Chromatin domains in higher eukaryotes: insights from genome-wide mapping studies

In genomes of higher eukaryotes, adjacent genes often show coordinated regulation of their expression. Compartmentalization of multiple neighboring genes into a shared chromatin environment can facilitate this coordinated expression. New mapping techniques have begun to reveal that such multigene chromatin domains are a common feature of fly and mammalian genomes. Many different types of chromatin domains have been identified based on the genomic binding patterns of various proteins and histone modifications. In addition, maps of genome–nuclear lamina associations and of looping interactions between loci provide the first systematic views of the three-dimensional folding of interphase chromosomes. These genome-wide datasets uncover new architectural principles of eukaryotic genomes and indicate that multigene chromatin domains are prevalent and important regulatory units.     

Motif mismatches in microsatellites: insights from genome-wide investigation among 20 insect species

We present a detailed genome-wide comparative study of motif mismatches of microsatellites among 20 insect species representing five taxonomic orders. The results show that varying proportions (∼15–46%) of microsatellites identified in these species are imperfect in motif structure, and that they also vary in chromosomal distribution within genomes. It was observed that the genomic abundance of imperfect repeats is significantly associated with the length and number of motif mismatches of microsatellites. Furthermore, microsatellites with a higher number of mismatches tend to have lower abundance in the genome, suggesting that sequence heterogeneity of repeat motifs is a key determinant of genomic abundance of microsatellites. This relationship seems to be a general feature of microsatellites even in unrelated species such as yeast, roundworm, mouse and human. We provide a mechanistic explanation of the evolutionary link between motif heterogeneity and genomic abundance of microsatellites by examining the patterns of motif mismatches and allele sequences of single-nucleotide polymorphisms identified within microsatellite loci. Using Drosophila Reference Genetic Panel data, we further show that pattern of allelic variation modulates motif heterogeneity of microsatellites, and provide estimates of allele age of specific imperfect microsatellites found within protein-coding genes.     

Engineering drought and salinity tolerance in plants: lessons from genome-wide expression profiling in Arabidopsis

World food security is increasingly dependent on continuous crop improvement and, in particular, the development of crops with increased drought and salinity tolerance. The completed genomic sequence of the model plant Arabidopsis thaliana and the development of whole-genome microarrays, together with increasing repositories of publicly available data and data analysis tools, have opened new avenues to genome-wide systemic analysis of plant stress responses. Here we outline examples of how this full-genome expression profiling can contribute to our understanding of complex stress responses and the identification and evaluation of novel transgenes that could hold the key to the development of commercially viable and sustainable crop plants.     

MicroRNAs profiling in murine models of acute and chronic asthma: a relationship with mRNAs targets

Background

miRNAs are now recognized as key regulator elements in gene expression. Although they have been associated with a number of human diseases, their implication in acute and chronic asthma and their association with lung remodelling have never been thoroughly investigated.

Methodology/Principal Findings

In order to establish a miRNAs expression profile in lung tissue, mice were sensitized and challenged with ovalbumin mimicking acute, intermediate and chronic human asthma. Levels of lung miRNAs were profiled by microarray and in silico analyses were performed to identify potential mRNA targets and to point out signalling pathways and biological processes regulated by miRNA-dependent mechanisms. Fifty-eight, 66 and 75 miRNAs were found to be significantly modulated at short-, intermediate- and long-term challenge, respectively. Inverse correlation with the expression of potential mRNA targets identified mmu-miR-146b, -223, -29b, -29c, -483, -574-5p, -672 and -690 as the best candidates for an active implication in asthma pathogenesis. A functional validation assay was performed by cotransfecting in human lung fibroblasts (WI26) synthetic miRNAs and engineered expression constructs containing the coding sequence of luciferase upstream of the 3′UTR of various potential mRNA targets. The bioinformatics analysis identified miRNA-linked regulation of several signalling pathways, as matrix metalloproteinases, inflammatory response and TGF-β signalling, and biological processes, including apoptosis and inflammation.

Conclusions/Significance

This study highlights that specific miRNAs are likely to be involved in asthma disease and could represent a valuable resource both for biological makers identification and for unveiling mechanisms underlying the pathogenesis of asthma.     

Viscous drag as the source of active site perturbation during protease translocation: insights into how inhibitory processes are controlled by serpin metastability

The native form of serine protease inhibitors (serpins) is kinetically trapped in a metastable state, which is thought to play a central role in the inhibitory mechanism. The initial binding complex between a serpin and a target protease undergoes a conformational change that forces the protease to translocate toward the opposite pole. Although structural determination of the final stable complex revealed a detailed mechanism of keeping the bound protease in an inactive conformation, it has remained unknown how the serpin exquisitely translocates a target protease with an acyl-linkage unhydrolyzed. We previously suggested that the acyl-linkage hydrolysis is strongly suppressed by active site perturbation during the protease translocation. Here, we address what induces the transient perturbation and how the serpin metastability contributes to the perturbation. Inhibitory activity of alpha1-antitrypsin (alpha1AT) toward elastase showed negative correlations with medium viscosity and Stokes radius of elastase moiety, indicating that viscous drag directly affects the protease translocation. Stopped-flow measurements revealed that the change in the inhibitory activity is primarily caused by the change in the translocation rate. The native stability of alpha1AT cavity mutants showed a negative correlation with the translocation rate but a positive correlation with the acyl-linkage hydrolysis rate, suggesting that the two kinetic steps are not independent but closely related. The degree of active site perturbation was probed by amino acid nucleophiles, supporting the view that the changes in the acyl-linkage hydrolysis rate are due to different perturbation states. These results suggest that the active site perturbation is caused by local imbalance between a pulling force driving protease translocation and a counteracting viscous drag force. The structural architecture of serpin metastability seems to be designed to ensure the active site perturbation by providing a sufficient pulling force, so the undesirable hydrolytic activity of protease is strongly suppressed during the translocation.     

The role of chemokines in Schistosoma mansoni infection: insights from human disease and murine models

Chemokines are a superfamily of low-molecular-weight cytokines that were initially described for their chemoattractant activity. It is now clear chemokines have several other activities that modulate immune processes. More than 50 chemokines ligands and at least 19 receptors have been described to date. Depending on the number of N-terminal cysteine residues, chemokines are grouped in the subfamilies CXC, CC, C or CX3C. A growing body of evidence suggests a role for chemokines in the pathogenesis of several inflammatory diseases. Our studies involving mice and humans infected with Schistosoma mansoni suggest an important role of the chemokine CCL3 and its receptors (CCR1 and CCR5) in the pathogenesis of severe schistosomiasis. We suggest that the differential activation of CCR1 or CCR5 during the course of schistosomiasis may dictate the outcome of the disease.     

Reasons for lower transformation efficiency in indica rice using Agrobacterium tumefaciens-mediated transformation: lessons from transformation assays and genome-wide expression profiling

Agrobacterium tumefaciens-mediated genetic transformation has been routinely used in rice for more than a decade. However, the transformation efficiency of the indica rice variety is still unsatisfactory and much lower than that of japonica cultivars. Further improvement on the transformation efficiency lies in the genetic manipulation of the plant itself, which requires a better understanding of the underlying process accounting for the susceptibility of plant cells to Agrobacterium infection as well as the identification of plant genes involved in the transformation process. In this study, transient and stable transformation assays using different japonica and indica cultivars showed that the lower transformation efficiency in indica rice was mainly due to the low efficiency in T-DNA integration into the plant genome. Analyses of the global gene expression patterns across the transformation process in different varieties revealed major differences in the expression of genes responding to Agrobacterium within the first 6 h after infection and more differentially expressed genes were observed in the indica cultivar Zhenshan 97 (ZS), with a number of genes repressed early during infection. Microarray analysis revealed an important effect of plant defense response on Agrobacterium-mediated transformation. It has been shown that some genes which may be necessary for the transformation process were down-regulated in the indica cultivar ZS. This dataset provided a versatile resource for plant genomic research to understand the regulatory network of transformation process, and showed great promise for improving indica rice transformation using genetic manipulation of the rice genome.     

Novel missense mutations in gidB gene associated with streptomycin resistance in Mycobacterium tuberculosis: insights from molecular dynamics

Streptomycin was the first antibiotic used for the treatment of tuberculosis by inhibiting translational proof reading. Point mutation in gidB gene encoding S-adenosyl methionine (SAM)-dependent 7-methylguanosine (m7G) methyltransferase required for methylation of 16S rRNA confers streptomycin resistance. As there was no structural substantiation experimentally, gidB protein model was built by threading algorithm. In this work, molecular dynamics (MD) simulations coupled with binding free energy calculations were performed to outline the mechanism underlying high-level streptomycin resistance associated with three novel missense mutants including S70R, T146M, and R187M. Results from dynamics analyses suggested that the structure distortion in the binding pocket of gidB mutants modulate SAM binding affinity. At the structural level, these conformational changes bring substantial decrease in the number of residues involved in hydrogen bonding and dramatically reduce thermodynamic stability of mutant gidB–SAM complexes. The outcome of comparative analysis of the MD simulation trajectories revealed lower conformational stability associated with higher flexibility in mutants relative to the wild-type, turns to be major factor driving the emergence of drug resistance toward antibiotic. This study will pave way toward design and development of resistant defiant gidB inhibitors as potent anti-TB agents.     

Rapid-eye movement sleep and muscarinic receptor binding in rats are augmented during withdrawal from chronic scopolamine treatment

The purpose of this study was to determine whether a seven day, once-daily morning administration of scopolamine produces upregulation of muscarinic receptors and augments REMS during withdrawal. After obtaining two, six-hour baseline sleep recordings, beginning at 0900, independent groups of rats were administered either scopolamine or saline every morning for seven days. Six hour sleep recordings were obtained following the first and seventh day of injection and during the two subsequent withdrawal days. After obtaining the last sleep recording the rats were sacrificed and the following brain areas removed: cerebral cortex, hippocampii, caudate nuclei, brainstem, and cerebellum. 3H-QNB was used as the ligand to assess for changes in muscarinic receptor binding. Compared to baseline, scopolamine produced a significant decrease in REMS during the period of drug administration. During the withdrawal days, however, REMS increased during the morning period. Compared to the saline group, the scopolamine treated animals had increased muscarinic receptor binding in the caudate and hippocampus; no significant change in receptor density was observed in the cortex, brainstem or cerebellum.