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Answer added in Cell Cycle Analysis5 Do you know any cell cycle detection kit assay which doesn't require flow cytometry?Maybe you could use EdU labelling (it incorporates during S-phase), and count by eye under the microscope the proportion of fluorescent cells.This kit... [more]
Publications (11) View all
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Article: Evidence for a role of Arabidopsis CDT1 proteins in gametophyte development and maintenance of genome integrity.
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ABSTRACT: Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase ε, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability.The Plant Cell 07/2012; 24(7):2779-91. · 8.99 Impact Factor -
Article: Oxidative DNA damage bypass in Arabidopsis thaliana requires DNA polymerase λ and proliferating cell nuclear antigen 2.
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ABSTRACT: The oxidized base 7,8-oxoguanine (8-oxo-G) is the most common DNA lesion generated by reactive oxygen species. This lesion is highly mutagenic due to the frequent misincorporation of A opposite 8-oxo-G during DNA replication. In mammalian cells, the DNA polymerase (pol) family X enzyme DNA pol λ catalyzes the correct incorporation of C opposite 8-oxo-G, together with the auxiliary factor proliferating cell nuclear antigen (PCNA). Here, we show that Arabidopsis thaliana DNA pol λ, the only member of the X family in plants, is as efficient in performing error-free translesion synthesis past 8-oxo-G as its mammalian homolog. Arabidopsis, in contrast with animal cells, possesses two genes for PCNA. Using in vitro and in vivo approaches, we observed that PCNA2, but not PCNA1, physically interacts with DNA pol λ, enhancing its fidelity and efficiency in translesion synthesis. The levels of DNA pol λ in transgenic plantlets characterized by overexpression or silencing of Arabidopsis POLL correlate with the ability of cell extracts to perform error-free translesion synthesis. The important role of DNA pol λ is corroborated by the observation that the promoter of POLL is activated by UV and that both overexpressing and silenced plants show altered growth phenotypes.The Plant Cell 02/2011; 23(2):806-22. · 8.99 Impact Factor -
Article: The nucleus-encoded trans-acting factor MCA1 plays a critical role in the regulation of cytochrome f synthesis in Chlamydomonas chloroplasts.
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ABSTRACT: Organelle gene expression is characterized by nucleus-encoded trans-acting factors that control posttranscriptional steps in a gene-specific manner. As a typical example, in Chlamydomonas reinhardtii, expression of the chloroplast petA gene encoding cytochrome f, a major subunit of the cytochrome b(6)f complex, depends on MCA1 and TCA1, required for the accumulation and translation of the petA mRNA. Here, we show that these two proteins associate in high molecular mass complexes that also contain the petA mRNA. We demonstrate that MCA1 is degraded upon interaction with unassembled cytochrome f that transiently accumulates during the biogenesis of the cytochrome b(6)f complex. Strikingly, this interaction relies on the very same residues that form the repressor motif involved in the Control by Epistasy of cytochrome f Synthesis (CES), a negative feedback mechanism that downregulates cytochrome f synthesis when its assembly within the cytochrome b(6)f complex is compromised. Based on these new findings, we present a revised picture for the CES regulation of petA mRNA translation that involves proteolysis of the translation enhancer MCA1, triggered by its interaction with unassembled cytochrome f.The Plant Cell 01/2011; 23(1):333-49. · 8.99 Impact Factor -
Article: The Arabidopsis MCM2 gene is essential to embryo development and its over-expression alters root meristem function.
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ABSTRACT: * Minichromosome maintenance (MCM) proteins are subunits of the pre-replication complex that probably function as DNA helicases during the S phase of the cell cycle. Here, we investigated the function of AtMCM2 in Arabidopsis. * To gain an insight into the function of AtMCM2, we combined loss- and gain-of-function approaches. To this end, we analysed two null alleles of AtMCM2, and generated transgenic plants expressing AtMCM2 downstream of the constitutive 35S promoter. * Disruption of AtMCM2 is lethal at a very early stage of embryogenesis, whereas its over-expression results in reduced growth and inhibition of endoreduplication. In addition, over-expression of AtMCM2 induces the formation of additional initials in the columella root cap. In the plt1,2 mutant, defective for root apical meristem maintenance, over-expression of AtMCM2 induces lateral root initiation close to the root tip, a phenotype not reported in the wild-type or in plt1,2 mutants, even when cell cycle regulators, such as AtCYCD3;1, were over-expressed. * Taken together, our results provide evidence for the involvement of AtMCM2 in DNA replication, and suggest that it plays a crucial role in root meristem function.New Phytologist 08/2009; 184(2):311-22. · 6.64 Impact Factor -
Article: Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis.
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ABSTRACT: Although it is a crucial cellular process required for both normal development and to face stress conditions, the control of programmed cell death in plants is not fully understood. We previously reported the isolation of ATXR5 and ATXR6, two PCNA-binding proteins that could be involved in the regulation of cell cycle or cell death. A yeast two-hybrid screen using ATXR5 as bait captured AtIPS1, an enzyme which catalyses the committed step of myo-inositol (MI) biosynthesis. atips1 mutants form spontaneous lesions on leaves, raising the possibility that MI metabolism may play a role in the control of PCD in plants. In this work, we have characterised atips1 mutants to gain insight regarding the role of MI in PCD regulation. - lesion formation in atips1 mutants depends of light intensity, is due to PCD as evidenced by TUNEL labelling of nuclei, and is regulated by phytohormones such as salicylic acid - MI and galactinol are the only metabolites whose accumulation is significantly reduced in the mutant, and supplementation of the mutant with these compounds is sufficient to prevent PCD - the transcriptome profile of the mutant is extremely similar to that of lesion mimic mutants such as cpr5, or wild-type plants infected with pathogens. Taken together, our results provide strong evidence for the role of MI or MI derivatives in the regulation of PCD. Interestingly, there are three isoforms of IPS in Arabidopsis, but AtIPS1 is the only one harbouring a nuclear localisation sequence, suggesting that nuclear pools of MI may play a specific role in PCD regulation and opening new research prospects regarding the role of MI in the prevention of tumorigenesis. Nevertheless, the significance of the interaction between AtIPS1 and ATXR5 remains to be established.PLoS ONE 01/2009; 4(10):e7364. · 4.09 Impact Factor
