CYTOPLASMIC DYNEIN HEAVY-CHAIN IS AN ESSENTIAL GENE-PRODUCT IN DICTYOSTELIUM

We describe here three different approaches to perturb cytoplasmic dyn ein heavy chain (DHC) gene function in Dictyostelium: integration of a marker Into the heavy chain coding sequence by homologous recombinati on to disrupt transcription, expression of antisense RNA to inhibit tr anslation, and expression of a 158 kDa amino-terminal coding region to perturb the native protein organization. By homologous recombination; we fail to obtain cells that lack an intact DHC gene product. Cells c ontaining antisense orientation plasmids (but not sense) appear to die 4 to 6 days following transformation. Plasmids designed to overexpres s an amino-terminal region of the DHC result in substantially reduced transformation efficiency. When expressed at low levels, the truncated amino-terminal product appears capable of dimerizing with an intact h eavy chain or with itself, essentially producing a cargo-binding domai n lacking mechanochemical activity. This, in turn, likely competes wit h the native protein's function. These three approaches taken together indicate that the dynein heavy chain is an essential gene in Dictyost elium. (C) 1998 Wiley-Liss, Inc.