Efficient autointegration of avian retrovirus DNA in vitro.

We have developed a cell-free system for an avian retrovirus that promotes autointegration, one-long-terminal-repeat (LTR) circle formation, and correct integration into exogenous target DNA. In this system, autointegration and one-LTR circle formation occurred far more frequently than integration into exogenous target DNA. Autointegration had the same characteristics of normal integration into target DNA except in its selection of target. Highly efficient autointegration as well as one-LTR circle formation in vitro suggest that there may be a mechanism to prevent these processes in vivo.     

Truncated gag-related proteins are produced by large deletion mutants of Rous sarcoma virus and form virus particles.

Large deletion (LD) mutants of Prague strain Rous sarcoma virus subgroup B (PrB), derived by serial undiluted passage through chicken (C/E) cells, contain two deletions relative to wild-type virus. One of these joins gag sequences in the p12 coding region to env sequences in region encoding gp37; the other deletion spans the src region. Analysis of the viral proteins of QT6 cell clones containing only LD proviruses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major truncated gag-related phosphoprotein of 60,000 to 66,000 daltons (P63LD). P63LD was stable, but could be cleaved in vitro to the predicted products by p15gag. A second gag-related LD protein of about 68,000 to 74,000 molecular weight (P70LD) was also found which often reacted with an anti-gp37 serum. P70LD was unstable and may represent a short-lived gag-gp37 fusion protein. Finally, immunoprecipitation indicated that particles containing P63LD were shed from QT6-LD clones. Thin section preparations of these clones viewed in an electron microscope showed enveloped budding particles of "immature" morphology. Thus, the synthesis and release of particles from infected cells does not require cleavage of the gag precursor, nor does it require the presence of p15 or (most of) p12.     

Chlamydia trachomatis RNA polymerase alpha subunit: sequence and structural analysis.

We describe the cloning and sequence analysis of the region surrounding the gene for the alpha subunit of RNA polymerase from Chlamydia trachomatis. This region contains genes for proteins in the order SecY, S13, S11, alpha, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins. The incorporation of chlamydial alpha subunit protein into the E. coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among alpha subunits from distantly related genera.     

Immunologic correlates of protection against rotavirus challenge after intramuscular immunization of mice.

The capacity of intramuscular (i.m.) inoculation of mice with homologous or heterologous host rotaviruses to induce protection from challenge was evaluated. i.m. inoculation with live, wild-type rotavirus (murine strain EDIM) induced complete protection from viral shedding after challenge for at least 6 weeks after inoculation; protection was correlated with production of virus-specific immunoglobulin A (IgA) by lamina propria (LP) lymphocytes. i.m. inoculation with inactivated EDIM, cell culture-adapted EDIM, or simian strain RRV was associated with partial protection, characterized by reduced viral shedding after challenge. Partial protection after challenge was not associated with production of virus-specific IgA by LP lymphocytes. The mechanisms by which i.m. inoculation induces virus-specific humoral immune responses in the small intestinal LP were examined.     

Development of microtopography in a semi-arid grassland: Effects of disturbance size and soil texture

Our objective was to evaluate effects of disturbance size and soil texture on the development of microtopography for a shortgrass plant community in north central Colorado USA. Disturbances, defined as the death of individual plants, were created in 1984 and 1985 to evaluate development through time of the small-scale pattern of perennial bunchgrasses and bare soil openings that characterize this semiarid grassland. Disturbed plots of three sizes (50, 100, 150 cm-diameter) comparable in size to naturally-occurring disturbances were produced by killing plants at two sites differing in soil texture (sandy loam, clay loam). Disturbed plots were not manipulated after being created. In 1993, a laser surveying instrument was used to measure heights of crowns of individual plants of the dominant species, the perennial bunchgrass Bouteloua gracilis ([H.B.K.] Lag. ex Griffiths), and bare soil openings between plants for two locations: within each disturbance and in the surrounding undisturbed landscape.Differences between crown heights of plants and bare soil openings were comparable for both the undisturbed landscape and inside disturbances indicating that small-scale microtopography had recovered within nine years after disturbance occurred. However, complete recovery to the undisturbed state had not occurred since crown heights of plants relative to bare soil openings were significantly less on disturbed than undisturbed locations. Differences in height between plant crowns and bare soil openings on disturbed plots increased as disturbance size increased, indicating greater soil redistribution with increasing plot size. Larger differences in height were also found on plots on the fine- than the coarse-textured soil, indicating the importance of soil particle size and plant cover type to the development of microtopography. Differences in height between microsites on disturbed plots were positively related to total plant cover and negatively related to cover of B. gracilis indicating the importance of this species to reducing erosion on disturbed areas.In this semiarid grassland, patterns in microtopography were heterogeneous, likely as a result of the small-scale redistribution of soil between bare soil openings and B. gracilis plants through time. Our results indicate that this redistribution of soil is affected by disturbance size, soil texture, and patchy plant cover. The major effect of small-scale disturbances on patterns in microtopography of the shortgrass steppe are causing plant death and exposing soil to erosional and depositional processes.     

Potential for Production of Perennial Biofuel Feedstocks in Conservation Buffers on the Coastal Plain of Georgia,USA

With global increases in the production of cellulosic biomass for fuel, or “biofuel,” concerns over potential negative effects of using land for biofuel production have promoted attention to concepts of agricultural landscape design that sustainably balance tradeoffs between food, fuel, fiber, and conservation. The Energy Independence Security Act (EISA) of 2007 mandates an increase in advanced biofuels to 21 billion gallons in 2022. The southeastern region of the USA has been identified as a contributor to meeting half of this goal. We used a GIS-based approach to estimate the production and N-removal potential of three perennial biofeedstocks planted as conservation buffers (field borders associated with riparian buffers, and grassed waterways) on the Coastal Plain of Georgia, USA. Land cover, hydrology, elevation, and soils data were used to identify locations within agricultural landscapes that are most susceptible to runoff, erosion, and nutrient loss. We estimated potential annual biomass production from these areas to be: 2.5–3.5 Tg for giant miscanthus (Miscanthus?×?giganteus), 2–8.6 Tg for “Merkeron” napier grass (Pennisetum purpureum), and 1.9–7.5 Tg for “Alamo” switchgrass (Panicum virgatum). When production strategies were taken into consideration, we estimated total biomass yield of perennial grasses for the Georgia Coastal Plain at 2.2–9.4 Tg year^?1. Using published rates of N removal and ethanol conversion, we calculated the amount of potential N removal by these systems as 8100–51,000 Mg year^?1 and ethanol fuel production as 778–3296 Ml year^?1 (206 to 871 million gal. US).     

A new approach to the isolation of RNA-DNA hybrids and its application to the quantitative determination of labeled tumor virus RNA

A procedure has been developed by which the hybrid formed between a labeled RNA and complementary DNA can be selectively separated from all other single and double-stranded nucleic acids. We describe the application of this procedure to the quantitative determination of labeled avian tumor virus RNA. Purified DNA complementary to avian myeloblastosis virus RNA is extended at its 3′ terminus with 40 to 60 dCMP residues, using terminal deoxynucleotidyl-transferase. The elongated DNA is annealed with the labeled nucleic acid preparation and the mixture is passed through a column of Sephadex to which poly(I) has been covalently bound. The poly(I) retains the specific RNA-DNA hybrids by virtue of their poly(C) extension. The column is washed with RNAase to degrade nonhybridized RNA, the RNA retained on the column is eluted with formamide and its radioactivity is determined. The background hybridization was reduced to 0.003 to 0.008% by addition of oligo(C)_5.20 to the hybridization mixture and by carrying out the adsorption to the poly(I)-Sephadex column in the presence of poly(U). The hybridization efficiency was about 50%. The content of radioactive Rous sarcoma virus-specific RNA was determined in infected and uninfected cells after labeling with [³H]uridine for two hours. The content of labeled virus-specific RNA in infected cells was 0.6 to 0.9% and 0.05% in uninfected cells. The value found for monkey cell RNA was 0.009%. This method can be used for the detection of hybrids between labeled RNA and complementary DNAs too short to allow quantitation by conventional methods. If the RNAase step is omitted the procedure can be used for the isolation of any RNA for which a complementary DNA is available, as well as for its precursor.     

5-Aryl-4-carboxamide-1,3-oxazoles: potent and selective GSK-3 inhibitors

5-Aryl-4-carboxamide-1,3-oxazoles are a novel, potent and selective series of GSK-3 inhibitors. The optimization of the series to yield compounds with cell activity and brain permeability is described.