Complementation of myosin null mutants in Dictyostelium discoideum by direct functional selection.
The eukaryotic slime mold Dictyostelium discoideum contains a single conventional myosin heavy chain gene (mhcA). Cell lines in which this gene was deleted via homologous recombination have been previously reported. These myosin null cells were shown to be defective for cytokinesis and for sporogenesis. We demonstrate here that the cloned mhcA gene can be reintroduced into these cells by the use of a direct functional selection. This selection was imposed by demanding that cells be capable of growth in suspension. The resulting transformants appear normal for cytokinesis, and also are fully competent for sporogenesis, confirming that reintroduction of the myosin gene is sufficient to restore these properties. These results demonstrate a method for rescuing mutants in Dictyostelium which may be generally applicable for genetically created mutations as well as for mutations which have been engineered.
Department of Cell Biology, Stanford University School of Medicine, California 94305.
T T
TT
Egelhoff
D J
DJ
Manstein
J A
JA
Spudich
D015139:Blotting, Southern; D015153:Blotting, Western; D004271:DNA, Fungal / Q000235:genetics; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D005455:Fluorescent Antibody Technique; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D009154:Mutation; D015879:Myosin Subfragments / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic
D004271:DNA, Fungal; D015879:Myosin Subfragments