Selection of cDNAs for phosphodiesterases that hydrolyze guanosine 3';5'-monophosphate in Escherichia coli.
A genetic selection procedure has been developed which makes the growth of E. coli dependent on expression of a cGMP phosphodiesterase cDNA. E. coli does not contain a cGMP phosphodiesterase, and guanine auxotrophs cannot extract the guanine from cGMP. If a functional cGMP phosphodiesterase is introduced, then guaA auxotrophs will grow on cGMP as a guanine source. The method also selects GMP synthetase cDNAs, which complement the guanine auxotrophy directly. Expression of a Dictyostelium discoideum or human heart cyclic nucleotide phosphodiesterase cDNA permits growth of the E. coli guaA auxotroph in the presence of cGMP. Several commercial cDNA libraries were corrupt and contained phosphodiesterase and/or GMP synthetase sequences that were from a contaminating DNA source.
Department of Anatomy and Cell Biology, Columbia University, New York 10032.
M M
MM
Van Lookeren Campagne
F
F
Villalba-Díaz
E
E
Meacci
V C
VC
Manganiello
R H
RH
Kessin
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D006152:Cyclic GMP / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D004023:Dictyostelium / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006157:Guanosine Monophosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D009206:Myocardium / Q000201:enzymology; D010957:Plasmids; D012641:Selection, Genetic
D006157:Guanosine Monophosphate; D004247:DNA; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP