, van der Ley P, van der Ende A, Bergmans H, Lugtenberg B (1982) Cloning of ompf, the structural gene for an outer membrane pore protein of E. coli K12: physical localization and homology with the phoE gene. Mol Gen Genet 185:105–110
Yang L, Somerville RL (1976) Prufication and properties of a new aminopeptidase from E. coli K12. Biochim Biophys Acta, 445:406–419
Yan C, Green L, Miller CG (1980) Degradation of intracellular protein in Salmonella typhimurium peptidase mutants. J Mol Biol 143:21–23
Author information
Authors and Affiliations
Additional information
Communicated by H. Böhme
Rights and permissions
About this article
Cite this article
Bally, M., Murgier, M. & Lazdunski, A. Cloning and orientation of the gene encoding aminopeptidase N in Escherichia coli . Mol Gen Genet 195, 507–510 (1984). https://doi.org/10.1007/BF00341454
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00341454