title: Homologous gene replacement in Physarum.
abstract: The protist Physarum polycephalum is useful for analysis of several aspects of cellular and developmental biology. To expand the opportunities for experimental analysis of this organism, we have developed a method for gene replacement. We transformed Physarum amoebae with plasmid DNA carrying a mutant allele, ardD delta 1, of the ardD actin gene; ardD delta 1 mutates the critical carboxy-terminal region of the gene product. Because ardD is not expressed in the amoeba, replacement of ardD+ with ardD delta 1 should not be lethal for this cell type. Transformants were obtained only when linear plasmid DNA was used. Most transformants carried one copy of ardD delta 1 in addition to ardD+, but 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, development and meiosis. We found no effect of ardD delta 1 on viability, growth, or development of any of the various cell types of Physarum. Thus, the carboxy-terminal region of the ardD product appears not to perform a unique essential role in growth or development. Nevertheless, this method for homologous gene replacement can be applied to analyze the function of any cloned gene.
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
authors.forename: T G
authors.initials: TG
authors.lastname: Burland
authors.forename: D
authors.initials: D
authors.lastname: Pallotta
mesh_terms: D000199:Actins / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005800:Genes, Fungal / Q000235:genetics*; D017125:Genes, Protozoan / Q000235:genetics*; D005822:Genetic Vectors; D008540:Meiosis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010804:Physarum / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
chemical_list: D000199:Actins; D012333:RNA, Messenger