Screening costramid libraries for chromosomal genes: an alternative interspecific hybridization method

Abstract Broad host-range RK2-based cosmid vectors ('costramids') are increasingly used in molecular genetic studies of Gram-negative soil bacteria such as Rhizobium spp. we describe a simple modification of existing methods, whereby a genomic library constructed in a stringently replicated vector can be screened for genes which are undetectable by colony hybridization due to background cross-hybridization. This method allows the use of 'heterologous' probes (interspecies hybridization) to isolate several presumptive genes of interest from a gene bank of Rhizobium sp. NGR234 made in the costramid pRK7813. These are a gene with homology to the citrate synthase gene ( gltA ) or Escherichia coli , the gene encoding δ-aminol evulinic acid synthase ( hemA ), and a gene or genes regulating dicarboxylate transport.     

Separating the climatic signal from tree-ring width and maximum latewood density records

We propose a technique for separating the climatic signal which is contained in two tree-ring parameters widely used in dendroclimatology. The method is based on the removal of the relationship between tree-ring width (TRW) and maximum latewood density (MXD) observed for narrow tree rings from high latitudes. The new technique is tested on data from three larch stands located along the northern timberline in Eurasia. Correlations were calculated between the temperatures of pentads (five consecutive days), TRW chronologies and MXD chronologies calculated according to the standard and proposed methods. The analysis confirms the great importance of summer temperature for tree radial growth and tree-ring formation. TRW is positively correlated with the temperature of four to eight pentads (depending on the region) at the beginning of the growth season, but MXD as obtained by the standard technique is correlated with temperature over a much longer period. For maximum density series from which the relationship between MXD and TRW has been removed (MXD′), there is a clear correlation with temperatures in the second part of the growing season. These results are consistent with the known dynamics of tree-ring growth in high latitudes and mechanisms of tree-ring formation.     

OGR1/GPR68 Modulates the Severity of Experimental Autoimmune Encephalomyelitis and Regulates Nitric Oxide Production by Macrophages

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a proton-sensing molecule that can detect decreases in extracellular pH that occur during inflammation. Although OGR1 has been shown to have pro-inflammatory functions in various diseases, its role in autoimmunity has not been examined. We therefore sought to determine whether OGR1 has a role in the development of T cell autoimmunity by contrasting the development of experimental autoimmune encephalomyelitis between wild type and OGR1-knockout mice. OGR1-knockout mice showed a drastically attenuated clinical course of disease that was associated with a profound reduction in the expansion of myelin oligodendrocyte glycoprotein 35-55-reactive T helper 1 (Th1) and Th17 cells in the periphery and a reduced accumulation of Th1 and Th17 effectors in the central nervous system. We determined that these impaired T cell responses in OGR1-knockout mice associated with a reduced frequency and number of dendritic cells in draining lymph nodes during EAE and a higher production of nitric oxide by macrophages. Our studies suggest that OGR1 plays a key role in regulating T cell responses during autoimmunity.     

The catalytic activities of DNA topoisomerase II are most closely associated with the DNA cleavage/religation steps.

DNA topoisomerase II regulates the three-dimensional organisation of DNA and is the principal target of many important anticancer and antimicrobial agents. These drugs usually act on the DNA cleavage/religation steps of the catalytic cycle resulting in accumulation of covalent DNA-topoisomerase II complexes. We have studied the different steps of the catalytic cycle as a function of salt concentration, which is a classical way to evaluate the biochemical properties of proteins. The results show that the catalytic activity of topoisomerase II follows a bell-shaped curve with optimum between 100 and 225 mM KCl. No straight-forward correlation exists between DNA binding and catalytic activity. The highest levels of drug-induced covalent DNA-topoisomerase II complexes are observed between 100 and 150 mM KCl. Remarkably, at salt concentrations between 150 mM and 225 mM KCl, topoisomerase II is converted into a drug-resistant form with greatly reduced levels of drug-induced DNA-topoisomerase II complexes. This is due to efficient religation rather than to absence of DNA cleavage as witnessed by relaxation of the supercoiled DNA substrate. In the absence of DNA, ATP hydrolysis is strongest at low salt concentrations. Unexpectedly, the addition of DNA stimulates ATP hydrolysis at 100 and 150 mM KCl, but has little or no effect below 100 mM KCl in spite of strong non-covalent DNA binding at these salt concentrations. Therefore, DNA-stimulated ATP hydrolysis appears to be associated with covalent rather than non-covalent binding of DNA to topoisomerase II. Taken together, the results suggest that it is the DNA cleavage/religation steps that are most closely associated with the catalytic activities of topoisomerase II providing a unifying theme for the biological and pharmacological modulation of this enzyme.     

Biopolymer of alginate nature with a predominance of L-guluronic acid

Highly viscous polysaccharide (250–350 kDa) of an alginate nature with a predominance of α-L-guluronic acid (M/G = 0.22) was obtained from Azotobacter vinelandi. The yield of polysaccharide was 20.5 ± 0.5 g/l when cultured in a medium containing molasses at a viscosity of the cultural liquid of over 30000 cSt. The biopolymer is stable at pH 4.0–9.0 in a wide temperature range and well soluble in highly mineralized water; it retains a high viscosity level and can be used in the petroleum industry for enhanced oil recovery.     

Fragment of the gene encoding chymotrypsin and trypsin inhibitor protein of potato tubers

Product of polymerase chain reaction designated as PKPIJ-B was isolated after amplification from genomic DNA of potato (Solarium tuberosum L., Zhukov Jubilee cultivar) using the designed primers. Nucleotide sequence of PKPIJ-B was determined and amino acid sequence of protein was restored. Analysis of this sequence has allowed us to suggest that isolated gene fragment encodes chymotrypsin and trypsin inhibitor protein (PKCI-23 potato Kunitz-type chymotrypsin inhibitor) of potato tubers.