Genetic Characterization of the M RNA Segment of Crimean Congo Hemorrhagic Fever Virus Strains, China

We report the genetic characterization of the M RNA segment of Crimean Congo hemorrhagic fever virus (CCHFV). Two CCHFV strains isolated in Xinjiang Province, a region endemic for CCHF in northwestern China, were studied. These strains, designated BA66019 and BA8402, were isolated in 1965 and 1984 from a CCHF patient and Hyalomma ticks, respectively. Viral RNA was extracted from suckling mouse brains infected with these two strains, amplified, and sequenced. The full-length M RNA, consisting of 5.3 kb, was determined for both strains. The coding nucleotide sequences of the two strains differed from each other by 17.5% and from the reference CCHFV strain IbAr10200 by a mean of 22%, suggesting that the genus Nairovirus comprises a group of genetically highly diverse strains

Heterodimeric DNA methyltransferases as a platform for creating designer zinc finger methyltransferases for targeted DNA methylation in cells

The ability to target methylation to specific genomic sites would further the study of DNA methylation’s biological role and potentially offer a tool for silencing gene expression and for treating diseases involving abnormal hypomethylation. The end-to-end fusion of DNA methyltransferases to zinc fingers has been shown to bias methylation to desired regions. However, the strategy is inherently limited because the methyltransferase domain remains active regardless of whether the zinc finger domain is bound at its cognate site and can methylate non-target sites. We demonstrate an alternative strategy in which fragments of a DNA methyltransferase, compromised in their ability to methylate DNA, are fused to two zinc fingers designed to bind 9 bp sites flanking a methylation target site. Using the naturally heterodimeric DNA methyltransferase M.EcoHK31I, which methylates the inner cytosine of 5′-YGGCCR-3′, we demonstrate that this strategy can yield a methyltransferase capable of significant levels of methylation at the target site with undetectable levels of methylation at non-target sites in Escherichia coli. However, some non-target methylation could be detected at higher expression levels of the zinc finger methyltransferase indicating that further improvements will be necessary to attain the desired exclusive target specificity

Inheritance of an Epigenetic Mark: The CpG DNA Methyltransferase 1 Is Required for De Novo Establishment of a Complex Pattern of Non-CpG Methylation

Site-specific methylation of cytosines is a key epigenetic mark of vertebrate DNA. While a majority of the methylated residues are in the symmetrical (meC)pG:Gp(meC) configuration, a smaller, but significant fraction is found in the CpA, CpT and CpC asymmetric (non-CpG) dinucleotides. CpG methylation is reproducibly maintained by the activity of the DNA methyltransferase 1 (Dnmt1) on the newly replicated hemimethylated substrates (meC)pG:GpC. On the other hand, establishment and hereditary maintenance of non-CpG methylation patterns have not been analyzed in detail. We previously reported the occurrence of site- and allele-specific methylation at both CpG and non-CpG sites. Here we characterize a hereditary complex of non-CpG methylation, with the transgenerational maintenance of three distinct profiles in a constant ratio, associated with extensive CpG methylation. These observations raised the question of the signal leading to the maintenance of the pattern of asymmetric methylation. The complete non-CpG pattern was reinstated at each generation in spite of the fact that the majority of the sperm genomes contained either none or only one methylated non-CpG site. This observation led us to the hypothesis that the stable CpG patterns might act as blueprints for the maintenance of non-CpG DNA methylation. As predicted, non-CpG DNA methylation profiles were abrogated in a mutant lacking Dnmt1, the enzymes responsible for CpG methylation, but not in mutants defective for either Dnmt3a or Dnmt2

MethylViewer: computational analysis and editing for bisulfite sequencing and methyltransferase accessibility protocol for individual templates (MAPit) projects

Bisulfite sequencing is a widely-used technique for examining cytosine DNA methylation at nucleotide resolution along single DNA strands. Probing with cytosine DNA methyltransferases followed by bisulfite sequencing (MAPit) is an effective technique for mapping protein–DNA interactions. Here, MAPit methylation footprinting with M.CviPI, a GC methyltransferase we previously cloned and characterized, was used to probe hMLH1 chromatin in HCT116 and RKO colorectal cancer cells. Because M.CviPI-probed samples contain both CG and GC methylation, we developed a versatile, visually-intuitive program, called MethylViewer, for evaluating the bisulfite sequencing results. Uniquely, MethylViewer can simultaneously query cytosine methylation status in bisulfite-converted sequences at as many as four different user-defined motifs, e.g. CG, GC, etc., including motifs with degenerate bases. Data can also be exported for statistical analysis and as publication-quality images. Analysis of hMLH1 MAPit data with MethylViewer showed that endogenous CG methylation and accessible GC sites were both mapped on single molecules at high resolution. Disruption of positioned nucleosomes on single molecules of the PHO5 promoter was detected in budding yeast using M.CviPII, increasing the number of enzymes available for probing protein–DNA interactions. MethylViewer provides an integrated solution for primer design and rapid, accurate and detailed analysis of bisulfite sequencing or MAPit datasets from virtually any biological or biochemical system

Anxiety Level of Graduate Students in Social Work

This study was designed to determine (1) the trend of anxiety level of social work students, term by term, over the academic year; (2) the cyclical trend of anxiety level of social work students within each term and (3) the effects of age and sex on level of anxiety among social work students. Anxiety was measured with the IPAT - 8 Parallel Form Anxiety Battery. This test was administered to twenty randomly selected first year students in the School of Social Work during the 1969-1970 academic year at Portland State University. Data was collected from six test administrations which took place at the beginning and the end of each term. Analysis of variance in a 2x2x2x3 factorial design simultaneously investigated all four variables. Some variation among these variables and their interactions was found, but only the time of quarter main effect reached statistical significance. A cyclical pattern of anxiety following a high-in-the-beginning, low-at-the-end of each term trend was observed. Anxiety, however, remained quite level over the three terms of the academic year. Nor was anxiety level related to differences in age or sex. These findings have led the authors to speculate that the uncertainty of a new situation at the beginning of each new term created more anxiety than did the final field evaluations, classroom examinations, papers or other outside influences such as the Kent State incidents etc., and that increased structuring at the beginning of each term might help to allay that anxiety. Perhaps it could be said that each individual\u27s role as a social work graduate student had a greater effect on his anxiety level than did sex, age, or important events not directly related to school expectations

DNA methylation and specific sequience motifs: association with genetics instability in p53 in cancer, and other loci in degenerative disorders and aging

Analysis of the p53 somatic mutation database reveals that genetic instability is strongly associated with methylation and repeated sequences which contribute to the formation of alternative DNA conformations. Similar genetic instability is observed in neurological disorders and aging. In the present analysis we identified common sequence elements associated with genetic instability in the above disorders. The formation of new epigenetic/genotoxic modification sites, like CC, GG, CpG, CCC and GGG sites due to AT>GC transitions and AT>CG transversions, next to TG and CA sequences can induce p53 mutations, whereas genetic instability at repeated sequences, such as CGG and CTG, in association with CpG methylation and other specific sequence elements like TG, consist the basis of various neurological disorders. Previous reports have also shown that in aging tissues, GT/TG, CA/AC sequences as well as DNA repeats and methylation are common motifs, whereas CA microsatellites can cause DNA conformational changes. These common sequence elements probably represent universal sites related to genetic instability in various genes including cancer related p53, development of various neurological diseases and organ -specific genome deterioration and dysfunction at old age