Construction and analysis of monomobile DNA junctions

Immobile DNA junctions are complexes of oligomeric DNA strands that interact to yield branched structures in which the branch point cannot migrate. This is achieved by minimizing the sequence symmetry in the flanking arms, so that base pairs lock at the branch site. Here, we report the design, synthesis, and analysis of two semimobile junctions, structures in which a controlled extent of branch point migratory freedom is deliberately introduced. We have constructed two minimally symmetric four-arm semimobile junctions from synthetic deoxy 17-mers. These junctions, termed "monomobile", contain a single pair of base pairs (A-T or C-G) which can migrate at the site of branching, while the rest of the junction is immobile. We have demonstrated by gel electrophoresis techniques that these junctions form and that they have the predicted 1:1:1:1 stoichiometry. We have compared these junctions with the immobile junction on which they are based, by means of hydroxyl radical protection experiments. From these data, both migratory conformers can be seen to coexist in solution. The semimobile junction with the C-G base pair has the same crossover and stacking pattern observed for the immobile junction, while the junction with the A-T base pair has the opposite pattern. We conclude that crossover and stacking patterns are a direct consequence of the base pairs which flank the junction. In addition, the data indicate that the crossover pattern biases for these junctions are much greater than are the migratory biases.     

An imputed genotype resource for the laboratory mouse

We have created a high-density SNP resource encompassing 7.87 million polymorphic loci across 49 inbred mouse strains of the laboratory mouse by combining data available from public databases and training a hidden Markov model to impute missing genotypes in the combined data. The strong linkage disequilibrium found in dense sets of SNP markers in the laboratory mouse provides the basis for accurate imputation. Using genotypes from eight independent SNP resources, we empirically validated the quality of the imputed genotypes and demonstrated that they are highly reliable for most inbred strains. The imputed SNP resource will be useful for studies of natural variation and complex traits. It will facilitate association study designs by providing high-density SNP genotypes for large numbers of mouse strains. We anticipate that this resource will continue to evolve as new genotype data become available for laboratory mouse strains. The data are available for bulk download or query at /. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium

Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder caused by mutations in the acidic compartment (which we define as the late endosome and the lysosome) protein, NPC1. The function of NPC1 is unknown, but when it is dysfunctional, sphingosine, glycosphingolipids, sphingomyelin and cholesterol accumulate. We have found that NPC1-mutant cells have a large reduction in the acidic compartment calcium store compared to wild-type cells. Chelating luminal endocytic calcium in normal cells with high-affinity Rhod-dextran induced an NPC disease cellular phenotype. In a drug-induced NPC disease cellular model, sphingosine storage in the acidic compartment led to calcium depletion in these organelles, which then resulted in cholesterol, sphingomyelin and glycosphingolipid storage in these compartments. Sphingosine storage is therefore an initiating factor in NPC1 disease pathogenesis that causes altered calcium homeostasis, leading to the secondary storage of sphingolipids and cholesterol. This unique calcium phenotype represents a new target for therapeutic intervention, as elevation of cytosolic calcium with curcumin normalized NPC1 disease cellular phenotypes and prolonged survival of the NPC1 mouse.     

Theophylline-induced protection in myoglobinuric acute renal failure: further characterization

We have reported previously that aminophylline has an ameliorating effect on the course and severity of glycerol-induced myoglobinuric acute renal failure in rats. Since aminophylline dissociates into theophylline in biological fluids and since theophylline is an adenosine receptor antagonist, we attributed the ameliorating effects to antagonism of the hemodynamic effects of endogenous adenosine. However, theophylline blocks tubuloglomerular feedback and produces natriuresis, and either of these effects might have accounted for the beneficial effects in acute renal failure. Therefore, this study was designed to further characterize the effects of theophylline in glycerol-induced acute renal failure in rats. Aminophylline had dose-dependent beneficial effects, as judged by the peak serum creatinine during the 3 days following induction of acute renal failure, by the number of animals with peak serum creatinine greater than 1 mg/dL, and by the mortality rate. Both furosemide and theophylline block tubuloglomerular feedback and produce natriuresis, but aminophylline had protective effects, whereas furosemide actually increased mortality, compared with aminophylline, following induction of myoglobinuric acute renal failure. Therefore, aminophylline's protective effects are independent of tubuloglomerular feedback and natriuresis. These results offer further support for the hypothesis that adenosine-induced hemodynamic changes play a pathogenic role in glycerol-induced acute renal failure in rats.     

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.     

Integration of QTL and bioinformatic tools to identify candidate genes for triglycerides in mice

To identify genetic loci influencing lipid levels, we performed quantitative trait loci (QTL) analysis between inbred mouse strains MRL/MpJ and SM/J, measuring triglyceride levels at 8 weeks of age in F2 mice fed a chow diet. We identified one significant QTL on chromosome (Chr) 15 and three suggestive QTL on Chrs 2, 7, and 17. We also carried out microarray analysis on the livers of parental strains of 282 F2 mice and used these data to find cis-regulated expression QTL. We then narrowed the list of candidate genes under significant QTL using a "toolbox" of bioinformatic resources, including haplotype analysis; parental strain comparison for gene expression differences and nonsynonymous coding single nucleotide polymorphisms (SNP); cis-regulated eQTL in livers of F2 mice; correlation between gene expression and phenotype; and conditioning of expression on the phenotype. We suggest Slc25a7 as a candidate gene for the Chr 7 QTL and, based on expression differences, five genes (Polr3 h, Cyp2d22, Cyp2d26, Tspo, and Ttll12) as candidate genes for Chr 15 QTL. This study shows how bioinformatics can be used effectively to reduce candidate gene lists for QTL related to complex traits.     

CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA

Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1–H3/H4 or H3/H4–DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)₂ tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.     

FLUXNET and modelling the global carbon cycle

Measurements of the net CO₂ flux between terrestrial ecosystems and the atmosphere using the eddy covariance technique have the potential to underpin our interpretation of regional CO₂ source–sink patterns, CO₂ flux responses to forcings, and predictions of the future terrestrial C balance. Information contained in FLUXNET eddy covariance data has multiple uses for the development and application of global carbon models, including evaluation/validation, calibration, process parameterization, and data assimilation. This paper reviews examples of these uses, compares global estimates of the dynamics of the global carbon cycle, and suggests ways of improving the utility of such data for global carbon modelling. Net ecosystem exchange of CO₂ (NEE) predicted by different terrestrial biosphere models compares favourably with FLUXNET observations at diurnal and seasonal timescales. However, complete model validation, particularly over the full annual cycle, requires information on the balance between assimilation and decomposition processes, information not readily available for most FLUXNET sites. Site history, when known, can greatly help constrain the model‐data comparison. Flux measurements made over four vegetation types were used to calibrate the land‐surface scheme of the Goddard Institute for Space Studies global climate model, significantly improving simulated climate and demonstrating the utility of diurnal FLUXNET data for climate modelling. Land‐surface temperatures in many regions cool due to higher canopy conductances and latent heat fluxes, and the spatial distribution of CO₂ uptake provides a significant additional constraint on the realism of simulated surface fluxes. FLUXNET data are used to calibrate a global production efficiency model (PEM). This model is forced by satellite‐measured absorbed radiation and suggests that global net primary production (NPP) increased 6.2% over 1982–1999. Good agreement is found between global trends in NPP estimated by the PEM and a dynamic global vegetation model (DGVM), and between the DGVM and estimates of global NEE derived from a global inversion of atmospheric CO₂ measurements. Combining the PEM, DGVM, and inversion results suggests that CO₂ fertilization is playing a major role in current increases in NPP, with lesser impacts from increasing N deposition and growing season length. Both the PEM and the inversion identify the Amazon basin as a key region for the current net terrestrial CO₂ uptake (i.e. 33% of global NEE), as well as its interannual variability. The inversion's global NEE estimate of −1.2 Pg [C] yr⁻¹ for 1982–1995 is compatible with the PEM‐ and DGVM‐predicted trends in NPP. There is, thus, a convergence in understanding derived from process‐based models, remote‐sensing‐based observations, and inversion of atmospheric data. Future advances in field measurement techniques, including eddy covariance (particularly concerning the problem of night‐time fluxes in dense canopies and of advection or flow distortion over complex terrain), will result in improved constraints on land‐atmosphere CO₂ fluxes and the rigorous attribution of mechanisms to the current terrestrial net CO₂ uptake and its spatial and temporal heterogeneity. Global ecosystem models play a fundamental role in linking information derived from FLUXNET measurements to atmospheric CO₂ variability. A number of recommendations concerning FLUXNET data are made, including a request for more comprehensive site data (particularly historical information), more measurements in undisturbed ecosystems, and the systematic provision of error estimates. The greatest value of current FLUXNET data for global carbon cycle modelling is in evaluating process representations, rather than in providing an unbiased estimate of net CO₂ exchange.