Analysis of a complex plasmid insertion in a phototaxis-deficient transformant of Dictyostelium discoideum selected on a Micrococcus luteus lawn.
A novel method for clonal selection of G418-resistant Dictyostelium discoideum transformants on lawns of Micrococcus luteus was developed. The procedure was used to isolate transformants deficient in phototaxis after nontargeted insertion of shuttle vector DNA. Southern blot analysis as well as restriction, T-tracking, and sequencing analysis of plasmids rescued from the genomic DNA of one of the putative phototaxis gene disruptants showed that it contained a complex multicopy insertion of the vector. While insertions of such plasmid vectors might typically be in tandem multicopy format, they can be much more complex, containing, as in this case, inverse as well as tandemly duplicated copies and various deletions.
Wilczynska
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Department of Microbiology, La Trobe University, Bundoora, Melbourne, Australia.
Fisher
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D000818:Animals; D001483:Base Sequence; D002465:Cell Movement / Q000528:radiation effects; D004271:DNA, Fungal / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics*; D004023:Dictyostelium / Q000235:genetics* / Q000254:growth & development / Q000528:radiation effects; D004352:Drug Resistance, Microbial; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D019868:Kanamycin Kinase; D008027:Light; D016982:Micrococcus luteus; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D012641:Selection, Genetic; D014170:Transformation, Genetic
D004271:DNA, Fungal; D016054:DNA, Protozoan; D005839:Gentamicins; C010680:antibiotic G 418; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase