Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA.
Introduction of restriction enzyme along with linearized plasmid results in integration of plasmid DNA at genomic restriction sites in a high proportion of the resulting transformants. We have found that electroporating BamHI or EcoRI together with pyr5-6 plasmids cut with the same enzyme stimulates the efficiency of transformation in Dictyostelium discoideum more than 20-fold over the rate seen when plasmid DNA alone is introduced. Restriction enzyme-mediated integration generates insertions into genomic restriction sites in an apparently random manner, some of which cause mutations. About 1 in 400 of the Dictyostelium transformants displayed arrested or aberrant development. The integrated plasmid, along with flanking genomic DNA, was excised from some of these mutants, cloned in Escherichia coli, and used to transform other Dictyostelium cells. Homologous recombination within the flanking sequences resulted in the same phenotypes displayed by the original mutants, directly demonstrating that the affected genes were responsible for the specific morphological phenotypes. This method of insertional mutagenesis should be useful for tagging, and subsequent cloning, of many developmentally important genes that can be identified by their mutant phenotypes.
Kuspa
A
A
Department of Biology, University of California, San Diego, La Jolla 92093.
Loomis
W F
WF
D000818:Animals; D002449:Cell Aggregation; D002872:Chromosome Deletion; D004252:DNA Mutational Analysis; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics*; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D005800:Genes, Fungal; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014162:Transfection
D004271:DNA, Fungal; D004262:DNA Restriction Enzymes