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Nucleic Acids Research, 1991, Vol. 19, No. 10 2693-2700
© 1991

MOLECULAR BIOLOGY

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

Holger Puchta and Barbara Hohn*

Friedrich Miescher-Institut PO Box 2543, CH-4002 Basel, Switzerland

* To whom correspondence should be addressed

Received February 11, 1991. Revised April 23, 1991. Accepted April 23, 1991.

An assay to monitor homologous recombination in plant cells has been established by cotransfecting Nicotiana plumbaginifolia protoplasts with different topological forms of plasmlds of various deletion mutants of a non-selectable marker gene, the ß-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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