This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 146/1/22    most recent pp.107.105213v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Van der Auwera, G. Articles by Depicker, A. Search for Related Content PubMed PubMed Citation Articles by Van der Auwera, G. Articles by Depicker, A. Agricola Articles by Van der Auwera, G. Articles by Depicker, A.

BREAKTHROUGH TECHNOLOGIES

Development and Application of Novel Constructs to Score C:G-to-T:A Transitions and Homologous Recombination in Arabidopsis^1,[W]

Department of Plant Systems Biology, Flanders Institute for Biotechnology, and Department of Molecular Genetics, Ghent University, 9052 Ghent, Belgium

We report on the development of five missense mutants and one recombination substrate of the β-glucuronidase (GUS)-encoding gene of Escherichia coli and their use for detecting mutation and recombination events in transgenic Arabidopsis (Arabidopsis thaliana) plants by reactivation of GUS activity in clonal sectors. The missense mutants were designed to find C:G-to-T:A transitions in a symmetrical sequence context and are in that respect complementary to previously published GUS point mutants. Small peptide tags (hemagglutinin tag and Strep tag II) and green fluorescent protein were translationally fused to GUS, which offers possibilities to check for mutant GUS production levels. We show that spontaneous mutation and recombination events took place. Mutagenic treatment of the plants with ethyl methanesulfonate and ultraviolet-C increased the number of mutations, validating the use of these constructs to measure mutation and recombination frequencies in plants exposed to biotic or abiotic stress conditions, or in response to different genetic backgrounds. Plants were also subjected to heavy metals, methyl jasmonate, salicylic acid, and heat stress, for which no effect could be seen. Together with an ethyl methanesulfonate mutation induction level much higher than previously described, the need is illustrated for many available scoring systems in parallel. Because all GUS missense mutants were cloned in a bacterial expression vector, they can also be used to score mutation events in E. coli.

² Present address: Department of Parasitology, Institute of Tropical Medicine Antwerp, 2000 Antwerp, Belgium.

³ Present address: Laboratory of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.

⁴ Present address: Department of Biology, Vrije Universiteit Brussel, 1050 Brussels, Belgium.

⁵ Present address: Department of Biology, Ghent University, 9000 Ghent, Belgium.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Anna Depicker (ann.depicker{at}psb.ugent.be).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.105213

^* Corresponding author; e-mail ann.depicker{at}psb.ugent.be.

Received July 6, 2007; accepted September 27, 2007; published October 5, 2007.