title: Efficient transformation of Dictyostelium discoideum amoebae.
abstract: We have transformed Dictyostelium discoideum amoebae by using derivatives of a plasmid, pAG60, which was designed for transformation of mammalian cells. The plasmid carries the promoter region of the herpes simplex virus type 1 thymidine kinase gene linked to the bacterial gene kan, which codes for the enzyme aminoglycoside 3'-phosphotransferase. kan is derived from the Tn5 transposon. Expression of the phosphotransferase permits direct selection of transformed cells by their resistance to the antibiotic G-418. pAG60 is incapable of transforming D. discoideum but is made transformation proficient by cloning D. discoideum sequences into the tetracycline resistance gene. The majority of transformed cells grow and develop normally and differentiate to give G-418-resistant spores. These transformants are unstable and rapidly lose their G-418-resistance during growth in the absence of antibiotic selection. Southern blots show that these unstable G-418-resistant transformants carry the pBR322 and kan sequences of pAG60. The pAG60-D. discoideum recombinant plasmids used for transformation were constructed in a way that might make them mutagenic. We have isolated several developmental mutants after transformation of D. discoideum with libraries of pAG60-D. discoideum recombinant plasmids. These mutants are G-418 resistant and carry pAG60 in their nuclear DNA. We recovered a pAG60-D. discoideum recombinant plasmid from several developmental mutants. This plasmid transforms D. discoideum at an elevated frequency and integrates into the nuclear genome. We speculate that integration can result in insertional inactivation of genes that are essential for differentiation but not for growth. Mutagenic transformation occurred only if the transforming plasmid had homology with D. discoideum nuclear DNA. A mammalian cell transformation vector, pSV2-neo, carried no D. discoideum sequences and was able to transform. However, pSV2-neo transformation was not mutagenic. These results suggest that direct inactivation and recovery of genes that are essential for differentiation of D. discoideum will be possible.
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004262:DNA Restriction Enzymes; D004023:Dictyostelium / Q000187:drug effects / Q000235:genetics*; D004351:Drug Resistance; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005839:Gentamicins / Q000494:pharmacology; D019868:Kanamycin Kinase; D009876:Operon; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D005839:Gentamicins; D003513:Cycloheximide; C010680:antibiotic G 418; D010770:Phosphotransferases; D013937:Thymidine Kinase; D019868:Kanamycin Kinase; D004262:DNA Restriction Enzymes