Fig. 1. Isolation of a mutant defective in DIF responses. (A) The 8-Br-cAMP monolayer assay as an enrichment for DIF-1 non-responsive mutants. (B) The dimA^– mutant shows no DIF-1 response in monolayer assays; compare with wild type (AX4). DIF-1 responses were measured in 8-Br-cAMP or cAMP removal monolayer assays. Results shown are the mean of three experiments. (C) The dimA^– mutant shows no DIF-1 response in a dissociated cell assay. Wild-type AX4 or dimA^– mutant cells were harvested at the mound stage of development and disaggregated. Cells were shaken for 4 hours in buffer containing 1 mM cAMP, with or without 100 nM DIF-1. Total RNA was extracted and, following northern transfer, probed with the ecmA and ecmB (prestalk), and cotB (prespore), markers. Methylene Blue staining of ribosomal RNA (rRNA) is shown as a loading control. (D) Prestalk (ecmAO/lacZ and ecmB/lacZ) and prespore (cotB/lacZ) reporter constructs are DIF-1 non-responsive in the dimA^– mutant in monolayer assays. Results are averages and standard deviations of two biological replicates, where each assay was performed in triplicate.