Recombination of selectable marker DNA in Nicotiana tabacum.
A chimeric neomycin phosphotransferase II (NPT II) gene, which normally provides kanamycin resistance to transformed plant cells, was inactivated by in vitro deletions. Repair plasmids not containing plant-specific transcription signals but containing only the NPT II coding region (or parts of it) were used in co-transformation experiments involving direct DNA uptake into protoplasts isolated from Nicotiana tabacum. Recombination, or gene conversion mediated by homologous sequences produced active NPT II genes in about 1% of transformants, rendering these cells resistant to kanamycin. Analysis of the size of the active enzyme indicated that recombination had occurred producing an NPT II gene indistinguishable from the wild-type gene. Southern blot analysis revealed that the bulk of co-transformed donor plasmid DNA had suffered structural modifications; however, kanamycin resistance was inherited in a Mendelian fashion, indicating that at least one functional and structurally intact copy of the regenerated NPT II gene is integrated into the host genome.
D004274:DNA, Recombinant / Q000378:metabolism*; D004351:Drug Resistance; D005785:Gene Conversion; D005796:Genes; D005809:Genes, Regulator; D005819:Genetic Markers; D007612:Kanamycin / Q000494:pharmacology; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000235:genetics*; D010947:Plants, Toxic; D010957:Plasmids; D011523:Protoplasts / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D012641:Selection, Genetic; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000166:cytology / Q000235:genetics*; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007612:Kanamycin; D010770:Phosphotransferases; D019868:Kanamycin Kinase