title: A transient assay in plant cells reveals a positive correlation between extrachromosomal recombination rates and length of homologous overlap.
abstract: An assay to monitor homologous recombination in plant cells has been established by cotransfecting Nicotiana plumbaginifolia protoplasts with different topological forms of plasmids of various deletion mutants of a non-selectable marker gene, the beta-glucuronidase (GUS) gene. Transient GUS enzyme activities were measured by a sensitive assay. In the nuclear DNA of the cotransfected protoplasts the recombined complete GUS gene could be detected by a specially modified PCR analysis. In comparison to the standard assay, which monitors homologous recombination by integration of a selectable marker, the described assay avoids position effects of gene expression, is fast, easy to handle and large numbers of samples can be processed simultaneously. We were able to demonstrate a positive correlation between the length of overlapping homology (up to 1200 base pairs) of the transfected supercoiled circular or linearized plasmids and the respective GUS activities. We found a significant drop in the recombination rates when the overlap of both substrates was reduced to 456 basepairs or less. The requirement for such a long stretch of homology for efficient recombination might ensure the stability of the rather repetitive plant genome.
authors.affiliation: Friedrich Miescher-Institut, Basel, Switzerland.
authors.forename: H
authors.initials: H
authors.lastname: Puchta
authors.forename: B
authors.initials: B
authors.lastname: Hohn
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D005111:Extrachromosomal Inheritance / Q000235:genetics*; D005816:Genetic Complementation Test; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D011523:Protoplasts / Q000378:metabolism; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000235:genetics*; D014162:Transfection
chemical_list: D004247:DNA; D005966:Glucuronidase