Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.     

The Genetic Structure and Evolutionary Fate of Parthenogenetic Amphibian Populations as Determined by Markovian Analysis

SYNOPSIS. One-locus, two-allele models are presented which describethe genetic consequences of naturally occurring andexperimentallyinduced parthenogesis in triploid and diploid amphibians. Themodels may in general be used to investigate genetic changeresulting from apomictic (ameiotic) and automictic (meiotic)parthenogenetic reproduction. These models quantify the influence of mutation, segregation,and selection upon genetic variability in parthenogeneticpopulations.They also allow an estimate of the relative importance of stochasticforces in altering this variability. They thus provide a basisfor understanding evolution in these populations. Some of the conclusions derived from this study contradict previouspredictions regarding genetic variability in parthenogeneticpopulations. First, if mutation is the sole source of geneticchange (i.e., strict apomixis), parthenogenetic populationsshould not become completely heterozygous. Second, small amountsof segregation occurring in apomictic populations have enormouseffects upon the genetic variability of these populations, i.e.,they should lose much of their heterozygosity. In addition to these conclusions, the results of this studysuggest that studies of protein variability in parthenogeneticspecies should contribute toward answering the question: Howmuch of the genetic variability observed in nature is evolutionarilyrelevant?     

Dating of graptolite zones by sedimentation rates: implications for rates of evolution

Preliminary determinations of ancient pelagic sedimentation rates agree with modern rates at about 4 meters per million years. By combining data on the thickness of graptolite zones from the North American Cordillera with data from other parts of the world, we have refined the Early Silurian time scale and obtained much better resolution than is possible for radiometric dates. The new Early Silurian time scale allows estimation of true rates of change in graptolite diversity. The Llandoverian diversity explosion is twice as rapid as was previously thought. The brevity of diversity lows and rapidity of speciation support modern theories of quantum evolution.     

Investigation of the Molecular Nature of Low-molecular-mass Cobalt(II) Ions in Isolated Osteoarthritic Knee-joint Synovial Fluid

High field ¹H NMR spectroscopy demonstrated that addition of Co(II) ions to osteoarthritic knee-joint synovial fluid (SF) resulted in its complexation by a range of biomolecules, the relative efficacies of these complexants/chelators being citrate ? histidine ～ threonine?glycine ～ glutamate ～ glutamine ～ phenylalanine ～ tyrosine > formate > lactate?alanine > valine > acetate > pyruvate > creatinine, this order reflecting the ability of these ligands to compete for the available Co(II) in terms of (1) thermodynamic equilibrium constants for the formation of their complexes and (2) their SF concentrations. Since many of these SF Co(II) complexants (e.g. histidinate) serve as powerful ^?OH scavengers, the results acquired indicate that any of this radical generated from the Co(II) source in such complexes via Fenton or pseudo-Fenton reaction systems will be “site-specifically” scavenged. The significance of these observations with regard to cobalt toxicity and the in vivo corrosion of cobalt-containing metal alloy joint prostheses (e.g. CoCr alloys) is discussed.     

The impact of two non-native plant species on native flora performance: potential implications for habitat restoration

Both Impatiens glandulifera and Fallopia japonica are highly invasive plant species that have detrimental impacts on native biodiversity in areas where they invade and form dense monocultures. Both species are weakly dependent on arbuscular mycorrhizal fungi (AMF) for their growth and, therefore, under monotypic stands, the AMF network can become depauperate. We evaluated the impact of I. glandulifera and F. japonica on the performance (expressed as shoot biomass) of three UK native species (Plantago lanceolata, Lotus corniculatus and Trifolium pratense) grown in soil collected from under stands of both invasive plants and compared to plants grown in soil from under stands of the corresponding native vegetation. All native species had a higher percentage colonisation of AMF when grown in uninvaded soil compared to the corresponding invaded soil. P. lanceolata and L. corniculatus had a higher biomass when grown in uninvaded soil compared to corresponding invaded soil indicating an indirect impact from the non-native species. However, for T. pratense there was no difference in biomass between soil types related to I. glandulifera, suggesting that the species is more reliant on rhizobial bacteria. We conclude that simply managing invasive populations of non-native species that are weakly, or non-dependent, on AMF is inadequate for habitat restoration as native plant colonisation and establishment may be hindered by the depleted levels of AMF in the soil below invaded monocultures. We suggest that the reintroduction of native plants to promote AMF proliferation should be incorporated into future management plans for habitats degraded by non-native plant species.     

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

Endocannabinoids and some phytocannabinoids bind to CB₁ and CB₂ cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 ^tm1Zim) CB₂ cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB₂ receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB₂ (Cnr2 ^Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 ^tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB₂ receptor deletion was lost. Likewise CB₁ receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB₁ receptor rather than a CB₂ receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.