A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo,Orthologue of Human Progeroid WRN Exonuclease,and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain TA1

Rhizobium leguminosarum bv. trifolii strain TA1 is an aerobic, motile, Gram-negative, non-spore-forming rod that is an effective nitrogen fixing microsymbiont on the perennial clovers originating from Europe and the Mediterranean basin. TA1 however is ineffective with many annual and perennial clovers originating from Africa and America. Here we describe the features of R. leguminosarum bv. trifolii strain TA1, together with genome sequence information and annotation. The 8,618,824 bp high-quality-draft genome is arranged in a 6 scaffold of 32 contigs, contains 8,493 protein-coding genes and 83 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

Genome sequence of Ensifer meliloti strain WSM1022; a highly effective microsymbiont of the model legume Medicago truncatula A17

Ensifer meliloti WSM1022 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1022 was isolated in 1987 from a nodule recovered from the roots of the annual Medicago orbicularis growing on the Cyclades Island of Naxos in Greece. WSM1022 is highly effective at fixing nitrogen with M. truncatula and other annual species such as M. tornata and M. littoralis and is also highly effective with the perennial M. sativa (alfalfa or lucerne). In common with other characterized E. meliloti strains, WSM1022 will nodulate but fixes poorly with M. polymorpha and M. sphaerocarpos and does not nodulate M. murex. Here we describe the features of E. meliloti WSM1022, together with genome sequence information and its annotation. The 6,649,661 bp high-quality-draft genome is arranged into 121 scaffolds of 125 contigs containing 6,323 protein-coding genes and 75 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

Antibody Maturation and Viral Diversification in HIV-Infected Women

Introduction

The Post-exposure Prophylaxis in Infants (PEPI)-Malawi trial evaluated infant antiretroviral regimens for prevention of post-natal HIV transmission. A multi-assay algorithm (MAA) that includes the BED capture immunoassay, an avidity assay, CD4 cell count, and viral load was used to identify women who were vs. were not recently infected at the time of enrollment (MAA recent, N = 73; MAA non-recent, N = 2,488); a subset of the women in the MAA non-recent group known to have been HIV infected for at least 2 years before enrollment (known non-recent, N = 54). Antibody maturation and viral diversification were examined in these women.

Methods

Samples collected at enrollment (N = 2,561) and 12–24 months later (N = 1,306) were available for serologic analysis using the BED and avidity assays. A subset of those samples was used for analysis of viral diversity, which was performed using a high resolution melting (HRM) diversity assay. Viral diversity analysis was performed using all available samples from women in the MAA recent group (61 enrollment samples, 38 follow-up samples) and the known non-recent group (43 enrollment samples, 22 follow-up samples). Diversity data from PEPI-Malawi were also compared to similar data from 169 adults in the United States (US) with known recent infection (N = 102) and known non-recent infection (N = 67).

Results

In PEPI-Malawi, results from the BED and avidity assays increased over time in the MAA recent group, but did not change significantly in the MAA non-recent group. At enrollment, HIV diversity was lower in the MAA recent group than in the known non-recent group. HRM diversity assay results from women in PEPI-Malawi were similar to those from adults in the US with known duration of HIV infection.

Conclusions

Antibody maturation and HIV diversification patterns in African women provide additional support for use of the MAA to identify populations with recent HIV infection.     

Genome Analysis and Physiological Comparison of Alicycliphilus denitrificans Strains BC and K601T

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601^T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601^T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601^T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601^T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601^T. Genes involved in cyclohexanol degradation were only found in strain K601^T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.     

Development and characterization of ten novel microsatellite loci for the emu (Dromaius novaehollandiae) and genetic diversity of Japanese farm populations

Molecular Biology Reports - The emu (Dromaius novaehollandiae) is a useful poultry animal farmed for fat, meat, and eggs. Genetic structure and relationships among farmed emu populations in Japan...     

Distance-based analysis of variance for brain connectivity

The field of neuroimaging dedicated to mapping connections in the brain is increasingly being recognized as key for understanding neurodevelopment and pathology. Networks of these connections are quantitatively represented using complex structures, including matrices, functions, and graphs, which require specialized statistical techniques for estimation and inference about developmental and disorder-related changes. Unfortunately, classical statistical testing procedures are not well suited to high-dimensional testing problems. In the context of global or regional tests for differences in neuroimaging data, traditional analysis of variance (ANOVA) is not directly applicable without first summarizing the data into univariate or low-dimensional features, a process that might mask the salient features of high-dimensional distributions. In this work, we consider a general framework for two-sample testing of complex structures by studying generalized within-group and between-group variances based on distances between complex and potentially high-dimensional observations. We derive an asymptotic approximation to the null distribution of the ANOVA test statistic, and conduct simulation studies with scalar and graph outcomes to study finite sample properties of the test. Finally, we apply our test to our motivating study of structural connectivity in autism spectrum disorder.     

Evidence and hypothesis in biogeography

Evidence can provide support for or against a particular biogeographical hypothesis. Treating a hypothesis as if it were evidence or an empirical observation confounds many biogeographical analyses. We focus on two recent publications that address, in part, the evolution of the biota of Sulawesi, the large Indonesian island in the centre of the Indo‐Australian Archipelago. Many biogeographical explanations are hampered by invoking simple notions of mechanism or process – dispersal and vicariance – or constraints, such as dispersal from a centre of origin, and, in so doing, dismiss more complex geological phenomena such as emergent volcanoes within island chains or composite areas as irrelevant. Moreover, they do not search for, therefore never discover, biogeographical patterns that may better explain the distribution of biota through time.