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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.
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