Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung+ host proficient in uracil excision repair

Papillary carcinoma of the thyroid: methylation is not involved in the regulation of MET expression

Hypomethylation has been reported to be responsible for the activation of several oncogenes. The possibility that hypomethylation is involved in the regulation of MET transcription was investigated through the analysis of the methylation status of one CpG island containing 43 CpGs in six cases of papillary carcinoma, in the corresponding normal thyroid tissue, and in two cases of hyperplastic goitre. Evidence of methylation was not found in any of the analysed CpG. © 2004 Cancer Research UK

Accuracy and completeness of patient pathways – the benefits of national data linkage in Australia

Background - The technical challenges associated with national data linkage, and the extent of cross-border population movements, are explored as part of a pioneering research project. The project involved linking state-based hospital admission records and death registrations across Australia for a national study of hospital related deaths. Methods - The project linked over 44 million morbidity and mortality records from four Australian states between 1st July 1999 and 31st December 2009 using probabilistic methods. The accuracy of the linkage was measured through a comparison with jurisdictional keys sourced from individual states. The extent of cross-border population movement between these states was also assessed. Results - Data matching identified almost twelve million individuals across the four Australian states. The percentage of individuals from one state with records found in another ranged from 3-5 %. Using jurisdictional keys to measure linkage quality, results indicate a high matching efficiency (F measure 97 to 99 %), with linkage processing taking only a matter of days. Conclusions - The results demonstrate the feasibility and accuracy of undertaking cross jurisdictional linkage for national research. The benefits are substantial, particularly in relation to capturing the full complement of records in patient pathways as a result of cross-border population movements. The project identified a sizeable ‘mobile’ population with hospital records in more than one state. Research studies that focus on a single jurisdiction will under-enumerate the extent of hospital usage by individuals in the population. It is important that researchers understand and are aware of the impact of this missing hospital activity on their studies. The project highlights the need for an efficient and accurate data linkage system to support national research across Australia

A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples

The bisulfite genomic sequencing protocol is a widely used method for analyzing DNA methylation. It relies on the deamination of unmethylated cytosine residues to uracil; however, its high rates of DNA degradation and incomplete cytosine to uracil conversion often lead to failed experiments, uninformative results, and false positives. Here, we report the addition of a single-step multiple restriction enzyme digestion (MRED) designed to differentially digest polymerase chain reaction products amplified from unconverted DNA while leaving those of converted DNA intact. We show that for our model system, RARB2 P2 promoter, use of MRED increased informative sequencings ninefold, and MRED did not alter the clonal representation in one fully methylated cell line, H-596, treated or not with 5-azadeoxycytidine, a methylation inhibitor. We believe that this method may easily be adapted for analyzing other genes and provide guidelines for selecting the most appropriate MRED restriction enzymes

CaZF, a Plant Transcription Factor Functions through and Parallel to HOG and Calcineurin Pathways in Saccharomyces cerevisiae to Provide Osmotolerance

Salt-sensitive yeast mutants were deployed to characterize a gene encoding a C2H2 zinc finger protein (CaZF) that is differentially expressed in a drought-tolerant variety of chickpea (Cicer arietinum) and provides salinity-tolerance in transgenic tobacco. In Saccharomyces cerevisiae most of the cellular responses to hyper-osmotic stress is regulated by two interconnected pathways involving high osmolarity glycerol mitogen-activated protein kinase (Hog1p) and Calcineurin (CAN), a Ca2+/calmodulin-regulated protein phosphatase 2B. In this study, we report that heterologous expression of CaZF provides osmotolerance in S. cerevisiae through Hog1p and Calcineurin dependent as well as independent pathways. CaZF partially suppresses salt-hypersensitive phenotypes of hog1, can and hog1can mutants and in conjunction, stimulates HOG and CAN pathway genes with subsequent accumulation of glycerol in absence of Hog1p and CAN. CaZF directly binds to stress response element (STRE) to activate STRE-containing promoter in yeast. Transactivation and salt tolerance assays of CaZF deletion mutants showed that other than the transactivation domain a C-terminal domain composed of acidic and basic amino acids is also required for its function. Altogether, results from this study suggests that CaZF is a potential plant salt-tolerance determinant and also provide evidence that in budding yeast expression of HOG and CAN pathway genes can be stimulated in absence of their regulatory enzymes to provide osmotolerance

Electrical modalities beyond pacing for the treatment of heart failure

In this review, we report on electrical modalities, which do not fit the definition of pacemaker, but increase cardiac performance either by direct application to the heart (e.g., post-extrasystolic potentiation or non-excitatory stimulation) or indirectly through activation of the nervous system (e.g., vagal or sympathetic activation). The physiological background of the possible mechanisms of these electrical modalities and their potential application to treat heart failure are discussed

A Gammaherpesviral Internal Repeat Contributes to Latency Amplification

BACKGROUND: Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. CONCLUSIONS/SIGNIFICANCE: These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression

Epigenetics of lung cancer

Epigenetics is the study of heritable changes in gene expression that occur without changes in DNA sequence. It has a role in determining when and where a gene is expressed during development. Perhaps the most well known epigenetic mechanism is DNA methylation whereby cytosines at position 5 in CpG dinucleotides are methylated. Histone modification is another form of epigenetic control, which is quite complex and diverse. Histones and DNA make up the nucleosome which is the structural unit of chromatin which are involved in packaging DNA. Apart from the crucial role epigenetics plays in embryonic development, transcription, chromatin structure, X chromosome inactivation and genomic imprinting, its role in an increasing number of human diseases is more and more recognized. These diseases include cancer, and lung cancer in particular has been increasingly studied for the potential biological role of epigenetic changes with the promise of better and novel diagnostic and therapeutic tools