Adaptive evolution of the vertebrate skeletal muscle sodium channel

Tetrodotoxin (TTX) is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Nav). The skeletal muscle Nav (Nav1.4) channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Nav1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Nav1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Nav1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement

Production of Recombinant Human DNA Polymerase Delta in a Bombyx mori Bioreactor

Eukaryotic DNA polymerase δ (pol δ) plays a crucial role in chromosomal DNA replication and various DNA repair processes. It is thought to consist of p125, p66 (p68), p50 and p12 subunits. However, rigorous isolation of mammalian pol δ from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. While recombinant pol δ isolated from infected insect cells have some problems of consistency in the quality of the preparations, and the yields are much lower. To address these deficiencies, we have constructed recombinant BmNPV baculoviruses using MultiBac system. This method makes the generation of recombinant forms of pol δ containing mutations in any one of the subunits or combinations thereof extremely facile. From about 350 infected larvae, we obtained as much as 4 mg of pol δ four-subunit complex. Highly purified enzyme behaved like the one of native form by rigorous characterization and comparison of its activities on poly(dA)/oligo(dT) template-primer and singly primed M13 DNA, and its homogeneity on FPLC gel filtration. In vitro base excision repair (BER) assays showed that pol δ plays a significant role in uracil-intiated BER and is more likely to mediate LP BER, while the trimer lacking p12 is more likely to mediate SN BER. It seems likely that loss of p12 modulates the rate of SN BER and LP BER during the repair process. Thus, this work provides a simple, fast, reliable and economic way for the large-scale production of human DNA polymerase δ with a high activity and purity, setting up a new platform for our further research on the biochemical properties of pol δ, its regulation and the integration of its functions, and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability

Molecular Evolution of the Primate Antiviral Restriction Factor Tetherin

Background: Tetherin is a recently identified antiviral restriction factor that restricts HIV-1 particle release in the absence of the HIV-1 viral protein U (Vpu). It is reminiscent of APOBEC3G and TRIM5a that also antagonize HIV. APOBEC3G and TRIM5a have been demonstrated to evolve under pervasive positive selection throughout primate evolution, supporting the redqueen hypothesis. Therefore, one naturally presumes that Tetherin also evolves under pervasive positive selection throughout primate evolution and supports the red-queen hypothesis. Here, we performed a detailed evolutionary analysis to address this presumption. Methodology/Principal Findings: Results of non-synonymous and synonymous substitution rates reveal that Tetherin as a whole experiences neutral evolution rather than pervasive positive selection throughout primate evolution, as well as in non-primate mammal evolution. Sliding-window analyses show that the regions of the primate Tetherin that interact with viral proteins are under positive selection or relaxed purifying selection. In particular, the sites identified under positive selection generally focus on these regions, indicating that the main selective pressure acting on the primate Tetherin comes from virus infection. The branch-site model detected positive selection acting on the ancestral branch of the New World Monkey lineage, suggesting an episodic adaptive evolution. The positive selection was also found in duplicated Tetherins in ruminants. Moreover, there is no bias in the alterations of amino acids in the evolution of the primate Tetherin, implyin

P50, the Small Subunit of DNA Polymerase Delta, Is Required for Mediation of the Interaction of Polymerase Delta Subassemblies with PCNA

Mammalian DNA polymerase δ (pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and various DNA repair processes. Its function as a chromosomal DNA polymerase is dependent on the association with proliferating cell nuclear antigen (PCNA) which functions as a molecular sliding clamp. All four of the pol δ subunits (p125, p50, p68, and p12) have been reported to bind to PCNA. However, the identity of the subunit of pol δ that directly interacts with PCNA and is therefore primarily responsible for the processivity of the enzyme still remains controversial. Previous model for the network of protein-protein interactions of the pol δ-PCNA complex showed that pol δ might be able to interact with a single molecule of PCNA homotrimer through its three subunits, p125, p68, and p12 in which the p50 was not included in. Here, we have confirmed that the small subunit p50 of human pol δ truthfully interacts with PCNA by the use of far-Western analysis, quantitative ELISA assay, and subcellular co-localization. P50 is required for mediation of the interaction between pol δ subassemblies and PCNA homotrimer. Thus, pol δ interacts with PCNA via its four subunits

The JCMT BISTRO Survey: The Magnetic Field Strength in the Orion A Filament

We determine the magnetic field strength in the OMC 1 region of the Orion A filament via a new implementation of the Chandrasekhar-Fermi method using observations performed as part of the James Clerk Maxwell Telescope (JCMT) B-Fields In Star-Forming Region Observations (BISTRO) survey with the POL-2 instrument. We combine BISTRO data with archival SCUBA-2 and HARP observations to find a plane-of-sky magnetic field strength in OMC 1 of B_pos=6.6±4.7 mG, where δB_pos=4.7 mG represents a predominantly systematic uncertainty. We develop a new method for measuring angular dispersion, analogous to unsharp masking. We find a magnetic energy density of ~1.7×10^-7 Jm^-3 in OMC 1, comparable both to the gravitational potential energy density of OMC 1 (~10^-7 Jm^-3), and to the energy density in the Orion BN/KL outflow (~10^-7 Jm^-3). We find that neither the Alfvén velocity in OMC 1 nor the velocity of the super-Alfvénic outflow ejecta is sufficiently large for the BN/KL outflow to have caused large-scale distortion of the local magnetic field in the ~500-year lifetime of the outflow. Hence, we propose that the hour-glass field morphology in OMC 1 is caused by the distortion of a primordial cylindrically-symmetric magnetic field by the gravitational fragmentation of the filament and/or the gravitational interaction of the BN/KL and S clumps. We find that OMC 1 is currently in or near magnetically-supported equilibrium, and that the current large-scale morphology of the BN/KL outflow is regulated by the geometry of the magnetic field in OMC 1, and not vice versa

Magnetic Fields toward Ophiuchus-B Derived from SCUBA-2 Polarization Measurements

We present the results of dust emission polarization measurements of Ophiuchus-B (Oph-B) carried out using the Submillimetre Common-User Bolometer Array 2 (SCUBA-2) camera with its associated polarimeter (POL-2) on the James Clerk Maxwell Telescope in Hawaii. This work is part of the B-fields in Star-forming Region Observations survey initiated to understand the role of magnetic fields in star formation for nearby star-forming molecular clouds. We present a first look at the geometry and strength of magnetic fields in Oph-B. The field geometry is traced over ~0.2 pc, with clear detection of both of the sub-clumps of Oph-B. The field pattern appears significantly disordered in sub-clump Oph-B1. The field geometry in Oph-B2 is more ordered, with a tendency to be along the major axis of the clump, parallel to the filamentary structure within which it lies. The degree of polarization decreases systematically toward the dense core material in the two sub-clumps. The field lines in the lower density material along the periphery are smoothly joined to the large-scale magnetic fields probed by NIR polarization observations. We estimated a magnetic field strength of 630 ± 410 μG in the Oph-B2 sub-clump using a Davis–Chandrasekhar–Fermi analysis. With this magnetic field strength, we find a mass-to-flux ratio λ = 1.6 ± 1.1, which suggests that the Oph-B2 clump is slightly magnetically supercritical