Transformation with vectors harboring the NEOR selection marker induces germination-specific adenylylcyclase activity in Dictyostelium cells.
Dictyostelium cells express an aggregative adenylylcyclase (ACA), responsible for oscillatory cAMP signaling, and a spore germination-specific adenylylcyclase (ACG). Overexpression of PKA regulatory (R) subunits blocks oscillatory cAMP signaling but increases basal cAMP levels, while neither ACA nor ACG mRNA could be detected. To test whether a novel type of adenylylcyclase (AC) was responsible for cAMP synthesis, dominant-negative PKA-R subunits (PKA-RM) and control R-subunits (PKA-RC) were overexpressed in ACA null mutants. Both transformations as well as transformation with an unrelated vector, carrying the same NEOR selection marker, induced an AC activity in growing cells with the biochemical characteristics of ACG. Similar vectors with a different URA selection marker did not increase AC activity. We conclude that the amino-glycoside phosphotransferase encoded by the very commonly used NEOR selection marker induces ectopic ACG activity in Dictyostelium cells.
Schulkes
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CC
Cell Biology Section, University of Leiden, The Netherlands.
Verkerke-van Wijk
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Schaap
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D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis; D004023:Dictyostelium / Q000166:cytology / Q000502:physiology*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D015870:Gene Expression; D005819:Genetic Markers; D005822:Genetic Vectors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D019868:Kanamycin Kinase; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D015398:Signal Transduction; D014162:Transfection
D005819:Genetic Markers; D012333:RNA, Messenger; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases