Separate roles of IQGAP Rng2p in forming and constricting the Schizosaccharomyces pombe cytokinetic contractile ring

Eukaryotic cells require IQGAP family multidomain adapter proteins for cytokinesis, but many questions remain about how IQGAPs contribute to the process. Here we show that fission yeast IQGAP Rng2p is required for both the normal process of contractile ring formation from precursor nodes and an alternative mechanism by which rings form from strands of actin filaments. Our work adds to previous studies suggesting a role for Rng2p in node and ring formation. We demonstrate that Rng2p is also required for normal ring constriction and septum formation. Systematic analysis of domain-deletion mutants established how the four domains of Rng2p contribute to cytokinesis. Contrary to a previous report, the actin-binding calponin homology domain of Rng2p is not required for viability, ring formation, or ring constriction. The IQ motifs are not required for ring formation but are important for ring constriction and septum formation. The GTPase-activating protein (GAP)–related domain is required for node-based ring formation. The Rng2p C-terminal domain is the only domain essential for viability. Our studies identified several distinct functions of Rng2 at multiple stages of cytokinesis.     

More Powerful Likelihood Ratio Tests for Isotonic Binomial Proportions

Binomial group testing involves pooling individuals into groups and observing a binary response on each group. Results from the group tests can then be used to draw inference about population proportions. Its use as an experimental design has received much attention in recent years, especially in public‐health screening experiments and vector‐transfer designs in plant pathology. We investigate the benefits of group testing in situations wherein one desires to test whether or not probabilities are increasingly ordered across the levels of an observed qualitative covariate, i.e., across strata of a population or among treatment levels. We use a known likelihood ratio test for individual testing, but extend its use to group‐testing situations to show the increases in power conferred by using group testing when operating in this constrained parameter space. We apply our methods to data from an HIV study involving male subjects classified as intraveneous drug users.     

Empirical Bayesian estimation of the disease transmission probability in multiple-vector-transfer designs

Plant disease is responsible for major losses in agriculture throughout the world. Diseases are often spread by insect organisms that transmit a bacterium, virus, or other pathogen. To assess disease epidemics, plant pathologists often use multiple-vector-transfers. In such contexts, groups of insect vectors are moved from an infected source to each of n test plants that will then be observed for developing symptoms of infection. The purpose of this paper is to present new estimators for p, the probability of pathogen transmission for an individual vector, motivated from an empirical Bayesian approach. We specifically investigate four such estimators, characterize their small-sample properties, and propose new credible intervals for p. These estimators remove the need to specify hyperparameters a priori and are shown to be easier to compute than the classical Bayes estimators proposed by Chaubey and Li (1995, Journal of Official Statistics 11, 1035-1046) and Chick (1996, Biometrics 52, 1055-1062). Furthermore, some of these estimators are shown to have better frequentist properties than the commonly used maximum likelihood estimator and to provide a smaller Bayes risk than the estimator proposed by Burrows (1987, Phytopathology 77, 363-365).     

Disparate requirements for the Walker A and B ATPase motifs of human RAD51D in homologous recombination

In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks (ICLs). Ectopic expression of wild-type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs.     

Informative Dorfman screening

Since the early 1940s, group testing (pooled testing) has been used to reduce costs in a variety of applications, including infectious disease screening, drug discovery, and genetics. In such applications, the goal is often to classify individuals as positive or negative using initial group testing results and the subsequent process of decoding of positive pools. Many decoding algorithms have been proposed, but most fail to acknowledge, and to further exploit, the heterogeneous nature of the individuals being screened. In this article, we use individuals' risk probabilities to formulate new informative decoding algorithms that implement Dorfman retesting in a heterogeneous population. We introduce the concept of "thresholding" to classify individuals as "high" or "low risk," so that separate, risk-specific algorithms may be used, while simultaneously identifying pool sizes that minimize the expected number of tests. When compared to competing algorithms which treat the population as homogeneous, we show that significant gains in testing efficiency can be realized with virtually no loss in screening accuracy. An important additional benefit is that our new procedures are easy to implement. We apply our methods to chlamydia and gonorrhea data collected recently in Nebraska as part of the Infertility Prevention Project.     

The initial step in human immunodeficiency virus type 1 GagProPol processing can be regulated by reversible oxidation

Background

Maturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of HIV-1 protease embedded within GagProPol precursors and cleavage of Gag and GagProPol polyproteins. Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol. In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.

Methodology/Principal Findings

To determine if oxidation influences this step, we created a GagProPol plasmid construct (pGPfs-1C) that encoded mutations at all cleavage sites except p2/NC, the initial cleavage site in GagProPol. pGPfs-1C was used in an in vitro translation assay to observe the behavior of this initial step without interference from subsequent processing events. Diamide, a sulfhydral oxidizing agent, inhibited processing at p2/NC by >60% for pGPfs-1C and was readily reversed with the reductant, dithiothreitol. The ability to regulate processing by reversible oxidation was lost when the cysteines of the embedded protease were mutated to alanine. Unlike mature protease, which requires only oxidation of cys95 for inhibition, both cysteines of the embedded protease contributed to this inhibition.

Conclusions/Significance

We developed a system that can be used to study the first step in the cascade of HIV-1 GagProPol processing and show that reversible oxidation of cysteines of HIV-1 protease embedded in GagProPol can block this initial GagProPol autoprocessing. This type of regulation may be broadly applied to the majority of retroviruses.     

Rescue of Xrcc1 knockout mouse embryo lethality by transgene-complementation

Xrcc1 knockout embryos show increased DNA breakage and apoptosis in tissues of the embryo proper prior to death at embryonic day E6.5. An additional deficiency in Trp53 allows Xrcc1(-/-) embryos to enlarge slightly and initiate gastrulation although ultimately death is delayed by less than 24h. Death presumably results from DNA damage that reaches toxic levels in the post-implantation mouse embryo. To investigate the level of XRCC1 protein needed for successful mouse development, we derived Xrcc1 transgene-complemented Xrcc1(-/-) mice that express Xrcc1 within the normal range or at a greatly reduced level (<10% normal). The greatly reduced XRCC1 protein level destabilized the XRCC1 partner protein DNA ligase III (LIG3) but still allowed for successful mouse development and healthy, fertile adults. Fibroblasts from these animals exhibited almost normal alkylation sensitivity measured by differential cytotoxicity. Thus, a large reduction of both XRCC1 and DNA ligase III has no observable effect on mouse embryogenesis and post-natal development, and no significant effect on cellular sensitivity to DNA alkylation. The presence of XRCC1, even at reduced levels of expression, is therefore capable of supporting mouse development and DNA repair.