Fig. 2. Developmental regulation of ACG transcription and ACG protein accumulation. (A,B). Dictyostelium wild-type (WT) cells (A), the acrA mutant (B) and both cell lines transformed with the ACG::ile-gal construct were incubated for 24 hours on PB agar. Every 2 hours, WT and acrA- cells were harvested, lysed in SDS-PAGE sample buffer and immunoblotted with ACG antibody. The ACG antibody reacts with a single band of around 98 kD, which is the predicted size of ACG. (C) The ACG-ile-gal transformed cells were lysed and assayed for ß-galactosidase activity using a spectrophotometric assay (black and white squares). The data are expressed as percentage of ß-galactosidase activity measured at 24 hours in wild-type cells. The means of two experiments assayed in triplicate are presented. This panel also shows data obtained from a densitometric scan of the ACG bands in the immunoblots in A,B (black and white circles). The optical density values of the scan are expressed as percentage of the value obtained for WT at 24 hours. (D) Total RNA was extracted from developing WT cells at 2-hour intervals and subjected to RT-PCR for 25, 30 and 35 cycles using primers that yield a product that spans the two introns of the ACG gene. Product was first detectable after 30 cycles (shown here) from both cDNA (lower band) and contaminating gDNA (upper band) amplification. The control reaction lacked RNA.