The rockefeller university

A large body of literature provides compelling evidence for the role of evolutionarily conserved core histone residues in various biological processes. However, site-directed mutagenesis of individual residues that are known to be sites of posttranslational modifications often does not result in clear phenotypic defects. In some cases, the combination of multiple mutations can give rise to stronger phenotypes, implying functional redundancy between distinct residues on histones. Here, we examined the "histone redundancy hypothesis" by characterizing double deletion of all pairwise combinations of amino-terminal tails (N-tails) from the four core histones encoded in budding yeast. First, we found that multiple lysine residues on the N-tails of both H2A and H4 are redundantly involved in cell viability. Second, simultaneous deletion of N-tails from H2A and H3 leads to a severe growth defect, which is correlated with perturbed gross chromatin structure in the mutant cells. Finally, by combining point mutations on H3 with deletion of the H2A N-tail, we revealed a redundant role for lysine 4 on H3 and the H2A N-tail in hydroxyurea-mediated response. Altogether, these data suggest that the N-tails of core histones share previously unrecognized, potentially redundant functions that, in some cases are different from those of the widely accepted H2A/H2B and H3/H4 dimer pairs.

The tuberculin skin test (TST) measures the intensity of antimycobacterial acquired immunity and is used to diagnose latent infection with Mycobacterium tuberculosis. We report evidence for a codominant gene explaining similar to 65% of the TST variability. Disregarding the host genetic background may lead to misclassifications of TST-based diagnosis of latent M. tuberculosis infection.

Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life.

This paper presents a search for standard model Higgs boson production in association with a W boson using events recorded by the CDF experiment in a data set corresponding to an integrated luminosity of 5.6 fb(-1). The search is performed using a matrix element technique in which the signal and background hypotheses are used to create a powerful discriminator. The discriminant output distributions for signal and background are fit to the observed events using a binned likelihood approach to search for the Higgs boson signal. We find no evidence for a Higgs boson, and 95% confidence level (C.L.) upper limits are set on sigma(p (p) over bar -> WH) X B(H -> b (b) over bar). The observed limits range from 3.5 to 37.6 relative to the standard model expectation for Higgs boson masses between m(H) = 100 GeV/c(2) and m(H) 150 GeV/c(2). The 95% C.L. expected limit is estimated from the median of an ensemble of simulated experiments and varies between 2.9 and 32.7 relative to the production rate predicted by the standard model over the Higgs boson mass range studied.

MicroRNAs (miRNAs) regulate the abundance of target mRNAs by guiding cleavage at sequence complementary regions. In this study, artificial miRNAs (amiRNAs) targeting conserved motifs of the L (replicase) gene of Watermelon silver mottle virus (WSMoV) were constructed using Arabidopsis pre-miRNA159a as the backbone. The constructs included six single amiRNAs targeting motifs A, B1, B2, C, D of E, and two triple amiRNAs targeting motifs AB1E or B2DC. Processing of pre-amiRNAs was confirmed by agro-infiltration, and transgenic Nicotiana benthamiana plants expressing each amiRNA were generated. Single amiRNA transgenic lines expressing amiR-LB2 or amiR-LD showed resistance to WSMoV by delaying symptom development. Triple amiRNA lines expressing amiR-LB2, amiR-LD and amiR-LC provided complete resistance against WSMoV, with no indication of infection 28 days after inoculation. Resistance levels were positively correlated with amiRNA expression levels in these single and triple amiRNA lines. The triple amiR-LAB1E line did not provide resistance to WSMoV. Similarly, the poorly expressed amiR-LC and amiR-LE lines did not provide resistance to WSMoV. The amiR-LA- and amiR-LB1-expressing lines were susceptible to WSMoV, and their additional susceptibility to the heterologous Turnip mosaic virus harbouring individual target sequences indicated that these two amiRNAs have no effect in vivo. Transgenic lines expressing amiR-LB2 exhibited delayed symptoms after challenge with Peanut bud necrosis virus having a single mismatch in the target site. Overall, our results indicate that two amiRNAs, amiR-LB2 and amiR-LD, of the six designed amiRNAs confer moderate resistance against WSMoV, and the triple construct including the two amiRNAs provides complete resistance.

Argonaute proteins (Ago1-4) are essential components of the microRNA-induced silencing complex and play important roles in both microRNA biogenesis and function. Although Ago2 is the only one with the slicer activity, it is not clear whether the slicer activity is a universally critical determinant for Ago2's function in mammals. Furthermore, functional specificities associated with different Argonautes remain elusive. Here we report that microRNAs are randomly sorted to individual Argonautes in mammals, independent of the slicer activity. When both Ago1 and Ago2, but not either Ago1 or Ago2 alone, are ablated in the skin, the global expression of microRNAs is significantly compromised and it causes severe defects in skin morphogenesis. Surprisingly, Ago3 is able to load microRNAs efficiently in the absence of Ago1 and Ago2, despite a significant loss of global microRNA expression. Quantitative analyses reveal that Ago2 interacts with a majority of microRNAs (60%) in the skin, compared with Ago1 (30%) and Ago3 (<10%). This distribution is highly correlated with the abundance of each Argonaute, as quantified by shotgun proteomics. The quantitative correlation between Argonautes and their associated microRNAs is conserved in human cells. Finally, we measure the absolute expression of Argonaute proteins and determine that their copy number is similar to 1.4 x 10(5) to 1.7 x 10(5) molecules per cell. Together, our results reveal a quantitative picture for microRNA activity in mammals.

We report the combination of recent measurements of the helicity of the W boson from top quark decay by the CDF and D0 collaborations, based on data samples corresponding to integrated luminosities of 2.7-5.4 fb(-1) of p (p) over bar collisions collected during Run II of the Fermilab Tevatron collider. Combining measurements that simultaneously determine the fractions of W bosons with longitudinal (f(0)) and right-handed (f(+)) helicities, we find f(0) = 0.722 +/- 0.081[+/- 0.062(stat) +/- 0.052(syst)] and f(+) = -0.033 +/- 0.046[+/- 0.034(stat) +/- 0.031(syst)]. Combining measurements where one of the helicity fractions is fixed to the value expected in the standard model, we find f(0) = 0.682 +/- 0.057[+/- 0.035(stat) +/- 0.046(syst)] for fixed f(+) and f(+) = -0.015 +/- 0.035[+/- 0.018(stat) +/- 0.030(syst)] for fixed f(0). The results are consistent with standard model expectations.

Populations vary in time and in space, and temporal variation may differ from spatial variation. Yet, in the past half century, field data have confirmed both the temporal and spatial forms of Taylor's power Law, a linear relationship between log(variance) and log(mean) of population size. Recent theory predicted that competitive species interactions should reduce the slope of the temporal version of Taylor's Law. We tested whether this prediction applied to the spatial version of Taylor's Law using simple, well-controlled laboratory populations of two species of bacteria that were cultured either separately or together for 24 h in media of widely varying nutrient richness. Experimentally, the spatial form of Taylor's Law with a slope of 2 held for these simple bacterial communities, but competitive interactions between the two species did not reduce the spatial Taylor's Law slope. These results contribute to the widespread usefulness of Taylor's Law in population ecology, epidemiology and pest control.

We look back on the discoveries that the tyrosine kinases TYK2 and JAK1 and the transcription factors STAT1, STAT2, and IRF9 are required for the cellular response to type I interferons. This initial description of the JAK-STAT pathway led quickly to additional discoveries that type II interferons and many other cytokines signal through similar mechanisms. This well-understood pathway now serves as a paradigm showing how information from protein-protein contacts at the cell surface can be conveyed directly to genes in the nucleus. We also review recent work on the STAT proteins showing the importance of several different post-translational modifications, including serine phosphorylation, acetylation, methylation, and sumoylation. These remarkably proficient proteins also provide noncanonical functions in transcriptional regulation and they also function in mitochondrial respiration and chromatin organization in ways that may not involve transcription at all.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.