Intrachromosomal Homologous Recombination in <I>Arabidopsis thaliana</I>

Abstract

Because of the availability of the complete sequence of the genome of the model plant Arabidopsis and of insertion mutants for most genes in public mutant collections, the elucidation of the particular role of different factors involved in DNA recombination and repair processes, an important task for plant biology, is becoming feasible. An assay system based on transgenes harboring homologous overlaps of the beta-glucuronidase (uidA) gene is available to determine recombination behavior in various mutant backgrounds. Restoration of the marker gene by homologous recombination can be detected by histochemical staining in planta. Inclusion of a site of the rare cutting restriction enzyme I-SceI in the transgene construct enables the determination of recombination frequencies after induction of double-strand breaks. In this chapter we describe how the respective transgene is transferred by transformation or crossing into the mutant background, how recombination frequencies are determined, and, if necessary, how cells carrying a restored uidA gene can be isolated and propagated for molecular analysis of the particular recombination event.

Two closely related RecQ helicases have antagonistic roles in homologous recombination and DNA repair in Arabidopsis thaliana

“…For HR assays, the seedlings were treated in the same manner as for the mutagen assays, but instead of transferring them to six-well plates after 7 days, 30-35 seedlings each were transferred into halved Petri dishes containing 10 ml of either pure liquid GM, or liquid GM with 5 g of bleomycin, respectively. After 5 additional days in liquid culture the seedlings were transferred to a staining solution (47). After 2 more days, the seedlings were incubated in 70% ethanol for 12 h, and subsequently, the number of blue sectors on each plant was determined by using a binocular microscope.…”

The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6 Complex Promotes Sister Chromatid Alignment and Homologous Recombination after DNA Damage inArabidopsis thaliana

“…Histochemical staining was performed as described (Schmidt-Puchta et al, 2004). Blue sectors were counted using a binocular microscope after the plants had been decolorized with 70% ethanol.…”

Routine Markerless Gene Replacement in Bacillus anthracis

“…cleave an introduced site that is essentially unique in a genome has been exploited in the promotion of homologous recombination in organisms as diverse as bacteria, Drosophila, and other higher eukaryotes (4,20,22). In one case, the use of I-SceI in promoting allelic exchange in Escherichia coli has been reported (17).…”