Figure 3

Generation and analyses of mef2A^-mutant strains. Panel A. Mutant AX2 and AX4 strains were generated by partially deleting the mef2A gene through homologous recombination. DNA was isolated from several clones and analyzed by PCR using oligonucleotides specific for the mef2A^- deleted region (mef2A) or for ribosomal DNA (rDNA) used as the internal control. The results obtained from non-mutated (AX4, AX2) and mutated (clones 37, 2 and 3) samples are shown. Panel B. Wild-type cells (AX4, AX2) and mef2A^- mutant cells (mef2A^-), derived from AX4 or AX2 cells were clonally grown on K. aerogenes for 4 days. Biological replicates were performed on several different plates. Pictures of the colonies were taken, and their size determined in three independent experiments. The average area of the colonies and the standard deviations are indicated under each picture. Scale bar: 5 mm. Panel C. Wild-type (AX4, AX2) and mutant cells (AX4/mef2A^-, AX2/mef2A^-) were collected and cultured under starvation conditions to study the multicellular development. The initial steps of aggregation, streaming and mound formation were assayed under submerged conditions (Mound column). For later stages of slug (Slug) and fruiting body (Culminant) formation, the cells were placed on nitrocellulose filters. Pictures were taken using a Leica stereomicroscope. Scale bar: 0.3 mm.