Abstract
Disruption of either the RDEA or REGA genes leads to rapid development in Dictyostelium. The RDEA gene product displays homology to certain H2-type phosphotransferases, while REGA encodes a cAMP phosphodiesterase with an associated response regulator. It has been proposed that RDEA activates REGA in a multistep phosphorelay. To test this proposal, we examined cAMP accumulation in rdeA and regA null mutants and found that these mutants show a pronounced accumulation of cAMP at the vegetative stage that is not observed in wild-type cells. This accumulation was due to a novel adenylyl cyclase and not to the known Dictyostelium adenylyl cyclases, aggregation stage adenylyl cyclase (ACA) or germination stage adenylyl cyclase (ACG), since it occurred in an acaA/rdeA double mutant and, unlike ACG, was inhibited by high osmolarity. The novel adenylyl cyclase was not regulated by G-proteins and was relatively insensitive to stimulation by Mn2+ ions. Addition of the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) permitted detection of the novel adenylyl cyclase activity in lysates of an acaA/acgA double mutant. The fact that disruption of the RDEA gene as well as inhibition of the REGA-phosphodiesterase by IBMX permitted detection of the novel AC activity supports the hypothesis that RDEA activates REGA.
Highlights
Disruption of either the RDEA or REGA genes leads to rapid development in Dictyostelium
Materials and Cell Lines—2Ј-Deoxyadenosine 3Ј,5Ј-monophosphate (2ЈH-cAMP), guanosine 5Ј-O-(2-thiodiphosphate) (GDPS), guanosine 5Ј-O-(3-thiotriphosphate) (GTP␥S), 3-isobutyl-1-methylxanthine (IBMX), dithiothreitol (DTT), and G418 were from Sigma, blasticidin was from ICN, and [3H]cAMP was from Amersham Pharmacia Biotech (Little Chalfont, United Kingdom)
In Vivo cAMP Accumulation in rdeA and regA Mutants—To investigate cAMP accumulation in rdeA and regA null mutants, we first performed a standard assay for the adenylyl cyclase (ACA)-mediated cAMP relay response [16]
Summary
Materials and Cell Lines—2Ј-Deoxyadenosine 3Ј,5Ј-monophosphate (2ЈH-cAMP), guanosine 5Ј-O-(2-thiodiphosphate) (GDPS), guanosine 5Ј-O-(3-thiotriphosphate) (GTP␥S), 3-isobutyl-1-methylxanthine (IBMX), dithiothreitol (DTT), and G418 were from Sigma, blasticidin was from ICN, and [3H]cAMP was from Amersham Pharmacia Biotech (Little Chalfont, United Kingdom). The acaA/acgA line was obtained by transforming the acaA line with the ACG gene, in which an internal XbaI-EcoRV fragment was replaced by a blasticidin expression cassette [15]. Assays for cAMP Accumulation by Intact Cells—Cells were harvested from growth medium, washed with 10 mM sodium/potassium phosphate buffer, pH 6.5 (PB), and either resuspended directly in PB at 108 methylxanthine; PDE, phosphodiesterase; 2ЈH-cAMP, 2Ј-deoxyadenosine 3Ј,5Ј-monophosphate; GDPS, guanosine 5Ј-O-(2-thiodiphosphate); GTP␥S, guanosine 5Ј-O-(3-thiotriphosphate); DTT, dithiothreitol; PB, phosphate buffer; ACB, adenylyl cyclase B.
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