Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases

DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.     

Altered expression of fibronectin gene in cells infected with human cytomegalovirus.

Human cytomegalovirus (HCMV) induces morphological changes in infected cells that are remarkably similar to those seen in oncogenically transformed cells. The molecular bases of these phenotypic alterations are not known but their occurrence in some transformed cells can be associated with abnormal fibronectin (FN) expression. In this report, we have compared FN levels in normal and HCMV-infected cells. In these studies, the HCMV-infected fibroblasts exhibited a progressive loss of cellular FN. Northern (RNA) blot analysis revealed that the decrease in FN levels resulted from a lowering of FN mRNA levels in HCMV-infected cells. We detected an initial decrease in FN mRNA of 25 to 30% at immediate-early and early times, whereas at late times after infection the levels of FN mRNA were lowered by greater than 80%. These results indicated that the HCMV-induced decrease in FN expression is due to a decrease in the quantity of FN mRNA and suggested that HCMV-encoded and/or -induced functions may be involved in producing these alterations.     

A statistical framework for quantitative trait mapping

We describe a general statistical framework for the genetic analysis of quantitative trait data in inbred line crosses. Our main result is based on the observation that, by conditioning on the unobserved QTL genotypes, the problem can be split into two statistically independent and manageable parts. The first part involves only the relationship between the QTL and the phenotype. The second part involves only the location of the QTL in the genome. We developed a simple Monte Carlo algorithm to implement Bayesian QTL analysis. This algorithm simulates multiple versions of complete genotype information on a genomewide grid of locations using information in the marker genotype data. Weights are assigned to the simulated genotypes to capture information in the phenotype data. The weighted complete genotypes are used to approximate quantities needed for statistical inference of QTL locations and effect sizes. One advantage of this approach is that only the weights are recomputed as the analyst considers different candidate models. This device allows the analyst to focus on modeling and model comparisons. The proposed framework can accommodate multiple interacting QTL, nonnormal and multivariate phenotypes, covariates, missing genotype data, and genotyping errors in any type of inbred line cross. A software tool implementing this procedure is available. We demonstrate our approach to QTL analysis using data from a mouse backcross population that is segregating multiple interacting QTL associated with salt-induced hypertension.     

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart

Ca²⁺-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca²⁺ responses in cardiac myocytes from Tpcn2^−/− mice, unlike myocytes from wild-type (WT) mice. Ca²⁺/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca²⁺ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2^−/− mice. Increases in amplitude of L-type Ca²⁺ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2^−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2^−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2^−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca²⁺-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.     

The collaborative cross: a recombinant inbred mouse population for the systems genetic era

The mouse is the most extensively used mammalian model for biomedical and aging research, and an extensive catalogue of laboratory resources is available to support research using mice: classical inbred lines, genetically modified mice (knockouts, transgenics, and humanized mice), selectively bred lines, consomics, congenics, recombinant inbred panels, outbred and heterogeneous stocks, and an expanding set of wild-derived strains. However, these resources were not designed or intended to model the heterogeneous human population or for a systematic analysis of phenotypic effects due to random combinations of uniformly distributed natural variants. The Collaborative Cross (CC) is a large panel of recently established multiparental recombinant inbred mouse lines specifically designed to overcome the limitations of existing mouse genetic resources for analysis of phenotypes caused by combinatorial allele effects. The CC models the complexity of the human genome and supports analyses of common human diseases with complex etiologies originating through interactions between allele combinations and the environment. The CC is the only mammalian resource that has high and uniform genomewide genetic variation effectively randomized across a large, heterogeneous, and infinitely reproducible population. The CC supports data integration across environmental and biological perturbations and across space (different labs) and time.