Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme From Thermus Thermophilus

DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the α polymerase, the β sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX ( and γ), and components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions

New Genus and Species of Aporocotylidae (Digenea) from a Basal Actinopterygian, the American Paddlefish, Polyodon spathula, (Acipenseriformes: Polyodontidae) from the Mississippi Delta

Acipensericola petersoni n. gen., n. sp. (Digenea: Aporocotylidae) infects the heart of the American paddlefish Polyodon spathula (Walbaum, 1792) in the Mississippi Delta. It has robust, spike-like body spines arranged in ventrolateral transverse rows; a bowl-shaped anterior sucker centered on the mouth and having minute spines on the inner anteroventral surface only; a pharynx; an inverse U-shaped ceca extending to near the posterior body end; intercecal testes comprising a pre-ovarian testicular column plus a single testis posteriorly; an extensively lobed ovary located medially and immediately posterior to the testicular column; a spherical ootype that is intercecal and post-ovarian; a Laurer’s canal; and a common genital pore. The new species is the first-named aporocotylid collected from a basal actinopterygian. It resembles the chondrichthyan aporocotylids Chimaerohemecus trondheimensis, Orchispirium heterovitellatum, and Hyperandrotrema cetorhini in having an inverse U-shaped ceca, but it is morphologically most similar to the anguilliform aporocotylid Paracardicoloides yamagutii in having that feature plus a comparable anterior sucker, a single testis posteriorly, an intertesticular ovary, and a common genital pore. Sequence data for the complete small subunit ribosomal DNA (18S) do not refute its membership within Aporocotylidae nor its affinity to 1 of those aforementioned aporocotylids: A. petersoni was basal to the few teleost aporocotylids analyzed, and C. trondheimensis was the only taxon basal to A. petersoni. We regard the specimens of Spirorchis sp. previously reported from the shortnose sturgeon Acipenser brevirostrum Lesueur, 1818 as congeneric with the new species

Toward Understanding the Molecular Bases of Stretch Activation: A Structural Comparison Of The Two Troponin C Isoforms Of Lethocerus

Muscles are usually activated by calcium binding to the calcium sensory protein troponin-C, which is one of the three components of the troponin complex. However, in cardiac and insect flight muscle activation is also produced by mechanical stress. Little is known about the molecular bases of this calcium-independent activation. In Lethocerus, a giant water bug often used as a model system because of its large muscle fibers, there are two troponin-C isoforms, called F1 and F2, that have distinct roles in activating the muscle. It has been suggested that this can be explained either by differences in structural features or by differences in the interactions with other proteins. Here we have compared the structural and dynamic properties of the two proteins and shown how they differ. We have also mapped the interactions of the F2 isoform with peptides spanning the sequence of its natural partner, troponin-I. Our data have allowed us to build a model of the troponin complex and may eventually help in understanding the specialized function of the F1 and F2 isoforms and the molecular mechanism of stretch activation

Towards Understanding the Molecular Bases of Stretch Activation: A Structural comparison of the Two Troponin C Isoforms of Lethocerus.

Muscles are usually activated by calcium binding to the calcium sensory protein troponin-C, which is one of the three components of the troponin complex. However, in cardiac and insect flight muscle activation is also produced by mechanical stress. Little is known about the molecular bases of this calcium-independent activation. In Lethocerus, a giant water bug often used as a model system because of its large muscle fibers, there are two troponin-C isoforms, called F1 and F2, that have distinct roles in activating the muscle. It has been suggested that this can be explained either by differences in structural features or by differences in the interactions with other proteins. Here we have compared the structural and dynamic properties of the two proteins and shown how they differ. We have also mapped the interactions of the F2 isoform with peptides spanning the sequence of its natural partner, troponin-I. Our data have allowed us to build a model of the troponin complex and may eventually help in understanding the specialized function of the F1 and F2 isoforms and the molecular mechanism of stretch activation

Completeness of cancer registration: a new method for routine use

We report a new method of estimating the completeness of cancer registration, in which the proportions of unregistered patients are derived from the time distributions of three probabilities, each of which can be directly estimated from the registry's own data – the probabilities of survival, of registration of the cancer during the patient's life, and of the mention of cancer on the death certificate of a cancer patient who dies. This method allows completeness to be assessed routinely by factors such as age, sex, geographical area and tumour type. © 2000 Cancer Research Campaig

The Sun's Preferred Longitudes and the Coupling of Magnetic Dynamo Modes

Observations show that solar activity is distributed non-axisymmetrically, concentrating at "preferred longitudes". This indicates the important role of non-axisymmetric magnetic fields in the origin of solar activity. We investigate the generation of the non-axisymmetric fields and their coupling with axisymmetric solar magnetic field. Our kinematic generation (dynamo) model operating in a sphere includes solar differential rotation, which approximates the differential rotation obtained by inversion of helioseismic data, modelled distributions of the turbulent resistivity, non-axisymmetric mean helicity, and meridional circulation in the convection zone. We find that (1) the non-axisymmetric modes are localised near the base of the convection zone, where the formation of active regions starts, and at latitudes around $30^{\circ}$; (2) the coupling of non-axisymmetric and axisymmetric modes causes the non-axisymmetric mode to follow the solar cycle; the phase relations between the modes are found. (3) The rate of rotation of the first non-axisymmetric mode is close to that determined in the interplanetary space.Comment: 22 pages, 18 figures. Accepted for publication in the Astrophysical Journa

Label-free electrochemical monitoring of DNA ligase activity

This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in the backbone of DNA and are of significant scientific interest due to their essential nature in DNA metabolism and their importance to a range of molecular biological methodologies. The electrochemical behavior of DNA at mercury and some amalgam electrodes is strongly influenced by its backbone structure, allowing a perfect discrimination between DNA molecules containing or lacking free ends. This variation in electrochemical behavior has been utilized previously for a sensitive detection of DNA damage involving the sugar-phosphate backbone breakage. Here we show that the same principle can be utilized for monitoring of a reverse process, i.e., the repair of strand breaks by action of the DNA ligases. We demonstrate applications of the electrochemical technique for a distinction between ligatable and unligatable breaks in plasmid DNA using T4 DNA ligase, as well as for studies of the DNA backbone-joining activity in recombinant fragments of E. coli DNA ligase

Replacing the detector mask with a structured scintillator in edge-illumination x-ray phase contrast imaging

We present a proof-of-concept edge illumination x-ray phase contrast system where the detector mask has been replaced by an indirect conversion detector in which sensitive and insensitive regions have been obtained by “patterning” the scintillator. This was achieved by creating a free-standing grid with period and aperture size matching that of a typical detector mask and filling the apertures with gadolinium oxysulfide. Images of various samples were collected with both the modified and the original edge illumination systems based on the use of two masks to characterize the performances of this detector design. We found that, despite the proof-of-concept nature of this attempt resulting in a structured detector with suboptimal performance, it allows effective separation of the attenuation and refraction channels through phase retrieval and the visualization of hard-to-detect features such as cartilage through the latter channel, thus demonstrating that the proposed approach holds the potential to lead to improved stability since it will use a single optical element facilitating the design of rotating phase contrast systems or the retrofitting of conventional x-ray systems