Figure 7

(A) Prespore and prestalk specific gene expression is altered in grlJ^-. Total RNA was isolated from A×2 and grlJ^- at the indicated time points. 20 μg RNA were separated on 1.2 % agarose gels under denaturing conditions (6% formaldehyde) and transferred to membranes as described [54]. cDNA probes specific for the indicated transcripts were used for hybridisation. A CAP cDNA probe was used as a control. The CAP specific message shows an increase during early aggregation and then returns to the level of growing cells [55]. Additionally, RNA from the above time course experiment was used to generate single stranded cDNA (see Methods) and RT-PCR was carried out for analysis of the expression of the stalk specific gene carB (cyclic AMP receptor 2). The actin8 gene was amplified as a control. (B) The grlJ^- strain has no pattern formation defect. grlJ^- cells were transfected using electroporation with the ecmB-Gal and pspA(D19)-Gal plasmids separately. The transformants were selected with 4 μg/ml G418 and 3 μg/ml of blasticidin. Single colonies were obtained by replica plating and they were developed on a nitrocellulose filter until the slug and culminant stages and then processed for LacZ expression as described [50]. Bar, 200 μm.