Solution hybridization of crosslinkable DNA oligonucleotides to bacteriophage M13 DNA. Effect of secondary structure on hybridization kinetics and equilibria

Several DNA oligonucleotides have been photochemically modified with the furocoumarin 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) such that each contained a single HMT furan side monoadduct to thymidine at a unique 5' TpA 3' sequence. When these oligonucleotides were hybridized to their respective complements, the HMT adduct could be driven to form an interstrand crosslink by irradiation of the hybrid with 360 nm light. The ability to crosslink probe-target complexes has allowed us to determine the kinetics and the extent of hybridization in solution between these oligonucleotides and their complementary sequences in single-stranded bacteriophage M13 DNA. Our data indicate that these parameters are strongly influenced by the existence of local as well as global secondary structure in the viral DNA. During hybridization, rearrangement of this secondary structure so as to expose the target sequence can be rate-limiting. Upon attainment of equilibrium, only a portion of the target sequence may be hybridized to the probe with the remainder involved in intrastrand base-pairing. Using crosslinkable oligonucleotide probes hybridized and irradiated near the melting temperature of the respective probe-target complex one can partially overcome these secondary structure effects.     

ATR kinase activation in G1 phase facilitates the repair of ionizing radiation-induced DNA damage

The kinase ATR is activated by RPA-coated single-stranded DNA generated at aberrant replicative structures and resected double strand breaks. While many hundred candidate ATR substrates have been identified, the essential role of ATR in the replicative stress response has impeded the study of ATR kinase-dependent signalling. Using recently developed selective drugs, we show that ATR inhibition has a significantly more potent effect than ATM inhibition on ionizing radiation (IR)-mediated cell killing. Transient ATR inhibition for a short interval after IR has long-term consequences that include an accumulation of RPA foci and a total abrogation of Chk1 S345 phosphorylation. We show that ATR kinase activity in G1 phase cells is important for survival after IR and that ATR colocalizes with RPA in the absence of detectable RPA S4/8 phosphorylation. Our data reveal that, unexpectedly, ATR kinase inhibitors may be more potent cellular radiosensitizers than ATM kinase inhibitors, and that this is associated with a novel role for ATR in G1 phase cells.     

GAR22β regulates cell migration,sperm motility,and axoneme structure

Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β^−/− Sertoli cells moved faster than wild-type cells. In addition, GAR22β^−/− cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β^−/− cells reduced cell motility and focal adhesion turnover. GAR22β–actin interaction was stronger than GAR22β–microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β–EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes.     

Impact of Postovulatory Food Deprivation on the Ova Transport, Hormonal Profiles and Metabolic Changes in Sows

The effect of food deprivation on ova transport, hormonal profiles and metabolic changes was studied in 20 crossbred multiparous sows during their second oestrus after weaning. To determine the time of ovulation, transrectal ultrasonographic examination was performed. The sows were divided into 2 groups, one control group (C-group), which was fed according to Swedish standards, and one experimental group (E-group). The E-group sows were deprived of food from the first morning meal after ovulation until slaughter. Blood samples were collected every second hour from about 12 h before expected ovulation in the second oestrus after weaning until slaughter and were analysed for progesterone, prostaglandin F_2α-metabolite, insulin, glucose, free fatty acids and triglycerides. All sows were slaughtered approximately 48 h after ovulation and the genital tract was recovered. The isthmic part of the oviduct was divided into 3 equally long segments and flushed separately with phosphate buffered saline (PBS). Uterine horns were also flushed with PBS. A significantly greater number of ova were found in the first and second part of the isthmus in the E-group (p = 0.05) while in the C-group most of the ova were found in the third part of the isthmus or the uterus (p = 0.01). The level of prostaglandin F_2α-metabolite was significantly higher in the E-group compared with the C-group. The concentration of progesterone increased in both groups after ovulation but there were no significant differences between the groups. The other blood parameters showed that the food-deprived sows were in a catabolic state. The 48 h period of fasting results, directly or indirectly in an delayed ova transport, which may be due to a delayed relaxation in the smooth circular muscle layer of the isthmus.     

Fat nerves keep pain at bay

Inflammatory pain is a debilitating condition and a severe health burden; physiologically, it is a complex phenomenon with multiple contributing mechanisms. A new study published in The EMBO Journal reports that tissue inflammation results in local depletion of cholesterol in nociceptive nerves, causing a loss of lipid raft localization of a sodium channel Na_v1.9 and, ultimately, resulting in potentiation of its activity. The discovered effect contributes to the inflammatory overexcitability of peripheral nociceptive nerve terminals resulting in inflammatory hyperalgesia. Topical application of cholesterol‐containing gels was able to offset this inflammatory pain mechanism thereby opening a novel avenue for therapeutic intervention.     

Whole-genome sequence analysis reveals differences in population management and selection of European low-input pig breeds

Background

A major concern in conservation genetics is to maintain the genetic diversity of populations. Genetic variation in livestock species is threatened by the progressive marginalisation of local breeds in benefit of high-output pigs worldwide. We used high-density SNP and re-sequencing data to assess genetic diversity of local pig breeds from Europe. In addition, we re-sequenced pigs from commercial breeds to identify potential candidate mutations responsible for phenotypic divergence among these groups of breeds.

Results

Our results point out some local breeds with low genetic diversity, whose genome shows a high proportion of regions of homozygosis (>50%) and that harbour a large number of potentially damaging mutations. We also observed a high correlation between genetic diversity estimates using high-density SNP data and Next Generation Sequencing data (r = 0.96 at individual level). The study of non-synonymous SNPs that were fixed in commercial breeds and also in any local breed, but with different allele, revealed 99 non-synonymous SNPs affecting 65 genes. Candidate mutations that may underlie differences in the adaptation to the environment were exemplified by the genes AZGP1 and TAS2R40. We also observed that highly productive breeds may have lost advantageous genotypes within genes involve in immune response – e.g. IL12RB2 and STAB1–, probably as a result of strong artificial in the intensive production systems in pig.

Conclusions

The high correlation between genetic diversity computed with the 60K SNP and whole genome re-sequence data indicates that the Porcine 60K SNP Beadchip provides reliable estimates of genomic diversity in European pig populations despite the expected bias. Moreover, this analysis gave insights for strategies to the genetic characterization of local breeds. The comparison between re-sequenced local pigs and re-sequenced commercial pigs made it possible to report candidate mutations to be responsible for phenotypic divergence among those groups of breeds. This study highlights the importance of low input breeds as a valuable genetic reservoir for the pig production industry. However, the high levels of ROHs, inbreeding and potentially damaging mutations emphasize the importance of the genetic characterization of local breeds to preserve their genomic variability.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-601) contains supplementary material, which is available to authorized users.     

arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger.

In the absence of oxygen and nitrate, Pseudomonas aeruginosa metabolizes arginine via the arginine deiminase pathway, which allows slow growth on rich media. The conversion of arginine to ornithine, CO2, and NH3 is coupled to the production of ATP from ADP. The enzymes of the arginine deiminase pathway are organized in the arcDABC operon. The arcD gene encodes a hydrophobic polytopic membrane protein. Translocation of arginine and ornithine in membrane vesicles derived from an Escherichia coli strain harboring a recombinant plasmid carrying the arcD gene was studied. Arginine and ornithine uptake was coupled to the proton motive force with a bias toward the transmembrane electrical potential. Accumulated ornithine was readily exchangeable for external arginine or lysine. The exchange was several orders of magnitude faster than proton motive force-driven transport. The ArcD protein was reconstituted in proteoliposomes after detergent solubilization of membrane vesicles. These proteoliposomes mediate a stoichiometric exchange between arginine and ornithine. It is concluded that the ArcD protein is a transport system that catalyzes an electroneutral exchange between arginine and ornithine to allow high-efficiency energy conversion in the arginine deiminase pathway.     

The genus Teliocrinus (Crinoidea,Echinodermata): a key taxon among pentacrinid stalked crinoids

The main characters of the stalked crinoids of the family Pentacrinitidae attributed to the genus Teliocrinus are re‐evaluated from a quantitative study of phenotype variation, new observations on arm and stalk articulations, and observation of ontogenetic trends. All of the specimens collected in the northern Indian Ocean belong to the same species, i.e. Teliocrinus springeri (Clark, 1909). However, two phenotypes living at different depths remain valid as subspecies: Teliocrinus springeri springeri (Clark, 1909) and Teliocrinus springeri liliaceus (Clark, 1909). Teliocrinus shares several ontogenetic trends with Endoxocrinus, especially in nonfunctional brachial articulations and stalk symplexies. Its assignment to the Diplocrininae is confirmed. A discussion of its affinities with pentacrinid fossil genera in which the crown is well preserved suggests that Diplocrininae could have first appeared during the Lower Cretaceous. A shortening of brachitaxes and a paedomorphic trend of stalk symplexies are the main other evolutionary traits. Nonfunctional articulations are frequently found at the paedomorphic pole of the heterochronic gradient, without clear derived characters. Classification of pentacrinids mainly based on such symplesiomorphy or paedomorphic characters must be definitively abandoned. However, in post‐Palaeozoic stalked crinoids the scarcity of well‐preserved fossils, the high frequency of paedomorphy, and convergent adaptive characters makes phylogenetic reconstruction only based on morphological characters very difficult and speculative. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 22–39.