Our objective is to characterize the adenylate cyclase of Bordetella pertussis, to clone its gene, and to use the gene to produce more of the protein than can be recovered from the bacterium. We have found that this enzyme is more complex than previously thought. Our biochemical studies allowed us to develop affinity procedures which we will use to purify this enzyme. Our initial experiments have also determined that binding of calmodulin is direct and we have developed methods to screen for the gene in recombinant DNA libraries based on the ability of the adenylate cyclase peptide to bind calmodulin. Cloned sequences will be characterized by sequencing and then manipulated for several purposes which include the production of enzyme in E. coli, the creation of specific Bordetella adenylate cyclase mutants, and the expression of the gene in eucaryotic cells. After physical characterization to resolve some of the conflicting results in the literature, we will study action of the adenylate cyclcase on mammalian target cells. Finally we will take advantage of the unusual evolution of the gene and attempt to use it for long term induction of adenylate cyclase and cAMP in mammalian cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI024240-04
Application #
3137097
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1986-12-01
Project End
1991-11-30
Budget Start
1989-12-01
Budget End
1991-11-30
Support Year
4
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Type
Schools of Medicine
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
Kessin, R H; Fleischmann, R D; Gottesman, M M et al. (1992) Use of the yeast low-Km cAMP-phosphodiesterase gene to control cyclic AMP levels in mammalian cells. Adv Second Messenger Phosphoprotein Res 25:13-27
Van Lookeren Campagne, M M; Franke, J; Kessin, R H (1991) Functional cloning of a Dictyostelium discoideum cDNA encoding GMP synthetase. J Biol Chem 266:16448-52
Van Lookeren Campagne, M M; Wu, E; Fleischmann, R D et al. (1990) Cyclic AMP responses are suppressed in mammalian cells expressing the yeast low Km cAMP-phosphodiesterase gene. J Biol Chem 265:5840-6
Van Lookeren Campagne, M M; Diaz, F V; Jastorff, B et al. (1990) Characterization of the yeast low Km cAMP-phosphodiesterase with cAMP analogues. Applications in mammalian cells that express the yeast PDE2 gene. J Biol Chem 265:5847-54
Van Lookeren Campagne, M M; Villalba Diaz, F; Chason, K W et al. (1990) Enzymatic synthesis of the cAMP antagonist (Rp)-adenosine 3',5'-monophosphorothioate on a preparative scale. Anal Biochem 188:86-90
Kessin, R H (1988) Genetics of early Dictyostelium discoideum development. Microbiol Rev 52:29-49