FIG. 4.

Disruption of the eppA gene and evaluation of development and growth of Dictyostelium cells. (A) Expression of the eppA gene during development. Wild-type cells (HS176) growing in suspension culture were collected by centrifugation, followed by a wash with phosphate buffer. The cell pellet was resuspended in the buffer, and 1 × 10⁷ cells (in 1 ml buffer) were placed on top of Ca/Mg-phosphate agar plates. RNA was isolated at the indicated times. RT-PCR was performed with eppA-specific primers after generation of first-strand cDNA by reverse transcriptase. PCR mixtures were subjected to electrophoresis on a 1% agarose gel. As mentioned in Materials and Methods, IG7 is a control constitutively expressed gene. com is an eppA⁻ disruptant completed by myc-EppA. (B) EppA is required for normal Dictyostelium development. A total of 10⁶ cells in 100 μl of phosphate buffer were placed on phosphate agar plates, and cells were allowed to develop at 22°C in a moist chamber. Pictures were taken at the indicated times. Bar, 1 mm. wt, wild-type parental cells; eppA⁻, cells with the eppA gene disrupted by targeting construct; wt/myc-EppA, wild-type cells overexpressing myc-EppA; wt/myc-EppA(S250A), wild-type cells overexpressing myc-EppA carrying the S250A point mutation; eppA⁻/myc-EppA, eppA knockout cells expressing myc-EppA. (C) Effect of EppA on Dictyostelium cell growth. Cells were grown in suspension, and the doubling time was calculated from cell density measurements made during the exponential-growth phase. Results are means ± standard errors of the means from three experiments. (D) Expression of cAR1 in early development of Dictyostelium. Cells were starved in PB and pulsed with 100 nM cAMP for varying times after 2 h of starvation. Aliquots of cells were taken at different time points, lysed by 2× SDS sample buffer, and Western blotted (WB) with an anti-cAR1 antibody. (E) cAMP-dependent activation of DdERK2. Cells were starved and pulsed for 7 h, treated with caffeine for 10 min, stimulated with 10 μM cAMP, lysed by an equal volume of 2× SDS sample buffer at 0 and 30 s after stimulation, and Western blotted with an anti-DdERK2 antibody. After stimulation, a higher-mobility band indicates the activated DdERK2 protein under these gel electrophoresis conditions.