Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora

A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain).     

A nuclear specific glycoprotein representative of a unique pattern of glycosylation

Whole rat liver nuclei were reacted with UDP-[14C]galactose in the presence of bovine beta(1----4) galactosyltransferase. The reaction mixture was electrophoresed on a reducing sodium dodecyl sulfate-polyacrylamide gel. Autoradiograms of the gel demonstrated a major labeled broad band migrating with an apparent molecular weight of 65,000-66,000. A number of other less prominently labeled bands were also present. The labeled 65,000-66,000 band when cut from the gel and subjected to alkaline reduction while in the gel matrix exclusively yielded a 14C-labeled disaccharide that co-migrated with a [14C]Gal-GlcNAcol standard in descending paper chromatography. Treatment of this disaccharide with beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) from Aspergillus niger removed all the [14C]galactose label. Treatment of the labeled 65,000-66,000 polypeptide with Endoglycosidase F, however, did not remove the [14C]galactose label. Western transfer blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels performed with horseradish peroxidase-labeled succinyl wheat germ agglutinin, a lectin specific for GlcNAc, on unlabeled nuclei revealed a dominant band at 63,000-64,000. Subjecting 14C-labeled nuclei to this procedure resulted in a shift of the major horseradish peroxidase-labeled succinyl wheat germ agglutinin band to 65,000-66,000. The shifted band was coincident with the [14C]galactose band as visualized on an autoradiogram. A survey of other rat tissue nuclei revealed the same spectrum of [14C]galactose acceptor proteins with a dominant 65,000-66,000 galactose-labeled band.     

Increased near-ultraviolet induced DNA fragmentation in xeroderma pigmentosum variants.

Immediate fragmentation of parental DNA by near-ultraviolet irradiation at 313 nm was measured in cultured skin fibroblasts from normal individuals, patients with Xeroderma pigmentosum of complementation group A (XPA) and Xeroderma pigmentosum variants (XPV) by the alkaline elution procedure. For a dose of 2.25 KJm^?2 given at O^o fragmentation was comparable in all cell strains. However, fragmentation was strongly increased relative to O^o in XPV but not in normal fibroblasts and the XPA strains when irradiation was carried out at 37^o. From our results it appears that a step in the repair of $\text{parental}$ DNA is abnormal in XPV.     

Water inputs across a tropical montane landscape in Veracruz,Mexico: synergistic effects of land cover,rain and fog seasonality,and interannual precipitation variability

Land‐cover change can alter the spatiotemporal distribution of water inputs to mountain ecosystems, an important control on land‐surface and land‐atmosphere hydrologic fluxes. In eastern Mexico, we examined the influence of three widespread land‐cover types, montane cloud forest, coffee agroforestry, and cleared areas, on total and net water inputs to soil. Stand structural characteristics, as well as rain, fog, stemflow, and throughfall (water that falls through the canopy) water fluxes were measured across 11 sites during wet and dry seasons from 2005 to 2008. Land‐cover type had a significant effect on annual and seasonal net throughfall (NTF <0=canopy water retention plus canopy evaporation; NTF >0=fog water deposition). Forest canopies retained and/or lost to evaporation (i.e. NTF<0) five‐ to 11‐fold more water than coffee agroforests. Moreover, stemflow was fourfold higher under coffee shade than forest trees. Precipitation seasonality and phenological patterns determined the magnitude of these land‐cover differences, as well as their implications for the hydrologic cycle. Significant negative relationships were found between NTF and tree leaf area index (R²=0.38, P<0.002), NTF and stand basal area (R²=0.664, P<0.002), and stemflow and epiphyte loading (R²=0.414, P<0.001). These findings indicate that leaf and epiphyte surface area reductions associated with forest conversion decrease canopy water retention/evaporation, thereby increasing throughfall and stemflow inputs to soil. Interannual precipitation variability also altered patterns of water redistribution across this landscape. Storms and hurricanes resulted in little difference in forest‐coffee wet season NTF, while El Niño Southern Oscillation was associated with a twofold increase in dry season rain and fog throughfall water deposition. In montane headwater regions, changes in water delivery to canopies and soils may affect infiltration, runoff, and evapotranspiration, with implications for provisioning (e.g. water supply) and regulating (e.g. flood mitigation) ecosystem services.