HOMOLOGOUS GENE REPLACEMENT IN PHYSARUM

The protist Physarum polycephalum is useful for analysis of several as pects of cellular and developmental biology. To expand the opportuniti es for experimental analysis of this organism, we have developed a met hod for gene replacement. We transformed Physarum amoebae with Plasmid DNA carrying a mutant allele, ardD Delta 1, of the ardD actin gene; a rdD Delta 1 mutates the critical carboxy-terminal region of the gene p roduct. Because ardD is not expressed in the amoeba, replacement of ar dD(+) with ardD Delta 1 should not be lethal for this cell type. Trans formants were obtained only when linear plasmid DNA was used. Most tra nsformants carried one copy of ardD Delta 1 in addition to ardD(+), bu t in two (5%), ardD(+) was replaced by a single copy of ardD Delta 1. This is the first example of homologous gene replacement in Physarum. ardD Delta 1 was stably maintained in the genome through growth, devel opment and meiosis. We found no effect of ardD Delta 1 on viability, g rowth, or development of any of the various cell types of Physarum. Th us, the carboxy-terminal region of the ardD product appears not to per form a unique essential role in growth or development. Nevertheless, t his method for homologous gene replacement can be applied to analyze t he function of any cloned gene.