Fig. 4. Prespore and spore differentiation in adenylyl cyclase mutants. (A) Slugs of wild-type, acrA-, acg- and acrA-/A15::ACGcat cells that had migrated for 2-3 hours were dissociated and stained with a spore-specific antiserum and GARFITC. Cell nuclei were counterstained with DAPI. The percentage of prespore cells (cells with at least 3-4 fluorescent vacuoles) relative to DAPI-stained cells was determined. Means and SE of four experiments are presented. Significant differences (P>0.95) between datasets connected by brackets, as determined by Kruskal-Wallis ANOVA on ranks using SigmaStat software (Systat, San Jose, US), are indicated by asterisks. (B) Wild-type, acrA-, acg- and acrA-/A15::ACGcat cells were developed on PB agar until fruiting bodies had formed. Total RNA was extracted at 2-hour intervals, size-fractionated on 1.5% agarose gels containing 2.2 M formaldehyde and transferred to nylon membranes (Nellen et al., 1987). The four northern blots were hybridized in the same batch to a [³²P]dATP-labelled CotB probe at 65^oC, then stripped and reprobed with the constitutively expressed gene 1G7 (Williams et al., 1987). (C) Three-day-old fruiting bodies of acrA-, acg- and acrA-/A15::ACGcat cells were transferred to a glass slide and stained with the cellulose dye Calcofluor at 0.03% (w/v) final concentration. The preparations were photographed under UV by fluorescence microscopy to visualize the Calcofluor-stained spores in the presence of a low level of transillumination to obtain a phase contrast image of the remaining amoebae. Scale bar: 10 µm.