A transient assay in plant cells reveals a positive correlation between extrachromosomal recombination rates and length of homologous overlap.

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RESUMO

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.

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