An easy protocol for studying chromatin and recombination protein dynamics during Arabidopsis thaliana meiosis: immunodetection of cohesins, histones and MLH1
2010
Chelysheva, Liudmila | Grandont, Laurie | Vrielynck, Nathalie | Le Guin, Sylvia | Mercier, Raphaël | Grelon, Mathilde | Institut Jean-Pierre Bourgin (IJPB) ; Institut National de la Recherche Agronomique (INRA)-AgroParisTech
Plant meiosis studies have enjoyed a fantastic boom in recent years with the use of Arabidopsis thaliana as a model not only for molecular genetics and genomics but also for cytogenetics. In this article we describe a new protocol for immunolabelling meiotic proteins that allows the detection of a large range of proteins on strongly spread chromosomes throughout the entire meiotic process. We used this method to immunodetect MLH1, a crucial component of the meiotic recombination machinery, and found that it can be visualised as foci from pachytene to diakinesis, where it co-localises with chiasmata. The mean MLH1 foci number per meiotic cell at diakinesis was 8.4 for WS-4 and 9.95 for Col-0, with the number of foci per bivalent ranging from 1 to 5. We also analysed MLH1 distribution within bivalents and found that they were not restricted to specific chromosomal regions. The analysis of MLH1 foci formation in the Atzip4 mutant, where class I crossover (CO) formation is prevented, revealed that residual chiasmata were not labelled by MLH1, strongly suggesting that MLH1 antibodies only label class I COs in Arabidopsis. It thus appears that the 'obligatory CO' is systematically labeled by MLH1 and is generated through the class I pathway
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