Genetics, Vol 139, 147-158, Copyright © 1995

INVESTIGATIONS

Homologous Gene Replacement in Physarum

T. G. Burland and D. Pallotta
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

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.

This article has been cited by other articles:

				G. Pierron, D. Pallotta, and M. Benard The One-Kilobase DNA Fragment Upstream of the ardC Actin Gene of Physarum polycephalum Is Both a Replicator and a Promoter Mol. Cell. Biol., May 1, 1999; 19(5): 3506 - 3514. [Abstract] [Full Text] [PDF]

				P. A. Lefebvre and C. D. Silflow Chlamydomonas: The Cell and Its Genomes Genetics, January 1, 1999; 151(1): 9 - 14. [Full Text]