ANCHOR: A Technical Approach to Monitor Single-Copy Locus Localization in Planta.
Ontology highlight
ABSTRACT:
SUBMITTER: Meschichi A
PROVIDER: S-EPMC8290188 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Bacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. In this study, we describe the development of a new library of mini-Tn7-lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro- and micro-scale imaging techniques. We demonstrate that a dual bioluminescence-fluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro- and microscopically and demonstrate that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris). We also show that bacterial bioluminescence can be used to study the impact of plant immune responses on bacterial multiplication, viability and spread within plant tissues. The constructs and approach described in this study can be used to study the spatiotemporal dynamics of bacterial colonization and to link population dynamics and cellular interactions in a wide range of biological contexts.
| S-EPMC8614114 | biostudies-literature
Project description:Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.
| S-EPMC5614340 | biostudies-literature
Project description:The chromatin at origins of replication is the template for DNA replication initiation in eukaryotic genomes. However, whether the chromatin composition instructs individual origins to fire early-efficiently (EE) or late-inefficiently (LI) remains to be investigated. Here, we used site-specific recombination and a single-locus chromatin isolation approach to purify selected EE and LI replication origins from chromosome III of Saccharomyces cerevisiae. Using mass spectrometry, we define the histone modification landscape and identify the protein composition of defined ~1kb native chromatin regions surrounding the EE and LI replication start sites. We confirm expected origin interactors but also find unexpected interactions, such as the Ask1 subunit of the kinetochore-associated DASH complex. Strikingly, we show that Ask1 is a critical factor in the replication timing control of specific origins in yeast. These results highlight that our unbiased approach can specifically identify the functionally-relevant proteome at a single-copy locus of interest and provide a detailed view of histone modification and protein interaction networks on replication origin chromatin. The current dataset contains proteomic data used for the histone PTM analysis.
2023-05-10 | PXD031984 | Pride
Project description:Copy number variation (CNV) is an important source of genomic diversity in humans, and influences disease susceptibility. The immunoglobulin-receptor genes FCGR3A and FCGR3B on chromosome 1q23.3 show CNV, and CNV of the FCGR3B gene is associated with glomerulonephritis in systemic lupus erythematosus and organ-specific autoimmunity. Large-scale case-control association studies of CNV require technologies that are amenable to high-throughput analysis with low error rates. Here we propose an integrated suite of five assays, four of them duplexed to reduce DNA usage, that assays for CNV at FCGR3A and FCGR3B, and genotype the polymorphic neutrophil antigen HNA1. We show how a maximum-likelihood (ML) approach to combining the results from these five assays allows estimation of statistical confidence for each individual copy number, and therefore an appropriate significance threshold to be set, controlling the error rate. This approach results in a high-throughput copy number genotyping system, with demonstrable precision and accuracy, that can be applied to large case-control cohort studies. We demonstrate Mendelian inheritance of this CNV, variation in frequency between Europeans and East Asians, and a lack of strong association between the CNV and flanking SNP genotypes, with important consequences for genome-wide association studies.
| S-EPMC3600569 | biostudies-literature
Project description:EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a recent single-cell genomic method based on a fusion-PCR allowing us to link a functional sequence of interest to a 16S rRNA gene fragment and use the mass sequencing of the resulting amplicons for taxonomic assignment of the functional sequence-carrying bacteria. Although it is interesting because it presents the highest efficiency for assigning a bacterial host to a marker, epicPCR remains a complex multistage procedure with technical difficulties that may easily impair the approach depth and quality. Here, we described how to adapt epicPCR to new gene targets and environmental matrices while identifying the natural host range of SXT/R391 integrative and conjugative elements in water microbial communities from the Meurthe River (France). We notably show that adding a supplementary PCR step allowed us to increase the amplicon yield and thus the number of reads obtained after sequencing. A comparison of operational taxonomic unit (OTU) identification approaches when using biological and technical replicates demonstrated that, although OTUs can be validated when obtained from three out of three technical replicates, up to now, results obtained from two or three biological replicates give a similar and even a better confidence level in OTU identification, while allowing us to detect poorly represented SXT/R391 hosts in microbial communities.
| S-EPMC8399275 | biostudies-literature
Project description:Multicolor single-molecule localization microscopy (λSMLM) is a powerful technique to reveal the relative nanoscale organization and potential colocalization between different molecular species. While several standard analysis methods exist for pixel-based images, λSMLM still lacks such a standard. Moreover, existing methods only work on 2D data and are usually sensitive to the relative molecular organization, a very important parameter to consider in quantitative SMLM. Here, we present an efficient, parameter-free colocalization analysis method for 2D and 3D λSMLM using tessellation analysis. We demonstrate that our method allows for the efficient computation of several popular colocalization estimators directly from molecular coordinates and illustrate its capability to analyze multicolor SMLM data in a robust and efficient manner.
| S-EPMC6542817 | biostudies-literature
Project description:Numerous techniques exist for arthroscopic subscapularis repair with varying degrees of complexity based on tear morphology, all of which have established satisfactory outcomes in function and patient satisfaction. Arthroscopic subscapularis repair can require several working portals and suture anchors, increasing both technical complexity and operative time. This Technical Note describes an arthroscopic repair of a superior one-third subscapularis tear using a self-punching knotless soft suture anchor through a single anterior working portal. Thus, we offer a unique approach to arthroscopic repair of superior one-third subscapularis tears that is time-saving, reproducible, and highly efficient while minimizing iatrogenic damage and postoperative complications.
| S-EPMC8416967 | biostudies-literature
Project description:Any given chromosomal activity (e.g., transcription) is governed predominantly by the local epiproteome. However, defining local epiproteomes has been limited by a lack of effective technologies to isolate discrete sections of chromatin and to identify with precision specific proteins and histone posttranslational modifications (PTMs). We report the use of the Cas9 and guide RNA (gRNA) components of the CRISPR system for gRNA-directed purification of a discrete section of chromatin. Quantitative mass spectrometry provides for unambiguous identification of proteins and histone PTMs specifically associated with the enriched chromatin. This CRISPR-based Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) approach revealed changes in the local epiproteome of a promoter during activation of transcription. CRISPR-ChAP-MS thus has broad applications for discovering molecular components and dynamic regulation of any in vivo activity at a given chromosomal location.
| S-EPMC4169012 | biostudies-literature
Project description:Two coding variants in the APOL1 gene (G1 and G2) explain most of the high rate of kidney disease in African Americans. APOL1-associated kidney disease risk inheritance follows an autosomal recessive pattern: The relative risk of kidney disease associated with inheritance of two high-risk variants is 7-30 fold, depending on the specific kidney phenotype. We wished to determine if the variability in phenotype might in part reflect structural differences in APOL1 gene. We analyzed sequence coverage from 1000 Genomes Project Phase 3 samples as well as exome sequencing data from African American kidney disease cases for copy number variation. 8 samples sequenced in the 1000 Genomes Project showed increased coverage over a ~100kb region that includes APOL2, APOL1 and part of MYH9, suggesting the presence of APOL1 copy number greater than 2. We reasoned that such duplications should be enriched in apparent G1 heterozygotes with kidney disease. Using a PCR-based assay, we observed the presence of this duplication in additional samples from apparent G0G1 or G0G2 individuals. The frequency of this APOL1 duplication was compared among cases (n = 123) and controls (n = 255) with apparent G0G1 heterozygosity. The presence of APOL1 duplication was observed in 4.06% of cases and 0.78% controls, preliminary evidence that this APOL1 duplication may alter susceptibility to kidney disease (p = 0.03). Taqman-based copy number assays confirmed the presence of 3 APOL1 copies in individuals positive for this specific duplication by PCR assay, but also identified a small number of individuals with additional APOL1 copies of presumably different structure. These observations motivate further studies to better assess the contribution of APOL1 copy number on kidney disease risk and on APOL1 function. Investigators and clinicians genotyping APOL1 should also consider whether the particular genotyping platform used is subject to technical errors when more than two copies of APOL1 are present.
| S-EPMC4416782 | biostudies-literature
Project description:Subcellular localization of mRNAs by cytoskeletal motors plays critical roles in the spatial control of protein function. However, optical limitations of studying mRNA transport in vivo mean that there is little mechanistic insight into how transcripts are packaged and linked to motors, and how the movement of mRNA-motor complexes on the cytoskeleton is orchestrated. Here, we have reconstituted transport of mRNPs containing specific RNAs in vitro. We show directly that mRNAs that are either apically localized or non-localized in Drosophila melanogaster embryos associate with the dynein motor and move bidirectionally on individual microtubules, with localizing mRNPs exhibiting a strong minus-end-directed bias. Single-molecule fluorescence measurements reveal that RNA localization signals increase the average number of dynein and dynactin components recruited to individual mRNPs. We find that, surprisingly, individual RNA molecules are present in motile mRNPs in vitro and provide evidence that this is also the case in vivo. Thus, RNA oligomerization is not obligatory for transport. Our findings lead to a model in which RNA localization signals produce highly polarized distributions of transcript populations through modest changes in motor copy number on single mRNA molecules.
| S-EPMC3343632 | biostudies-literature