Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis

Abstract

The single gene encoding calmodulin in the eukaryotic microorganism Dictyostelium discoideum was cloned and sequenced. The gene was found to contain three introns, one lying immediately after the translation initiation codon. The deduced amino acid sequence indicated that Dictyostelium calmodulin contains 19 amino acid differences from vertebrate calmodulin, including extensions at both termini. Northern blot analysis showed that similar levels of calmodulin mRNA are present throughout growth and development of wild-type cells. A complete copy of the calmodulin cDNA was prepared, and an 87-base pair fragment complementary to the 5'-end of the calmodulin mRNA was subcloned into the Dictyostelium transformation vector pVEII, such that expression of the antisense transcript was driven by the discoidin I gamma promoter. Transformed cells were selected and maintained at low cell density, a condition resulting in minimal activity of the discoidin I promoter. High level expression was induced by allowing the transformants to reach high cell density or by growing them in the presence of medium conditioned by high density cells. Under these conditions, in which calmodulin mRNA and protein levels were reduced about twofold, the calmodulin antisense transformants lost the ability to complete cytokinesis. A contractile ring formed and constricted, but the midbody linking daughter cells failed to break. The resulting cell population contained multinucleated cells and networks of cells connected by cytoplasmic bridges. Normal cell division was restored when the cells were diluted to low density. These observations have identified a new point at which calmodulin may regulate cell cleavage.