Project description:Enterotoxin-producing C. perfringens type A is a common cause of food poisonings. The cpe encoding the enterotoxin can be chromosomal (genotype IS1470) or plasmid-borne (genotypes IS1470-like-cpe or IS1151-cpe). The chromosomal cpe-carrying C. perfringens are a more common cause of food poisonings than plasmid-borne cpe-genotypes. The chromosomal cpe-carrying C. perfringens type A strains are generally more resistant to most food-processing conditions than plasmid-borne cpe-carrying strains. On the other hand, the plasmid-borne cpe-positive genotypes are more commonly found in human feces than chromosomal cpe-positive genotypes, and humans seem to be a reservoir for plasmid-borne cpe-carrying strains. Thus, it is possible that the epidemiology of C. perfringes type A food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains is different. A DNA microarray was designed for analysis of genetic relatedness between the different cpe-positive and cpe-negative genotypes of C. perfringens strains isolated from human, animal, environmental and food samples. The DNA microarray contained two probes for all protein-coding sequences in the three genome-sequenced strains (C. perfringens type A strains 13, ATCC13124, and SM101). The chromosomal and plasmid-borne C. perfringens genotypes were grouped into two distinct clusters, one consisting of the chromosomal cpe-genotypes and the other consisting of plasmid-borne cpe-genotypes. Analysis of the variable gene pool complemented with the growth studies demonstrate different carbohydrate and amine metabolism in the chromosomal and plasmid-borne cpe-carrying strains, suggesting different epidemiology of the cpe-positive C. perfringens strain groups.
Project description:We performed physiological recording and single cell RNAseq on mouse prefrontal cortical pyramidal and fast-spiking interneurons in the live brain slice preparation. One of the goals was to identify potential gene targets whose function might be disrupted in schizophrenia.
Project description:Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and in vivo and in vitro interaction analyses. We describe a Rep-STB bypass system in which a plasmid non-covalently associated with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.
Project description:Natural plasmids are common in prokaryotes, but few have been documented in eukaryotes. The natural 2µ plasmid present in the yeast Saccharomyces cerevisiae is one of these best-characterized exceptions. This highly stable genetic element has coexisted with its host for millions of years, faithfully segregating at each cell division through a mechanism that remains unclear. Using proximity ligation methods (such as Hi-C, Micro-C) to map the contacts between 2µ plasmid and yeast chromosomes under dozens of different biological conditions, we found that the plasmid is tethered preferentially to regions with low transcriptional activity, often corresponding to long, inactive genes. These contacts do not depend on common chromosome-structuring factors, such as members of the structural maintenance of chromosome complexes (SMC) but depend on a nucleosome-encoded signal associated with RNA Pol II depletion. They appear stable throughout the cell cycle and can be established within minutes. This chromosome hitchhiking strategy may extend beyond the 2µ plasmid/S. cerevisiae pair, as suggested by the binding pattern of the natural eukaryotic plasmid Ddp5 along silent chromosome regions of the amoeba Dictyostelium discoideum.
Project description:<p>Gut environments harbour dense microbial ecosystems in which plasmids are widely distributed. Plasmids facilitate the exchange of genetic material among microorganisms while enabling the transfer of a diverse array of accessory functions. However, their precise impact on microbial community composition and function remains largely unexplored. Here we identify a prevalent bacterial toxin and a plasmid-encoded resistance mechanism that mediates the interaction between Lactobacilli and Enterococci. This plasmid is widespread across ecosystems, including the rumen and human gut microbiota. Biochemical characterization of the plasmid revealed a defence mechanism against reuterin, a toxin produced by various gut microbes, such as Limosilactobacillus reuteri. Using a targeted metabolomic approach, we find reuterin to be prevalent across rumen ecosystems with impacts on microbial community structure. Enterococcus strains carrying the protective plasmid were isolated and their interactions with L. reuteri, the toxin producer, were studied in vitro. Interestingly, we found that by conferring resistance against reuterin, the plasmid mediates metabolic exchange between the defending and the attacking microbial species, resulting in a beneficial relationship or mutualism. Hence, we reveal here an ecological role for a plasmid-coded defence system in mediating a beneficial interaction. </p>
Project description:By following chromatin occupancy of RNAPII and TOP1 using ChIP-seq throughout mitosis, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity through its interaction with RNAPII during elongation, allowed RNAPII clearance from genes in prometaphase and enabled proper chromosomal segregation. Interference with the TOP1-RNAPII interaction or acute depletion of TOP1 at the onset of mitosis impaired RNAPII spiking at promoters, which is necessary for the first pioneering round of transcription during mitotic exit, and triggered defects in the transcriptional program of the post-mitotic cells.
Project description:Ventral subiculum (vSUB) is integral to the regulation of stress and reward, however the intrinsic connectivity and synaptic properties of the inhibitory microcircuit are poorly understood. Neurexin-3 (Nrxn3) is highly expressed in hippocampal inhibitory neurons, but its function at inhibitory synapses has remained elusive. Using slice electrophysiology, imaging, and single-cell RNA sequencing, we identify multiple roles for Nrxn3 at GABAergic parvalbumin (PV) interneuron synapses made onto vSUB regular spiking (RS) and burst spiking (BS) principal neurons. Surprisingly, we found that intrinsic connectivity of vSUB and synaptic function of Nrxn3 in vSUB are sexually dimorphic. We reveal that vSUB PVs make preferential contact with RS neurons in males, but BS neurons in females. Furthermore, we determined that despite comparable Nrxn3 isoform expression in male and female PV neurons, Nrxn3 maintains synapse density at PV-RS synapses in males, but suppresses presynaptic release at the same synapses in females.
Project description:Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract of global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases as plasmid-deficient organisms are highly attenuated. The plasmid encodes both noncoding RNAs and eight conserved ORFs of undefined function. To understand plasmid gene function we generated plasmid shuttle vectors with deletions in each of the eight ORFs. The individual deletion mutants were used to transform chlamydiae and the transformants were characterized in terms of plasmid biology and transcriptional profiling. We show that pgp1-2, -6 and -8 are essential for plasmid maintenance while the other ORFs can be deleted and the plasmid stably maintained. We further show that a pgp4 knockout mutant exhibits an in vitro phenotype similar to its isogenic plasmid-less strain in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that pgp4 is involved in transcriptional regulation of multiple chromosomal genes; including the glycogen synthase gene glgA. Based on our results, we propose that Pgp1 is a plasmid replicative helicase, Pgp2 is a plasmid replication protein, Pgp4 is a transcriptional regulator of virulence associated chromosomal genes, and Pgp6-8 are plasmid partitioning proteins. These findings have important implications for understanding the plasmid’s role in chlamydial pathogenesis and the development of novel antigenically multivalent live-attenuated chlamydial vaccines.
Project description:Low transfection efficiency poses a significant challenge to experimental success, making the improvement of gene-carrying plasmid delivery and expression essential across various research fields. The stability of transfected vector DNA or mRNA significantly impacts transgene expression, as the innate immune system may recognize plasmid DNA as foreign and initiate a degradation response. Currently, it is unclear whether RNA transcribed from plasmids can activate RNA sensors, affecting transfection efficiency. This study employed RNA sequencing to analyze cellular responses to various circular and linear DNAs across five cell lines—HEK293T, HCT116, HeLa, L02, and NCM460—revealing that the innate immune response is a primary contributor to low transfection efficiency. Additionally, we used ChIP-seq to detect H3K27ac histone marks to explore whether the immune response triggered by plasmid transfection is regulated by epigenetic mechanisms. Finally, we performed ChIP assays for IRF7 and p-IRF3 in HeLa cells transfected with the linear pcDNA3.1-neo plasmid to demonstrate that these two transcription factors can indeed bind to the RTV genes.