Homologous gene replacement in Physarum.
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.
Burland
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McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Pallotta
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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
D000199:Actins; D012333:RNA, Messenger