A segment of DNA containing cya, the gene for adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], has been isolated from Salmonella typhimurium. The phage Agt4 was used as a cloning vector and adenylate cyclase-positive hybrid phages were isolated that complemented adenylate cyclase-negative bacteria. The cloned DNA fragment encodes a polypeptide of molecular weight '81,000 that gives rise to adenylate cyclase activity. This protein represents a functional, mutant of the bacterial adenylate cyclase. When the cya gene was amplified by inserting into a multicopy plasmid, the enzyme activity was overproduced 20-fold, but the cyclic AMP level increased only 60%, suggesting several probable regulatory mechanisms. Overprod_uction ofenzymes by recombinant DNA techniques can be a useful probe of relationships in the metabolizing organism in vivo.Although many basic principles of regulation have been defined, it is usually quite difficult to make a thorough investigation of the interplays within particular systems, especially those in which a general "second messenger" modulates a variety of different functions. The best-established example of a second messenger is cyclic adenosine 3',5'-monophosphate (cAMP). In both eukaryotes and prokaryotes, cAMP initiates the response of cellular metabolism to external stimuli (1, 2). In mammalian systems, cAMP-dependent protein kinase is an essential component of the regulatory scheme (3, 4). However, mutants insensitive to exogenous cAMP have been found to contain this kinase activity at a normal level, suggesting the possibility ofother roles for cAMP (5). Regulatory protein phosphorylation has been found in prokaryotes (6-9), but cAMP in these cells acts largely at the level of transcription. Approximately 30 operons are under cAMP control in Escherichia coli (10). The expression of these genes-e.g., the lac, mel, orfla operons-requires cAMP and its binding protein as a positive effector. The inhibition ofcAMP synthesis by glucose is partially responsible for the state of "catabolite repression" (10-12).However, a group of mutants resistant to catabolite repression has been isolated, and, interestingly, they contain a lowslevel ofcAMP (13,14 The E. coli adenylate' cyclase-negative (Cya-) mutant used, obtained from A. J. Clark, was strain 5080.(Acya, str', suA, his-). CA8000 (Hfr, thi) and its Cya-derivative CA8306 (Acya) were given to us-by J. Beckwith (21). The Agt7 Salmonella gene pool was obtained from R. Davis. A phages were propagated with E. coli strain C600 as host.Synthesis of A-Encoded Proteins in Vivo. The proteins encoded by the hybrid phage were labeled with [3S]methionine in vivo by using the procedure of Jaskunas et aL (22). Strain N01406 was grown to early logarithmic phase (OD6w = 0.25) on 3-(N-morpholino)propanesulfonic acid (Mops) medium (23) containing 10 mM sodium phosphate, 1% glycerol, and 0.2% maltose. Cells were collected by centrifugation and washed twice with the growth medium without maltose. The washed cells were then resuspen...

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