Defining and Detecting Crossover-Interference Mutants in Yeast

The analysis of crossover interference in many creatures is complicated by the presence of two kinds of crossovers, interfering and noninterfering. In such creatures, the values of the traditional indicators of interference are subject not only to the strength of interference but also to the relative frequencies of crossing over contributed by the two kinds. We formalize the relationship among these variables and illustrate the possibilities and limitations of classical interference analysis with meiotic tetrad data from wild-type Saccharomyces cerevisiae and from mlh1 and ndj1 mutants

Aptamers for respiratory syncytial virus detection.

The identification of the infectious agents is pivotal for appropriate care of patients with viral diseases. Current viral diagnostics rely on selective detection of viral nucleic acid or protein components. In general, detection of proteins rather than nucleic acids is technically more suitable for rapid tests. However, protein-based virus identification methods depend on antibodies limiting the practical applicability of these approaches. Aptamers rival antibodies in target selectivity and binding affinity, and excel in terms of robustness and cost of synthesis. Although aptamers have been generated for virus identification in laboratory settings, their introduction into routine virus diagnostics has not been realized, yet. Here, we demonstrate that the rationally designed SELEX protocol can be applied on whole virus to select aptamers, which can potentially be applied for viral diagnostics. This approach does not require purified virus protein or complicated virus purification. The presented data also illustrate that corroborating the functionality of aptamers with various approaches is essential to pinpoint the most appropriate aptamer amongst the panel of candidates obtained by the selection. Our protocol yielded aptamers capable of detecting respiratory syncytial virus (RSV), an important pathogen causing severe disease especially in young infants, at clinically relevant concentrations in complex matrices

Observations of quiet-time moderate midlatitude L-band scintillation in association with plasma bubbles

Observations of moderate night time amplitude scintillation on the GPS L1C/A signal were recorded at the midlatitude station of Nicosia, corresponding geographic latitude and longitude of 35.18˚N and 33.38˚E respectively, on a geomagnetically quiet day. The variations of slant total electron content (STEC) and amplitude scintillation index (S4) on the night of June 12, 2014, indicate the presence of electron density depletions accompanying scintillation occurrence. The estimated apparent horizontal drift velocity and propagation direction of the plasma depletions are consistent with those observed for the equatorial plasma bubbles, thus suggesting that the moderate amplitude L-band scintillation observed over Nicosia may be associated with the extension of such plasma bubbles. The L-band scintillation occurrence was concurrent with the observations of range spread F on the ionograms recorded by the digisonde at Nicosia. The height–time–intensity plot generated using the ionogram data also showed features which can be attributed to off-angle reflections from electron density depletions, thus corroborating the STEC observations. This observation suggests that the midlatitude ionosphere is more active even during geomagnetically quiet days than previously thought and that further studies are necessary. This is particularly relevant for the GNSS user community and related applications

ReCombine: A Suite of Programs for Detection and Analysis of Meiotic Recombination in Whole-Genome Datasets

In meiosis, the exchange of DNA between chromosomes by homologous recombination is a critical step that ensures proper chromosome segregation and increases genetic diversity. Products of recombination include reciprocal exchanges, known as crossovers, and non-reciprocal gene conversions or non-crossovers. The mechanisms underlying meiotic recombination remain elusive, largely because of the difficulty of analyzing large numbers of recombination events by traditional genetic methods. These traditional methods are increasingly being superseded by high-throughput techniques capable of surveying meiotic recombination on a genome-wide basis. Next-generation sequencing or microarray hybridization is used to genotype thousands of polymorphic markers in the progeny of hybrid yeast strains. New computational tools are needed to perform this genotyping and to find and analyze recombination events. We have developed a suite of programs, ReCombine, for using short sequence reads from next-generation sequencing experiments to genotype yeast meiotic progeny. Upon genotyping, the program CrossOver, a component of ReCombine, then detects recombination products and classifies them into categories based on the features found at each location and their distribution among the various chromatids. CrossOver is also capable of analyzing segregation data from microarray experiments or other sources. This package of programs is designed to allow even researchers without computational expertise to use high-throughput, whole-genome methods to study the molecular mechanisms of meiotic recombination

The pch2Δ Mutation in Baker's Yeast Alters Meiotic Crossover Levels and Confers a Defect in Crossover Interference

Pch2 is a widely conserved protein that is required in baker's yeast for the organization of meiotic chromosome axes into specific domains. We provide four lines of evidence suggesting that it regulates the formation and distribution of crossover events required to promote chromosome segregation at Meiosis I. First, pch2Δ mutants display wild-type crossover levels on a small (III) chromosome, but increased levels on larger (VII, VIII, XV) chromosomes. Second, pch2Δ mutants show defects in crossover interference. Third, crossovers observed in pch2Δ require both Msh4-Msh5 and Mms4-Mus81 functions. Lastly, the pch2Δ mutation decreases spore viability and disrupts crossover interference in spo11 hypomorph strains that have reduced levels of meiosis-induced double-strand breaks. Based on these and previous observations, we propose a model in which Pch2 functions at an early step in crossover control to ensure that every homolog pair receives an obligate crossover

Pch2 Links Chromosome Axis Remodeling at Future Crossover Sites and Crossover Distribution during Yeast Meiosis

Segregation of homologous chromosomes during meiosis I depends on appropriately positioned crossovers/chiasmata. Crossover assurance ensures at least one crossover per homolog pair, while interference reduces double crossovers. Here, we have investigated the interplay between chromosome axis morphogenesis and non-random crossover placement. We demonstrate that chromosome axes are structurally modified at future crossover sites as indicated by correspondence between crossover designation marker Zip3 and domains enriched for axis ensemble Hop1/Red1. This association is first detected at the zygotene stage, persists until double Holliday junction resolution, and is controlled by the conserved AAA+ ATPase Pch2. Pch2 further mediates crossover interference, although it is dispensable for crossover formation at normal levels. Thus, interference appears to be superimposed on underlying mechanisms of crossover formation. When recombination-initiating DSBs are reduced, Pch2 is also required for viable spore formation, consistent with further functions in chiasma formation. pch2Δ mutant defects in crossover interference and spore viability at reduced DSB levels are oppositely modulated by temperature, suggesting contributions of two separable pathways to crossover control. Roles of Pch2 in controlling both chromosome axis morphogenesis and crossover placement suggest linkage between these processes. Pch2 is proposed to reorganize chromosome axes into a tiling array of long-range crossover control modules, resulting in chiasma formation at minimum levels and with maximum spacing

A high-throughput de novo sequencing approach for shotgun proteomics using high-resolution tandem mass spectrometry

<p>Abstract</p> <p>Background</p> <p>High-resolution tandem mass spectra can now be readily acquired with hybrid instruments, such as LTQ-Orbitrap and LTQ-FT, in high-throughput shotgun proteomics workflows. The improved spectral quality enables more accurate <it>de novo </it>sequencing for identification of post-translational modifications and amino acid polymorphisms.</p> <p>Results</p> <p>In this study, a new <it>de novo </it>sequencing algorithm, called Vonode, has been developed specifically for analysis of such high-resolution tandem mass spectra. To fully exploit the high mass accuracy of these spectra, a unique scoring system is proposed to evaluate sequence tags based primarily on mass accuracy information of fragment ions. Consensus sequence tags were inferred for 11,422 spectra with an average peptide length of 5.5 residues from a total of 40,297 input spectra acquired in a 24-hour proteomics measurement of <it>Rhodopseudomonas palustris</it>. The accuracy of inferred consensus sequence tags was 84%. According to our comparison, the performance of Vonode was shown to be superior to the PepNovo v2.0 algorithm, in terms of the number of <it>de novo </it>sequenced spectra and the sequencing accuracy.</p> <p>Conclusions</p> <p>Here, we improved <it>de novo </it>sequencing performance by developing a new algorithm specifically for high-resolution tandem mass spectral data. The Vonode algorithm is freely available for download at <url>http://compbio.ornl.gov/Vonode</url>.</p

Micro-spectroscopic investigation of selenium-bearing minerals from the Western US Phosphate Resource Area

Mining activities in the US Western Phosphate Resource Area (WPRA) have released Se into the environment. Selenium has several different oxidation states and species, each having varying degrees of solubility, reactivity, and bioavailability. In this study we are investigating the speciation of Se in mine-waste rocks. Selenium speciation was determined using bulk and micro-x-ray absorption spectroscopy (XAS), as well as micro-x-ray fluorescence mapping. Rocks used for bulk-XAS were ground into fine powders. Shale used for micro-XAS was broken along depositional planes to expose unweathered surfaces. The near edge region of the XAS spectra (XANES) for the bulk rock samples revealed multiple oxidation states, with peaks indicative of Se(-II), Se(IV), and Se(+VI) species. Micro-XANES analysis of the shale indicated that three unique Se-bearing species were present. Using the XANES data together with ab initio fitting of the extended x-ray absorption fine structure region of the micro-XAS data (micro-EXAFS) the three Se-bearing species were identified as dzharkenite, a di-selenide carbon compound, and Se-substituted pyrite. Results from this research will allow for a better understanding of the biogeochemical cycling of Se in the WPRA

Cross-platform comparability of microarray technology: Intra-platform consistency and appropriate data analysis procedures are essential

BACKGROUND: The acceptance of microarray technology in regulatory decision-making is being challenged by the existence of various platforms and data analysis methods. A recent report (E. Marshall, Science, 306, 630–631, 2004), by extensively citing the study of Tan et al. (Nucleic Acids Res., 31, 5676–5684, 2003), portrays a disturbingly negative picture of the cross-platform comparability, and, hence, the reliability of microarray technology. RESULTS: We reanalyzed Tan's dataset and found that the intra-platform consistency was low, indicating a problem in experimental procedures from which the dataset was generated. Furthermore, by using three gene selection methods (i.e., p-value ranking, fold-change ranking, and Significance Analysis of Microarrays (SAM)) on the same dataset we found that p-value ranking (the method emphasized by Tan et al.) results in much lower cross-platform concordance compared to fold-change ranking or SAM. Therefore, the low cross-platform concordance reported in Tan's study appears to be mainly due to a combination of low intra-platform consistency and a poor choice of data analysis procedures, instead of inherent technical differences among different platforms, as suggested by Tan et al. and Marshall. CONCLUSION: Our results illustrate the importance of establishing calibrated RNA samples and reference datasets to objectively assess the performance of different microarray platforms and the proficiency of individual laboratories as well as the merits of various data analysis procedures. Thus, we are progressively coordinating the MAQC project, a community-wide effort for microarray quality control

Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

Plasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of erythrocyte invasion are incompletely understood. P. falciparum depends heavily on sialic acid present on glycophorins to invade erythrocytes. However, a significant proportion of laboratory and field isolates are also able to invade erythrocytes in a sialic acid-independent manner. The identity of the erythrocyte sialic acid-independent receptor has been a mystery for decades. We report here that the complement receptor 1 (CR1) is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum. We show that soluble CR1 (sCR1) as well as polyclonal and monoclonal antibodies against CR1 inhibit sialic acid-independent invasion in a variety of laboratory strains and wild isolates, and that merozoites interact directly with CR1 on the erythrocyte surface and with sCR1-coated microspheres. Also, the invasion of neuraminidase-treated erythrocytes correlates with the level of CR1 expression. Finally, both sialic acid-independent and dependent strains invade CR1 transgenic mouse erythrocytes preferentially over wild-type erythrocytes but invasion by the latter is more sensitive to neuraminidase. These results suggest that both sialic acid-dependent and independent strains interact with CR1 in the normal red cell during the invasion process. However, only sialic acid-independent strains can do so without the presence of glycophorin sialic acid. Our results close a longstanding and important gap in the understanding of the mechanism of erythrocyte invasion by P. falciparum that will eventually make possible the development of an effective blood stage vaccine