EVIDENCE FOR THE TRANSFORMATION OF DICTYOSTELIUM DISCOIDEUM WITH HOMOLOGOUS DNA

Abstract

ABSTRACT A transformation system for Dictyostelium discoideum should facilitate the isolation and in vivo study of genes important for slime mold differentiation. We are developing a system in which mutant slime mold amoebae unable to utilize Bacillus subtilis as a food source are converted to Bacillus “prototrophy” by treatment with wild type slime mold DNA. The bsgA5 mutation, preventing growth of Dictyostelium amoebae on B. subtilis , is recessive and non-leaky, maps at a single locus, and reverts infrequently, all factors favorable for transformation. Mutant amoebae, after DNA treatment and brief permissive growth, are plated selectively upon lawns of B. subtilis . High molecular weight DNA preparations from a Bsg + slime mold strain generate Bsg + colonies at an average frequency of O.24 × 10–7, roughly one log above that of reversion of parallel cultures treated with calcium phosphate alone. We have ligated restricted wild type slime mold DNA to DNA from the pBR322-yeast-slime mold plasmid ARS22. We are now analyzing Bsg + colonies obtained after treatment of Bsg cells with this recombinant DNA. One transformant contained two DNA fragments that hybridize to pBR322; the untreated Bsg strain contains no such fragment. These data constitute molecular evidence for transformation and offer the possibility of recovery of the bsgA gene. Isolation of this gene should allow high frequency transformation of Dictyostelium .