Affiliation:
1. Département de Biologie, Centre d'Etudes Nucléaires de Saclay, Gif-sur Yvette, France.
Abstract
Several unknown Escherichia coli genes for different species of acid phosphatase were cloned in vivo with the plasmid Mu dII4042. When present in a multicopy state, each gene promoted hydrolysis of p-nitrophenyl-phosphate at acidic pH. Among seven recombinant clones that encoded periplasmic acid phosphatase activities, five different genes could be distinguished by the pH optimum and substrate preference for the enzyme and by the restriction enzyme pattern. A 1.7-kilobase recombinant DNA fragment, common to two clones, was inserted into plasmid pBR322 and shown to contain a new gene, agp, which leads to the overexpression of the periplasmic acid glucose-1-phosphatase, a dimer of a 44-kilodalton polypeptide. Fusions of agp to gene phoA deprived of its own signal sequence conferred an alkaline phosphatase-positive phenotype to bacteria, showing the presence of an export signal on agp. The resulting hybrid proteins were characterized by immunoprecipitation with an antiserum directed against purified acid phosphatase or against alkaline phosphatase, showing that agp is the structural gene of the acid phosphatase. The beginning, the orientation, and the end of gene agp on the cloned DNA fragment were determined by the characteristics of such hybrid proteins.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Reference31 articles.
1. A new cyclic phosphodiesterase having a 3' nucleotidase activity from Escherichia coli B. I. Purification and properties of the enzyme;Anraku Y.;J. Biol. Chem.,1964
2. A new cyclic phosphodiesterase having a 3' nucleotidase activity from Escherichia coli B. II. Further studies on substrate specificity and mode of action of the enzyme;Anraku Y.;J. Biol. Chem.,1964
3. Anraku Y. 1966. Cyclic phosphodiesterase of Escherichia coli p. 130-149. In D. Cantoni and B. Davis (ed.) Procedures in nucleic acid research. Harper & Row Publishers Inc. New York.
4. Periplasmic enzymes in gram-negative bacteria;Beacham I. R.;Int. J. Biochem.,1979
5. Isolation of Escherichia coli mutants (cpdB) deficient in periplasmic 2':3'-cyclic phosphodiesterase and genetic mapping of the cpdB locus;Beacham I. R.;J. Gen. Microbiol.,1980
Cited by
50 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献