DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal program. The ATR/Chk1 dependent replication checkpoint inhibits the activation of later firing origins. In the Xenopus in vitro system initiations are not sequence dependent and 2-5 origins are grouped in clusters that fire at different times despite a very short S phase. We have shown that the temporal program is stochastic at the level of single origins and replication clusters. It is unclear how the replication checkpoint inhibits late origins but permits origin activation in early clusters. Here, we analyze the role of Chk1 in the replication program in sperm nuclei replicating in Xenopus egg extracts by a combination of experimental and modelling approaches. After Chk1 inhibition or immunodepletion, we observed an increase of the replication extent and fork density in the presence or absence of external stress. However, overexpression of Chk1 in the absence of external replication stress inhibited DNA replication by decreasing fork densities due to lower Cdk2 kinase activity. Thus, Chk1 levels need to be tightly controlled in order to properly regulate the replication program even during normal S phase. DNA combing experiments showed that Chk1 inhibits origins outside, but not inside, already active clusters. Numerical simulations of initiation frequencies in the absence and presence of Chk1 activity are consistent with a global inhibition of origins by Chk1 at the level of clusters but need to be combined with a local repression of Chk1 action close to activated origins to fit our data.
The application of high-throughput SNP genotyping is a great challenge for many research projects in the plant genetics domain. The GOOD assay for mass spectrometry, Amplifluor and TaqMan are three methods that rely on different principles for allele discrimination and detection, specifically, primer extension, allele-specific PCR and hybridization, respectively. First, with the goal of assessing allele frequencies by means of SNP genotyping, we compared these methods on a set of three SNPs present in the herbicide resistance genes CSR, AXR1 and IXR1 of Arabidopsis thaliana. In this comparison, we obtained the best results with TaqMan based on PCR specificity, flexibility in primer design and success rate. We also used mass spectrometry for genotyping polyploid species. Finally, a combination of the three methods was used for medium- to high-throughput genotyping in a number of different plant species. Here, we show that all three genotyping technologies are successful in discriminating alleles in various plant species and discuss the factors that must be considered in assessing which method to use for a given application.
Meiosis is a special type of cell division present in all organisms that reproduce by sexual reproduction. It ensures the transition between the sporophytic and gametophytic state and allows gamete production through meiotic recombination and chromosome number reduction. In this paper, we describe a technique for the isolation of Arabidopsis thaliana male meiocytes. From this cellular material, it was then possible to develop large-scale transcriptome studies using CATMA microarrays and thus to obtain an overview of genes expressed during Arabidopsis meiosis. The expression profiles were studied with either stringent statistical criteria or by performing clustering. Both methods resulted in gene clusters enriched in meiosis-specific genes (from 14- to 55-fold). Analysis of these data provided a unique set of genes that will be pivotal to further analysis aimed at understanding the meiotic process.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.