Candida albicans repetitive elements display epigenetic diversity and plasticity

Transcriptionally silent heterochromatin is associated with repetitive DNA. It is poorly understood whether and how heterochromatin differs between different organisms and whether its structure can be remodelled in response to environmental signals. Here, we address this question by analysing the chromatin state associated with DNA repeats in the human fungal pathogen Candida albicans. Our analyses indicate that, contrary to model systems, each type of repetitive element is assembled into a distinct chromatin state. Classical Sir2-dependent hypoacetylated and hypomethylated chromatin is associated with the rDNA locus while telomeric regions are assembled into a weak heterochromatin that is only mildly hypoacetylated and hypomethylated. Major Repeat Sequences, a class of tandem repeats, are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Marker gene silencing assays and genome-wide RNA sequencing reveals that C. albicans heterochromatin represses expression of repeat-associated coding and non-coding RNAs. We find that telomeric heterochromatin is dynamic and remodelled upon an environmental change. Weak heterochromatin is associated with telomeres at 30?°C, while robust heterochromatin is assembled over these regions at 39?°C, a temperature mimicking moderate fever in the host. Thus in C. albicans, differential chromatin states controls gene expression and epigenetic plasticity is linked to adaptation

Mitochondria of the Yeasts Saccharomyces cerevisiae and Kluyveromyces lactis Contain Nuclear rDNA-Encoded Proteins

In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named TAR1 for Transcript Antisense to Ribosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the authentic product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the Saccharomyces cerevisiae TAR1 product, we detected the endogenous Tar1p polypeptides in S. cerevisiae (Sc) and Kluyveromyces lactis (Kl) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous Sc Tar1p protein sub-localizes in the inner membrane in a Nin-Cout topology. Plasmid-versions of 5′ end or 3′ end truncated TAR1 ORF were used to demonstrate that neither the N-terminus nor the C-terminus of Sc Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous Sc Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous Sc TAR1 transcripts were also found low abundant and consistently 5′ flanking regions of TAR1 ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous Sc TAR1 transcripts possess heterogeneous 5′ and 3′ ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene TAR1 constitutes a functional transcription unit within the nuclear rDNA repeats

МОКРОТА КАК ИСТОЧНИК АДИПОКИНОВ ПРИ БРОНХИАЛЬНОЙ АСТМЕ

Forty-four patients with allergic (ABA) and non-allergic (NABA) variants of bronchial asthma (BA) were examined to evaluate levels of key adipokines (leptin, resistin, adiponectin) in sputum in different variants of BA. Adipokines in sputum and blood plasma were measured by Enzyme-Linked Immunosorbent Assay (ELISA). The indices that reflect the percentage of adipokines in sputum regarding adipokines in plasma of the same patients were worked out to evaluate the ratio of levels of corresponding adipokines in plasma and sputum in patients with BA. Two regularities are clearly seen in the study: the first - levels of proinflammatory adipokines (leptin, resistin) in sputum in ABA correlate directly with indicators of respiratory function but levels of anti-inflammatory adipokines (adiponectin) in sputum correlate inversely with indicators of respiratory function; the second -correlation of levels of the studied adipokines with indicators of respiratory function are almost not revealed in NABA. The first regularity reflects the important fact that the content of adipokines in bronchial secretion is to a certain extent one of regulating local mechanisms in target organ controlled system levels of corresponding adipokines in exacerbation of BA.Для оценки уровней ключевых адипокинов (лептина, резистина, адипонектина) в мокроте при различных вариантах бронхиальной астмы (БА) обследованы 44 больных БА с аллергическим (АБА) и неаллергическим (НАБА) вариантами заболевания. Адипокины в мокроте и плазме крови определяли иммуноферментным методом (ELISA). Для оценки соотношения уровней соответствующих адипоки-нов в плазме и мокроте больных БА разработаны индексы, которые отражают процентное содержание адипокинов в мокроте по отношению к таковым в плазме у одних и тех же больных. В результате исследования четко прослеживаются две закономерности: первая - при АБА уровни провоспалительных адипокинов в мокроте (лептина и резистина) коррелируют с показателями ФВД с прямой зависимостью, а уровни противоспалительного адипокина в мокроте (адипонектина) - с обратной зависимостью; вторая - при НАБА корреляционная зависимость уровней исследуемых адипокинов с показателями ФВД практически не выявляется. Первая закономерность отражает тот важный факт, что содержание адипокинов в содержимом бронхов в определенной мере является одним из регуляторных местных механизмов в органе-мишени, участвующих в контроле за системными уровнями соответствующих адипокинов при обострении БА

The PolyA tail length of yeast histone mRNAs varies during the cell cycle and is influenced by Sen1p and Rrp6p

Yeast histone mRNAs are polyadenylated, yet factors such as Rrp6p and Trf4p, required for the 3′-end processing of non-polyadenylated RNAs, contribute to the cell cycle regulation of these transcripts. Here, we investigated the role of other known 3′-end processing/transcription termination factors of non-polyadenylated RNA in the biogenesis of histone mRNAs, specifically the Nab3p/Nrd1p/Sen1p complex. We also re-evaluated the polyadenylation status of these mRNAs during the cell cycle. Our analysis reveals that yeast histone mRNAs have shorter than average PolyA tails and the length of the PolyA tail varies during the cell cycle; S-phase histone mRNAs possess very short PolyA tails while in G1, the tail length is relatively longer. Inactivation of either Sen1p or Rrp6p leads to a decrease in the PolyA tail length of histone mRNAs. Our data also show that Sen1p contributes to 3′-end processing of histone primary transcripts. Thus, histone mRNAs are distinct from the general pool of yeast mRNAs and 3′-end processing and polyadenylation contribute to the cell cycle regulation of these transcripts

The mRNA encoding the yeast ARE-binding protein Cth2 is generated by a novel 3′ processing pathway

Microarray analyses of mRNAs over-expressed in strains lacking the nuclear exosome component Rrp6 identified the transcript encoding the ARE-binding protein Cth2, which functions in cytoplasmic mRNA stability. Subsequent northern analyses revealed that exosome mutants accumulate a 3′-extended transcript at the expense of the mature CTH2 mRNA. The 3′ ends of the CTH2 mRNA were mapped to a [GU3–5]5 repeat, unlike any previously characterized polyadenylation site. CTH2 mRNA accumulation was not inhibited by mutations in 3′-cleavage and polyadenylation factors, Rna14, Rna15 and Pap1, which block accumulation of other mRNAs. The 3′-extended CTH2 pre-mRNA strongly accumulated in strains with mutations in the TRAMP4 polyadenylation complex or the Nrd1/Nab3/Sen1 complex, and contains multiple Nrd1 and Nab3 binding sites. CTH2 carries a consensus ARE element and levels of the pre-mRNA and mRNA were elevated by mutation of the ARE or inactivation of the nuclear 5′-exonuclease Rat1. We propose that CTH2 mRNA is processed from a 3′-extended primary transcript by the exosome, TRAMP and Nrd1/Nab3/Sen1 complexes. This unusual pathway may allow time for nuclear, ARE-mediated regulation of CTH2 levels involving Rat1

Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control

Trf4 is the poly(A) polymerase component of TRAMP4, which stimulates nuclear RNA degradation by the exosome. We report that in Saccharomyces cerevisiae strains lacking Trf4, cryptic transcripts are detected from regions of repressed chromatin at telomeres and the rDNA intergenic spacer region (IGS1-R), and at CEN3. Degradation of the IGS1-R transcript was reduced in strains lacking TRAMP components, the core exosome protein Mtr3 or the nuclear-specific exosome component Rrp6. IGS1-R has potential binding sites for the RNA-binding proteins Nrd1/Nab3, and was stabilized by mutation of Nrd1. IGS1-R passes through the replication fork barrier, a region required for rDNA copy number control. Strains lacking Trf4 showed sporadic changes in rDNA copy number, whereas loss of both Trf4 and either the histone deacetylase Sir2 or the topoisomerase Top1 caused dramatic loss of rDNA repeats. Chromatin immunoprecipitation analyses showed that Trf4 is co-transcriptionally recruited to IGS1-R, consistent with a direct role in rDNA stability. Co-transcriptional RNA binding by Trf4 may link RNA and DNA metabolism and direct immediate IGS1-R degradation by the exosome following transcription termination

Permitted Oxygen Abundances and the Temperature Scale of Metal-Poor Turn-Off Stars

We use high quality VLT/UVES published data of the permitted OI triplet and FeII lines to determine oxygen and iron abundances in unevolved (dwarfs, turn-off, subgiants) metal-poor halo stars. The calculations have been performed both in LTE and NLTE, employing effective temperatures obtained with the new infrared flux method (IRFM) temperature scale by Ramirez & Melendez, and surface gravities from Hipparcos parallaxes and theoretical isochrones. A new list of accurate transition probabilities for FeII lines, tied to the absolute scale defined by laboratory measurements, has been used. We find a plateau in the oxygen-to-iron ratio over more than two orders of magnitude in iron abundance (-3.2 < [Fe/H] < -0.7), with a mean [O/Fe] = 0.5 dex (sigma = 0.1 dex), independent of metallicity, temperature and surface gravity. According to the new IRFM Teff scale, the temperatures of turn-off halo stars strongly depend on metallicity, a result that is in excellent qualitative and quantitative agreement with stellar evolution calculations, which predict that the Teff of the turn-off at [Fe/H] = -3 is about 600-700 K higher than that at [Fe/H] = -1.Comment: In press, Ap

HEMITSELLYULOZA AND THEIR NANOBIOCOMPOSITES - PERSPECTIVE NANOSTRUCTURED SINBIOTICS

Natural nanocomposites hemicellulose arabinogalactan and flavonoids isolated (from Siberian larch) and characterized. Additionally nitro- and sulfo-esters of arabinogalactan and its calcium salt are synthesized and. characterized. All of the derivatives of the beta-hemicellulose arabinogalactan. are water-soluble and are promising prebiotics on the example test-strain Bifidobacterium bifidum. (except for the nitrate esters of arabinogalactan)

Genome-wide search for breast cancer linkage in large Icelandic non-BRCA1/2 families

Abstract Introduction: A significant proportion of high-risk breast cancer families are not explained by mutations in known genes. Recent genome-wide searches (GWS) have not revealed any single major locus reminiscent of BRCA1 and BRCA2, indicating that still unidentified genes may explain relatively few families each or interact in a way obscure to linkage analyses. This has drawn attention to possible benefits of studying populations where genetic heterogeneity might be reduced. We thus performed a GWS for linkage on nine Icelandic multiple-case non-BRCA1/2 families of desirable size for mapping highly penetrant loci. To follow up suggestive loci, an additional 13 families from other Nordic countries were genotyped for selected markers. Methods: GWS was performed using 811 microsatellite markers providing about five centiMorgan (cM) resolution. Multipoint logarithm of odds (LOD) scores were calculated using parametric and nonparametric methods. For selected markers and cases, tumour tissue was compared to normal tissue to look for allelic loss indicative of a tumour suppressor gene. Results: The three highest signals were located at chromosomes 6q, 2p and 14q. One family contributed suggestive LOD scores (LOD 2.63 to 3.03, dominant model) at all these regions, without consistent evidence of a tumour suppressor gene. Haplotypes in nine affected family members mapped the loci to 2p23.2 to p21, 6q14.2 to q23.2 and 14q21.3 to q24.3. No evidence of a highly penetrant locus was found among the remaining families. The heterogeneity LOD (HLOD) at the 6q, 2p and 14q loci in all families was 3.27, 1.66 and 1.24, respectively. The subset of 13 Nordic families showed supportive HLODs at chromosome 6q (ranging from 0.34 to 1.37 by country subset). The 2p and 14q loci overlap with regions indicated by large families in previous GWS studies of breast cancer. Conclusions: Chromosomes 2p, 6q and 14q are candidate sites for genes contributing together to high breast cancer risk. A polygenic model is supported, suggesting the joint effect of genes in contributing to breast cancer risk to be rather common in non-BRCA1/2 families. For genetic counselling it would seem important to resolve the mode of genetic interaction

High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and Massively Parallel Sequencing

We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system