Radiation-induced strand breaks in phi X174 replicative form DNA: an improved experimental and theoretical approach

To determine the yield of radiation-induced single-strand, double-strand and potential breaks (breaks which are converted into actual breaks by alkali or heat treatment) oxygenated aqueous solutions of phi X174 supercoiled circular double-stranded (RFI) DNA were irradiated with increasing doses of gamma-irradiation and subjected to electrophoresis on agarose gels both before and after heat treatment. A complete separation was obtained of RFI, RFII (relaxed circle due to one or more single-strand breaks) and RFIII (linear DNA due to one double-strand break). A computer-assisted spectrophotometric procedure was developed, which enabled us to measure very accurately the amount of DNA present in the three DNA fractions. The quantitative changes of each fraction of DNA with dose could be fitted to a straightforward statistical model, which described the dose-dependent formation of the different types of breaks and from which the D37-values of single-strand, potential single-strand and double-strand breaks could be calculated to be 0.42 +/- 0.02, 1.40 +/- 0.25 and 57 +/- 36 Gy respectively. Potential double-strand breaks were not formed significantly under our conditions. In addition the maximum distance between two independently introduced single-strand breaks in opposite strands resulting in a double-strand break could be determined. The values before and after heat treatment are shown to be 29 +/- 6 and 102 +/- 13 nucleotides, respectively.     

Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.     

Influence of anoxic sensitizers on the radiation damage in biologically active DNA in aqueous solution

The competition between biologically active single-stranded phiX174 DNA and the anoxic radiosensitizers metronidazole, misonidazole, paranitroacetophenone or nifuroxime for OH radicals is studied. The results are compared with experiments in which the protection of the DNA by t-butanol is determined. Also the effects of the sensitizers on the chemical nature of the damage (immediate and potential break, immediate and potential base damage) is studied. It is found that in diluted aqueous solutions of DNA these radiosensitizers do not sensitize with respect to the biological inactivation. The only effect observed is a shift from potential to immediate breaks with misonidazole and also nifuroxime.     

Purine nucleoside phosphorylase (Np) in the mouse: Linkage relationship of Np-2 to esterase-10 (Es-10) and Np-1 on chromosome 14

An improved method for detecting four Np-1 (purine nucleoside phosphorylase) alleles in mouse erythrocytes by cellulose acetate electrophoresis is described. The previous linkage of Np-1 and Es-10 (esterase-10) was confirmed, with a map distance of 13.0±2.6 cM. Np-2 was detected by either specific activity assay or starch gel electrophoresis and shown to be linked to Es-10, 15.9 ± 3.1 cM, on chromosome 14. No recombinants between Np-1 and Np-2 were observed in 52 offspring, indicating either that these loci are either closely associated or that Np-2 represents simply a property of existing allelic products of the Np-1 locus.This research was supported by Medical Research Council of Canada grants to F.G.B. and F.F.S.     

Introduction of lanthanide(III) chelates to oligopeptides on solid phase

The synthesis of oligopeptide building blocks for the introduction of nonluminescent and luminescent lanthanide(III) chelates to the oligopeptide structure on the solid phase is described. The oligopeptide conjugates synthesized were used in DELFIA-based receptor binding assay (motilin) as well as in LANCE time-resolved fluorescence quenching assay (caspase-3).     

An X-encoded alloantigenicity between BALB/c and C57BL/6 strains of mice

To detect minor barriers to histocompatibility that might be encoded on the X chromosome in mice, we grafted reciprocal sets of (C57BL/6xBALB/c)F1, (C57BL/6xDBA/2)F1, and (BALB/cxDBA/2)F1 mice with tail skin from the respective paternal inbred strain. Our histogenic analysis suggests that, compared with the C57BL/6 mouse strain, the BALB/c strain generates X-linked antigen loss. In contrast, we detected no X-linked histogenic differences between strains C57BL/6 and DBA/2, or DBA/2 and BALB/c. To localize this X-linked barrier to histocompatibility, we produced a panel of 25 [(BALB/cxC57BL/6)F1xC57BL/6]N2 males that were grafted with C57BL/6 skin to determine which carried the BALB/c-derived component(s) necessary for graft rejection. DNA marker analysis showed one region of overlapping BALB/c-derived X-chromosomal segments among the graft rejecters, suggesting that this antigen-loss haplotype ( H-hix(c), for histoincompatibility on the X chromosome, c haplotype) may be restricted within the DXMit55 to the Xq telomere interval (which excludes only the centromeric tip of the X). Further backcrossing of H-hix(c) to C57BL/6 resulted in fewer rejecter mice than expected by the N4 generation, suggesting that a second, unlinked locus is also involved in this X-linked alloantigenicity. The vigorous rejection of male (C57BL/6xBALB)F1 and female (B6.C- H2(d)xC57BL/6)F1 skin by (BALB/cxC57BL/6)F1 males, as well as the assessment of markers on Chromosome 17 among N2 and N4 graft-recipient males, suggests that this second locus is H2, and that H-hix(b)-encoded alloantigens require both H2(b) and H2(d)-encoded presentation molecules for efficient graft rejection.     

Structural prediction and comparative docking studies of psychrophilic ��- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.     

Calculating orthologs in bacteria and Archaea: a divide and conquer approach

Among proteins, orthologs are defined as those that are derived by vertical descent from a single progenitor in the last common ancestor of their host organisms. Our goal is to compute a complete set of protein orthologs derived from all currently available complete bacterial and archaeal genomes. Traditional approaches typically rely on all-against-all BLAST searching which is prohibitively expensive in terms of hardware requirements or computational time (requiring an estimated 18 months or more on a typical server). Here, we present xBASE-Orth, a system for ongoing ortholog annotation, which applies a "divide and conquer" approach and adopts a pragmatic scheme that trades accuracy for speed. Starting at species level, xBASE-Orth carefully constructs and uses pan-genomes as proxies for the full collections of coding sequences at each level as it progressively climbs the taxonomic tree using the previously computed data. This leads to a significant decrease in the number of alignments that need to be performed, which translates into faster computation, making ortholog computation possible on a global scale. Using xBASE-Orth, we analyzed an NCBI collection of 1,288 bacterial and 94 archaeal complete genomes with more than 4 million coding sequences in 5 weeks and predicted more than 700 million ortholog pairs, clustered in 175,531 orthologous groups. We have also identified sets of highly conserved bacterial and archaeal orthologs and in so doing have highlighted anomalies in genome annotation and in the proposed composition of the minimal bacterial genome. In summary, our approach allows for scalable and efficient computation of the bacterial and archaeal ortholog annotations. In addition, due to its hierarchical nature, it is suitable for incorporating novel complete genomes and alternative genome annotations. The computed ortholog data and a continuously evolving set of applications based on it are integrated in the xBASE database, available at http://www.xbase.ac.uk/.     

Identification of Novel Imidazo[1,2-a]pyridine Inhibitors Targeting M. tuberculosis QcrB

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1–4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism ⁹³⁷ACC>⁹³⁷GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.     

Intraspecific competition in tadpoles of Rana arvalis: does it matter in nature? A field experiment

Tadpole growth and development are easily affected by intraspecific competition in tank experiments, provided treatment density is sufficiently high. Is this a phenomenon also observed in nature? A pond was divided into four tadpole-proof sections. Each year for 8 years, all spawn laid by moorfrogs (Rana arvalis) in this pond was relocated to create relative spawn and tadpole densities of 1 : 4 : 1 : 4. No direct effect of the density manipulation on survival, tadpole size, and development and metamorph timing and size could be demonstrated. However, I also measured actual tadpole density during the time of development. Apart from the experimental density manipulation, this measure included effects of between-year variation in amount of spawn, natural tadpole mortality, and pond drying (which concentrated the tadpoles by decreasing the area of the pond sections). Actual density had limited but significant effects on tadpole size and development. I suggest that density regulation, acting on the tadpole stage, may be present in the population but was of less short-term importance than abiotic factors and, possibly, adult density regulation. Consequences of the findings for conservation are discussed.