Phenotypic analysis of a temperature sensitive mutant in the large subunit of the vaccinia virus mRNA capping enzyme

AbstractThe heterodimeric vaccinia virus mRNA capping enzyme is a multifunctional enzyme, encoded by genes D1R and D12L. Published biochemical experiments demonstrate that, in addition to mRNA capping, the enzyme is involved in early viral gene transcription termination and intermediate viral gene transcription initiation. This paper presents the phenotypic characterization of Dts36, a temperature sensitive mutant in the large subunit of the mRNA capping enzyme (G705D), encoded by gene D1R. At the non-permissive temperature, Dts36 displays decreased steady state levels of some early RNAs, suggesting a defect in mRNA capping. Mutant infections also show decreased steady state levels of some early proteins, while DNA replication and post-replicative gene expression are absent. Under non-permissive conditions, the mutant directs synthesis of longer-than-normal early mRNAs from some genes, demonstrating that early gene transcription termination is defective. If mutant infections are initiated at the permissive temperature and shifted to the non-permissive temperature late during infection, steady state levels of intermediate gene transcripts decrease while the levels of late gene transcripts remain constant, consistent with a defect in intermediate gene transcription initiation. In addition to its previously described role in mRNA capping, the results presented in this study provide the first in vivo evidence that the vaccinia virus mRNA capping enzyme plays a role in early gene transcription termination and intermediate gene transcription

Mutation of Vaccinia Virus Gene G2R Causes Suppression of Gene A18R ts Mutants: Implications for Control of Transcription

AbstractThis report provides genetic evidence that two vaccinia virus genes, A18R and G2R, both of which affect the fidelity of viral transcriptionin vivo,interact with each other or act on a common biochemical pathway. Previous experiments with the antipoxviral drug isatin-β-thiosemicarbazone suggest that lethal mutation of gene G2R would compensate for mutations in gene A18R. We therefore tested the hypothesis that gene G2R is an extragenic suppressor of A18R mutations. First, we constructed a recombinant which contains both a G2R deletion mutation and an A18R temperature-sensitive mutation and found that this recombinant was viable. Second, we isolated both cold-sensitive and temperature-insensitive phenotypic revertants of A18R temperature-sensitive mutants and found in both cases that the revertants contained G2R mutations. In the case of the cold-sensitive revertants, we were able to prove that the cold-sensitive phenotype mapped to the G2R gene. Combined with the biochemical data on A18R and G2R, these results imply that the A18R and G2R genes interact with each other either directly or indirectly in a fashion which affects the fidelity of intermediate and late viral transcription

Media(ted) fabrications: How the science-media symbiosis helped ‘sell’ cord banking

This paper considers the problematic role of the science–media symbiosis in the dissemination of misleading and emotionally manipulative information regarding services offered by CordBank, New Zealand's only umbilical cord blood banking facility. As this case study illustrates, the growing reliance of health and science reporters on the knowledge capital of medical specialists, biogenetic researchers, and scientists potentially enhances the ability of ‘expert’ sources to set the agenda for media representations of emerging medical and scientific developments, and may undermine the editorial independence of journalists and editors, many of whom in this case failed to critically evaluate deeply problematic claims regarding the current and future benefits of cord banking. Heavy reliance on established media frames of anecdotal personalization and technoboosterism also reinforced a proscience journalistic culture in which claims by key sources were uncritically reiterated and amplified, with journalistic assessments of the value of cord banking emphasizing potential benefits for individual consumers. It is argued that use of these media frames potentially detracts from due consideration of the broader social, ethical, legal, and health implications of emerging biomedical developments, along with the professional, personal, and increasingly also financial interests at stake in their public promotion, given the growing commercialization of biogenetic technologies

The E6 protein from vaccinia virus is required for the formation of immature virions.

An IPTG-inducible mutant in the E6R gene of vaccinia virus was used to study the role of the E6 virion core protein in viral replication. In the absence of the inducer, the mutant exhibited a normal pattern DNA replication, concatemer resolution and late gene expression, but it showed an inhibition of virion structural protein processing it failed to produce infectious particles. Electron microscopic analysis showed that in the absence of IPTG viral morphogenesis was arrested before IV formation: crescents, aberrant or empty IV-like structures, and large aggregated virosomes were observed throughout the cytoplasm. The addition of IPTG to release a 12-h block showed that virus infectious particles could be formed in the absence of de novo DNA synthesis. Our observations show that in the absence of E6 the association of viroplasm with viral membrane crescents is impaired

Dating Metasomatism: Monazite and Zircon Growth during Amphibolite Facies Albitization

We present coupled textural observations and trace element and geochronological data from metasomatic monazite and zircon, to constrain the timing of high-grade Na-metasomatism (albitization) of an Archean orthogneiss in southwest Montana, USA. Field, mineral textures, and geochemical evidence indicate albitization occurred as a rind along the margin of a ~3.2 Ga granodioritic orthogneiss (Pl + Hbl + Kfs + Qz + Bt + Zrn) exposed in the Northern Madison range. The metasomatic product is a weakly deformed albitite (Ab + Bt + OAm + Zrn + Mnz + Ap + Rt). Orthoamphibole and biotite grew synkinematically with the regional foliation fabric, which developed during metamorphism that locally peaked at upper amphibolite-facies during the 1800–1710 Ma Big Sky orogeny. Metasomatism resulted in an increase in Na, a decrease in Ca, K, Ba, Fe, and Sr, a complete transformation of plagioclase and K-feldspar into albite, and loss of quartz. In situ geochronology on zoned monazite and zircon indicate growth by dissolution–precipitation in both phases at ~1750–1735 Ma. Trace element geochemistry of rim domains in these phases are best explained by dissolution–reprecipitation in equilibrium with Na-rich fluid. Together, these data temporally and mechanistically link metasomatism with high-grade tectonism and prograde metamorphism during the Big Sky orogeny.Funding for this work was provided by NSF grant (EAR1252295) to Maha

Forward-imaging optical coherence tomography (OCT) systems and probes

Provided are forward-imaging optical coherence tomography (OCT) systems and probes. In one embodiment, a scanning reflector surface is configured to be rotated about two axes in a single operating plane to direct light transmitted along the sample path to a sample to be imaged.Board of Regents, University of Texas Syste

Use of Lysolecithin-Permeabilized Infected-Cell Extracts to Investigate thein VitroBiochemical Phenotypes of Poxvirus ts Mutations Altered in Viral Transcription Activity

AbstractLysolecithin permeabilization of vaccinia virus-infected cells was employed to prepare extracts that support faithful transcription initiationin vitroon plasmids possessing early, intermediate, and late viral gene promoters. Conditions which optimize transcription from each promoter were defined. Thein vitrosystem was used to investigate the multifunctional viral mRNA capping enzyme, which also functions as the viral early gene transcription termination factor (VTF) and a viral intermediate gene transcription initiation factor. A low level of signal-dependent termination of early gene transcription was observedin vitrowhich could be elevated by the addition of pure mRNA capping enzyme. VTF-dependent transcription termination was found to be restricted to templates that possessed an early promoter. This restriction mimics that observedin vivoand demonstrates that transcription termination is limited to RNA polymerase molecules that recognize early rather than intermediate or late gene promoters. Extracts prepared from cells infected at the nonpermissive temperature with a virus containing a ts mutation in gene D12L, which encodes the small subunit of VTF, are incapable of supporting both early gene transcription termination and intermediate gene transcription initiation. Both activities are restored upon addition of the purified wild-type mRNA capping enzyme

Functional Traits of Tropical Trees and Lianas Explain Spatial Structure across Multiple Scales

Dispersal and density dependence are major determinants of spatial structure, population dynamics and coexistence for tropical forest plants. However, because these two processes can jointly influence spatial structure at similar scales, analysing spatial patterns to separate and quantify them is often difficult. Species functional traits can be useful indicators of dispersal and density dependence. However, few methods exist for linking functional traits to quantitative estimates of these processes that can be compared across multiple species. We analysed static spatial patterns of woody plant populations in the 50 ha Forest Dynamics Plot on Barro Colorado Island, Panama with methods that distinguished scale‐specific differences in species aggregation. We then tested how these differences related to seven functional traits: growth form, dispersal syndrome, tree canopy layer, adult stature, seed mass, wood density and shade tolerance. Next, we fit analytically tractable spatial moment models to the observed spatial structure of species characterized by similar trait values, which allowed us to estimate relationships of functional traits with the spatial scale of dispersal, and the spatial scale and intensity of negative density dependence. Our results confirm that lianas are more aggregated than trees, and exhibit increased aggregation within canopy gaps. For trees, increased seed mass, wood density and shade tolerance were associated with less intense negative density dependence, while higher canopy layers and increased stature were associated with decreased aggregation and better dispersal. Spatial structure for trees was also strongly determined by dispersal syndrome. Averaged across all spatial scales, zoochory was more effective than wind dispersal, which was more effective than explosive dispersal. However, at intermediate scales, zoochory was associated with more aggregation than wind dispersal, potentially because of differences in short‐distance dispersal and the intensity of negative density dependence. Synthesis. We develop new tools for identifying significant associations between functional traits and spatial structure, and for linking these associations to quantitative estimates of dispersal scale and the strength and scale of density dependence. Our results help clarify how these processes influence woody plant species on Barro Colorado, and demonstrate how these tools can be applied to other sites and systems