Project description:The plasmids introduced into the E. coli cells affect the expression of chromosomal genes. Therefore we aimed at comparing the protein expression profiles of a strain not containing any plasmid with strains that do carry plasmids.
Project description:Transient plasmid transfection is common approach for studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We found that plasmids generate different levels of transcripts virtually everywhere. Spurious transcription may have undesirable effects as some co-transfected plasmids inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to kan/neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression of transiently transfected plasmids highlights the importance of appropriate experimental controls.
Project description:Transient plasmid transfection is common approach for studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We found that plasmids generate different levels of transcripts virtually everywhere. Spurious transcription may have undesirable effects as some co-transfected plasmids inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to kan/neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression of transiently transfected plasmids highlights the importance of appropriate experimental controls. HEK293 cells (human origin) transiently transfected with 4 various plasmids
Project description:Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with Bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser2-phosphorylated Pol II (Pol II Ser2) at both enhancers and promoters of active genes. Disruption of bromodomain:histone acetylation interactions by JQ1, a small-molecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser2, and reduced expression of lineage-specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited di-acetylated H4 (lysine-5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancer BRD4 binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells. Examination of BRD4, total Pol II, serine 2 phosphorylated Pol II and serine 5 phosphorylated Pol II binding in CD4+ T cells (with and without JQ1 treatment) and BRD4 binding in human embryonic stems cell; PolyA RNA expression in CD4+ T cells( with and without JQ1 treatment) using RNA-seq
Project description:Transcriptional elongation by RNA polymerase II (Pol II) is regulated by positive transcription elongation factor b (P-TEFb) in association with Bromodomain-containing protein 4 (BRD4). We used genome-wide chromatin immunoprecipitation sequencing in primary human CD4+ T cells to reveal that BRD4 co-localizes with Ser2-phosphorylated Pol II (Pol II Ser2) at both enhancers and promoters of active genes. Disruption of bromodomain:histone acetylation interactions by JQ1, a small-molecule bromodomain inhibitor, resulted in decreased BRD4 binding, reduced Pol II Ser2, and reduced expression of lineage-specific genes in primary human CD4+ T cells. A large number of JQ1-disrupted BRD4 binding regions exhibited di-acetylated H4 (lysine-5 and -8) and H3K27 acetylation (H3K27ac), which correlated with the presence of histone acetyltransferases and deacetylases. Genes associated with BRD4/H3K27ac co-occupancy exhibited significantly higher activity than those associated with H3K27ac or BRD4 binding alone. Comparison of BRD4 binding in T cells and in human embryonic stem cells revealed that enhancer BRD4 binding sites were predominantly lineage-specific. Our findings suggest that BRD4-driven Pol II phosphorylation at serine 2 plays an important role in regulating lineage-specific gene transcription in human CD4+ T cells.
Project description:Antibiotic resistance is exacerbated by the exchange of antibiotic resistance genes (ARGs) between microbes from diverse habitats. Plasmids are important ARGs mobile elements and are spread by horizontal gene transfer (HGT). In this study, we demonstrated the presence of multi-resistant plasmids from inhalable particulate matter (PM) and its effect on gene horizontal transfer. Three transferable multi-resistant plasmids were identified from PM in a hospital, using conjugative mating assays and nanopore sequencing. pTAir-3 contained 26 horizontal transfer elements and 10 ARGs. Importantly pTAir-5 harbored carbapenem resistance gene (blaOXA) which shows homology to plasmids from human and pig commensal bacteria, thus indicating that PM is a media for antibiotic resistant plasmid spread. In addition, 125 μg/mL PM2.5 and PM10 significantly increased the conjugative transfer rate by 110% and 30%, respectively, and augmented reactive oxygen species (ROS) levels. Underlying mechanisms were revealed by identifying the upregulated expressional levels of genes related to ROS, SOS, cell membranes, pilus generation, and transposition via genome-wide RNA sequencing. The study highlights the airborne spread of multi-resistant plasmids and the impact of inhalable PM on the horizontal transfer of antibiotic resistance.
Project description:Plasmid-free Lactococcus lactis IL1403 is one of the best-characterized representatives of lactic acid bacteria (LAB), intensively used in broad microbiology worldwide. Its parent strain, L. lactis IL594, contains seven plasmids (pIL1-pIL7) with resolved DNA sequences and an indicated role for overall plasmid load in enhancing host adaptive potential. To determine how individual plasmids manipulate the expression of phenotypes and chromosomal genes, we conducted global comparative phenotypic analyses combined with transcriptomic studies in plasmid-free L. lactis IL1403, multi-plasmid L. lactis IL594 and its single-plasmid derivatives. The presence of pIL2, pIL4 and pIL5 led to the most pronounced phenotypic differences in the metabolism of several carbon sources, including some β-glycosides and organic acids. The pIL5 plasmid also contributed to increased tolerance to some antimicrobial compounds and heavy metal ions, especially those in the toxic cation group. Comparative transcriptomics showed significant variation in the expression levels of up to 189 chromosomal genes due to the presence of single plasmids, and 435 unique chromosomal genes that are resultant of the activity of all plasmids, which may suggest that the observed phenotypic changes are not only the result of direct action of their own genes, but also originate from indirect actions through cross-talk between plasmids and the chromosome. The data obtained here indicate that plasmid maintenance leads to the development of important mechanisms of global gene regulation that provide changes in the central metabolic pathways and adaptive properties of L. lactis, and suggest the possibility of a similar phenomenon among other groups of bacteria.