Figure 3.

 Differentiation of Dictyostelium with exogenous regulatory factors. (A) Differentiation in suspension cultures. Wild-type JH10 and car4 null cells were harvested from vegetative growth and resuspended for differentiation in culture. For the first 5 hr, cAMP was added at 6-min intervals to a final concentration of 30 nm. Subsequently, cultures were left untreated or incubated with additional cAMP, adenosine, ADA, and/or DIF-1, as described below. Samples were extracted for RNA and, in some cases, for DIF-1. (B) Regulation of cell-specific gene expression by cAMP and adenosine. Wild-type JH10 and car4 null cells were pulsed (P) with cAMP for 5 hr and then treated with 1 mm cAMP, 10 mm adenosine (Ado), and/or 2 U/ml of ADA for an additional 5 hr, as described in A. RNAs were isolated and hybridized on Northern blots to the prestalk probe ecmA and the prespore probe cotB. (C) Regulation of cell-specific gene expresion by DIF-1. Wild-type JH10 and car4 null cells were pulsed with cAMP and then treated with 300 μm cAMP and/or 300 nm DIF-1, as described in A. RNAs were isolated from cells during vegetative growth (V), after 5 hr of cAMP pulses (P), or following various incubations after pulsing as indicated. Corresponding Northern blots were hybridized to the prestalk probes ecmA and ecmB and the prespore probe cotB.