Project description:RNA silencing is a mechanism for regulating gene expression at the transcriptional and post-transcriptional levels. Its functions include regulating endogenous gene expression and protecting the cell against viruses and invading transposable elements (TEs). A key component of the mechanism is small RNAs (sRNAs) of 21-24 nucleotides (nt) in length, which direct the silencing machinery in a sequence specific manner to target nucleic acids. sRNAs of 24 nt are involved in methylation of cytosine residues of target loci in three sequence contexts (CG, CHG and CHH), referred to as RNA-directed DNA methylation (RdDM). We previously demonstrated that 24 nt sRNAs are mobile from shoot to root in Arabidopsis thaliana. In this study we demonstrated that methylation of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot. Furthermore, we found that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. These findings were made using base-resolution next generation sequencing approaches and genome wide analyses. Specific classes of short TEs are the predominant targets of mobile sRNA-dependent DNA methylation; classes typically found in gene-rich euchromatic regions. Mobile sRNA-regulated genes were also identified. Mechanistically, we demonstrate that mobile sRNA-dependent non-CG methylation is largely independent of the CMT2/3 RdDM pathway but dependent upon the DRM1/DRM2 RdDM pathway. This is in contrast to non-mobile sRNA-dependent DNA methylation, which predominantly depends upon the CMT2/3 RdDM pathway. These data are complementary to the small RNA sequencing data from Arabidopsis root grafts described in Molnar et al (Science, 2010 May 14;328(5980):872-5).
Project description:Staphylococcus aureus is the most common cause of hospital-acquired infection. In healthy hosts outside of the health care setting, S.aureus is a frequent colonizer of the human nose but rarely causes severe invasive infection such as bacteremia, endocarditis, or osteomyelitis. To identify genes associated with community-acquired invasive isolates, regions of genomic variability, and the S.aureus population structure, we compared 61 community-acquired invasive isolates of S.aureus and 100 nasal carriage isolates from healthy donors using a microarray spotted with PCR products representing every gene from the seven S.aureus sequencing projects. The core genes common to all strains were identified, and 10 dominant lineages of S.aureus were clearly discriminated. Each lineage carried a unique combination of hundreds of core variable (CV) genes scattered throughout the chromosome, suggesting a common ancestor but early evolutionary divergence. Many CV genes are regulators of virulence genes or known or predicted to be expressed on the bacterial surface and to interact with the host during nasal colonization and infection. Within each lineage, isolates showed substantial variation in the carriage of mobile genetic elements and their associated virulence and resistance genes, indicating frequent horizontal transfer. However, we were unable to identify any association between lineage or gene and invasive isolates. We suggest that the S.aureus gene combinations necessary for invasive disease may also be necessary for nasal colonization and that community-acquired invasive disease is strongly dependent on host factors. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-33
Project description:A silencing signal in plants with an RNA specificity determinant moves through plasmodesmata and the phloem. To identify the mobile RNA we grafted Arabidopsis thaliana shoots to roots that would be a recipient for the silencing signal. Using high throughput sequencing as a sensitive detection method and mutants to block small RNA (sRNA) biogenesis in either source or recipient tissue, we detected endogenous and transgene specific sRNA that moved across the graft union. Surprisingly we found that the mobile endogenous sRNAs account for a substantial proportion of the sRNA in roots and we provide evidence that 24nt mobile sRNAs direct epigenetic modifications in the genome of the recipient cells. Mobile sRNA thus represents a mechanism for transmitting the specification of epigenetic modification and could affect genome defence and responses to external stimuli that have persistent effects in plants. Keywords: Small RNA Analysis, Epigenetics
Project description:Impact of small molecules from different microbial gut community types on gene expression from preterm intestinal derived organoids
Project description:Approximatively 75% of the genome of S. aureus (“core” genome) are highly conserved between strains, whereas the remaining 25% (“accessory” genome) are composed of variable regions that are mostly composed of mobile genetic elements (MGE), containing virulence and resistance genes. We have developed a composite DNA-microarray (StaphVar Array) which selectively targets 403 genes located on the accessory or core variable genome. Target genes encode antimicrobial resistance factors (35 %), virulence factors (28 %) and adhesins (31%). This microarray was validated with reference strains and used to characterize the genomic DNA of 13 community-acquired methicillin resistant S. aureus (CA-MRSA) strains representative of all the Multilocus Sequence Types (STs) described to date in Belgium. Analysis of gene content of 8 reference strains by the StaphVar Array matched 96 to 99% of the theoretical results. Analysis of CA-MRSA strains showed that 54.4 % of the genes tested were strain-dependent. Strains presented specific exotoxin, enterotoxin, cytolysin and adhesin gene profile by multi-locus sequence typing (MLST) lineage. One exception to these “lineage-specific” gene profile was the variable presence of the Arginin Catabolic Mobile Element (ACME, characteristic of the USA300 clone) within ST8 strains. In conclusion, this novel StaphVar array enables characterization of more than 400 variable resistance and virulence determinants in S. aureus strains. CA-MRSA strains from Belgium displayed extensive diversity in virulence and resistance profile. The presence of the USA 300 clone in our country was confirmed. Although mainly located on MGE, association of virulence genes were highly conserved within strains of the same MLST lineage.
Project description:Small RNAs recently emerged as a new class of mobile instructive signals in development. Here, we investigate their mechanism of action and show that the gradients formed by mobile small RNAs generate sharply defined domains of target gene expression. By modulating the source of artificial miRNAs we show that boundary formation is an inherent property of the small RNA gradient itself. The threshold-based readout of such gradients is highly sensitive to small RNA levels at the source, allowing plasticity in the positioning of a target gene expression boundary. In addition to generating sharp expression domains of their immediate targets, the readouts of opposing small RNA gradients enable formation of stable and uniformly positioned developmental boundaries. These novel patterning properties of small RNAs are reminiscent of those of morphogens in animal systems. However, their exceptionally high specificity, direct mode of action, and the fully intrinsic nature of their gradients, distinguish mobile small RNAs from classical morphogens. Our findings present mobile small RNAs and their targets as highly portable and evolutionarily-tractable regulatory modules through which to create pattern in development and beyond.
Project description:Double-stranded RNA (dsRNA) can cause specific gene silencing upon ingestion in many animals and is being developed as a pesticide to target essential genes in animal pests. However, the organismal response to ingested dsRNA that leads to eventual gene silencing within animals is unknown. In the worm C. elegans, ingested dsRNA is recruited into the RNA interference pathway by the dsRNA-binding protein RDE-4 for eventual gene silencing by Argonaute proteins. We found that when RDE-4 was expressed at high levels within a tissue, silencing by ingested dsRNA could occur in rde-4(-) somatic tissues but not in the rde-4(-) germline. Such silencing by dsRNA-derived mobile RNA had different Argonaute requirements and could escape inhibition by expressed repetitive DNA. Thus, our results suggest that, when animals ingest dsRNA, the ingested dsRNA and dsRNA-derived mobile RNAs use distinct mechanisms to silence genes.
Project description:Introduction Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are increasingly isolated, with USA300-0114 being the predominant clone in the USA. Comparative whole genome sequencing of USA300 isolates collected in 2002, 2003 and 2005 showed a limited number of single nucleotide polymorphisms and regions of difference. This suggests that USA300 has undergone rapid clonal expansion without great genomic diversification. However, whole genome comparison of CA-MRSA has been limited to isolates belonging to USA300. The aim of this study was to compare the genetic repertoire of different CA-MRSA clones with that of HA-MRSA from the USA and Europe through comparative genomic hybridization (CGH) to identify genetic clues that may explain the successful and rapid emergence of CA-MRSA. Materials and Methods Hierarchical clustering based on CGH of 48 MRSA isolates from the community and nosocomial infections from Europe and the USA revealed dispersed clustering of the 19 CA-MRSA isolates. This means that these 19 CA-MRSA isolates do not share a unique genetic make-up. Only the PVL genes were commonly present in all CA-MRSA isolates. However, 10 genes were variably present among 14 USA300 isolates. Most of these genes were present on mobile elements. Conclusion The genetic variation present among the 14 USA300 isolates is remarkable considering the fact that the isolates were recovered within one month and originated from a confined geographic area, suggesting continuous evolution of this clone. Data is also available from <ahref=http://bugs.sgul.ac.uk/E-BUGS-108 target=_blank>BuG@Sbase</a>
Project description:The epidemic character of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA), especially the geographically widespread clone USA300, is poorly understood. USA300 isolates carry a type IV staphylococcal chromosomal cassette mec (SCCmec) element conferring -lactam antibiotic class resistance and a putative pathogenicity island, ACME (arginine catabolic mobile element). Physical linkage between SCCmec and ACME suggests that selection for antibiotic resistance and for pathogenicity may be interconnected. We constructed isogenic mutants containing deletions of SCCmec and ACME in a USA300 clinical isolate to determine the role of these elements in a rabbit model of bacteremia. We found that deletion of type IV SCCmec did not affect competitive fitness, whereas deletion of ACME significantly attenuated pathogenicity or fitness of USA300. These data are consistent with a model in which ACME enhances growth and survival of USA300, allowing for genetic "hitchhiking" of SCCmec. SCCmec in turn protects against exposure to β -lactams. Keywords: Wild type control vs mutant Wild type untreated in triplicate is compared to three mutants in triplicate in both exponential and stationary growth phases, totalling 24 samples