Project description:Using 4C-Seq experimental procedure we have characterized, in cultured chicken lymphoid and erythroid cells, genome-wide patterns of spatial contacts of several CpG islands scattered along the chromosome 14. A clear tendency for interaction of CpG islands present within the same and different chromosomes has been observed. Accordingly, preferential spatial contacts between Sp1 binding motifs, and other GC-rich genomic elements including DNA sequence motifs capable to form G-quadruplexes were demonstrated. On the other hand, an anchor placed in gene/CpG islands-poor area was found to form spatial contacts with other gene/CpG islands-poor areas within chromosome 14 and other chromosomes. These results corroborate the two compartments model of interphase chromosome spatial organization and suggest that clustering of CpG islands harboring promoters and origins of DNA replication constitutes an important determinant of the 3D organization of eukaryotic genome in the cell nucleus. Using ChIP-Seq experimental procedure we have mapped genome-wide the CTCF deposition sites in chicken lymphoid and erythroid cells subjected to the 4C analysis. A good correlation between the density of these sites and the level of 4C signals was observed for the anchors located in CpG islands. It is thus possible that CTCF contributes to the clustering of CpG islands revealed in our experiments. We applied 4C-seq to map long-range interactions of a CpG island harboring promoter of a housekeeping gene NPRL3 on a genome-wide scale in DT40 and HD3 cell lines in chicken (Gallus gallus). Two replicates per cell line were sequenced in paired-end mode with a depth of 45-64 million reads. We next performed additional 4C experiments on HD3 cells with different 4C anchors. In three experiments anchors were placed on different CpG island on the chromosome 14 that have demonstrated a strong interaction with the NPRL3 anchor (anchors near TSR3, TRAP1, PPL genes). In the forth experiment (anchor GENE-Des) the anchor was placed in a gene-poor area that did not interact with the NPRL3 promoter. The libraries for all anchors were pooled and sequenced in paired-end mode with a total depth of 75 million reads. Anchor NPRL3: two replicates for HD3 and two replicates for DT40 cell lines. Other anchors (TSR3, GENE-Des, TRAP1, PPL): two replicates, pooled library

Project description:Understanding the environmental factors that shape microbial communities is crucial, especially in extreme environments, like Antarctica. Two main forces were reported to influence Antarctic soil microbes: birds and plants. Both birds and plants are currently undergoing unprecedented changes in their distribution and abundance due to global warming. However, we need to clearly understand the relationship between plants, birds and soil microorganisms. We therefore collected rhizosphere and bulk soils from six different sampling sites subjected to different levels of bird influence and colonized by Colobanthus quitensis and Deschampsia antarctica in the Admiralty Bay, King George Island, Maritime Antarctic. Microarray and qPCR assays targeting 16S rRNA genes of specific taxa were used to assess microbial community structure, composition and abundance and analyzed with a range of soil physico-chemical parameters. The results indicated significant rhizosphere effects in four out of the six sites, including areas with different levels of bird influence. Acidobacteria were significantly more abundant in soils with little bird influence (low nitrogen) and in bulk soil. In contrast, Actinobacteria were significantly more abundant in the rhizosphere of both plant species. At two of the sampling sites under strong bird influence (penguin colonies), Firmicutes were significantly more abundant in D. antarctica rhizosphere but not in C. quitensis rhizosphere. The Firmicutes were also positively and significantly correlated to the nitrogen concentrations in the soil. We conclude that the microbial communities in Antarctic soils are driven both by bird and plants, and that the effect is taxa-specific. The study was carried out at the Brazilian Antarctic Station Comandante Ferraz (EACF, 62M-BM-004M-bM-^@M-^YS, 58M-BM-021M-bM-^@M-^YW), located in Martel Inlet, Admiralty Bay, King George Island, Antarctic Peninsula, which is part of the South Shetlands Archipelago in Maritime Antarctica. It is a medium sized research station with a population of 10 to 15 people during the winter months (March to November) and about 60 people during the austral summer months (November to March). During the austral summers of 2006 M-bM-^@M-^S 2007 and 2008 M-bM-^@M-^S 2009, the vascular plants D. antarctica or C. quitensis were sampled, where both plants were found, in triplicate at six different sites: A M-bM-^@M-^S Arctowski (2006 M-bM-^@M-^S 2007), Q M-bM-^@M-^S Quimica (2006 M-bM-^@M-^S 2007), I M-bM-^@M-^S Ipanema (2006 M-bM-^@M-^S 2007), M M-bM-^@M-^S North Mountain (2008 M-bM-^@M-^S 2009), D M-bM-^@M-^S Demay Point (2008 M-bM-^@M-^S 2009), C M-bM-^@M-^S Copacabana (2008 M-bM-^@M-^S 2009) (Figure 1). Points A, C and D were located inside an environmental protected area. Point A is close to the Arctowski Polish Station and next to a colony of Adelie penguins (Pygoscelis adeliae), point C is next to the USA summer station Copacabana in a Gentoo penguin (P. papua) colony, and point D is near to a Polish refuge next to a colony of Chinstrap penguins (P. antarcticus). At point I, there were no penguin colonies present, but this section was used as a nesting site by local species of flying birds. Point Q was located in the vicinity of the EACF; thus there has been (and continues to be) an intense anthropogenic influence on this spot, which is not the case at the other sampling sites. Point M was located at the top of North Mountain, around 200 m altitude. This site has no influence from penguin colonies and only a few nests of skua (Catharacta sp.) were observed. At each sampling site, triplicate soil samples were taken for chemical and biological analyses, with the exception of the Arctowski site (A) where we only took two replicates. Each vascular plant sample was frozen (-20M-BM-0C) at the EACF.

Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased itM-bM-^@M-^Ys ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice. 6 Samples in total, 3 each from VL and CL causing isolates were analyzed by Splice Leader RNASeq. These three samples from each of the isolates were grown to form one of the following three lifestages, Promastigotes, Macrophage derived Amastigotes, Axenic Amastigotes.

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Clustering of CpG islands constitutes an important determinant of the interphase chromosome 3D organization

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