Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

Expression of asparagine synthetase in response to carbohydrate supply in model callus cultures and shoot tips of asparagus (Asparagus officinalis L.)

Sugar uptake and metabolism were studied in callus cultures and shoot tips of asparagus. Asparagus callus cultures were used to model senescence in shoot tips. Callus cultures absorbed glucose from a nutrient medium, and accumulated sucrose, glucose and fructose. This uptake of glucose by the callus cultures down-regulated expression of asparagine synthetase and beta -galactosidase transcripts that otherwise accumulated when sugar was withheld. When 80 mm-long asparagus shoots were excised from growing plants and placed in 2% and 8% sucrose solutions, endogenous concentrations of sucrose, glucose, fructose, UDPglucose, and glucose-6-phosphate declined in the 30mm-long meristematic tip regions. At the same time, asparagine and asparagine synthetase gene transcripts began to accumulate in these tips. When 10 mm-long asparagus shoot tips were placed on glucose- or fructose-containing agar, the tips accumulated sucrose, glucose and fructose, and asparagine accumulation and expression of asparagine synthetase were marginally reduced. We concluded that in callus cultures, asparagine synthetase expression was sugar regulated, but that sugar regulation was not as pronounced in asparagus shoot tips. This may be due in part to slower rates of sugar uptake into shoot tips and in part to compartmentation of sugars in the tips. We suggest that callus cultures are not a suitable model for metabolic studies in asparagus shoot tips.

Exploiting the versatility of human cytochrome P450 enzymes: The promise of blue roses from biotechnology

The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries.

Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P4501B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009

A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium, tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450S (but not CYP1B1) (in T. ni cells), we found that CY1P1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.

Crystal structure of yeast acetohydroxyacid synthase: A target for herbicidal inhibitors

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are defined. The structure of AHAS provides a starting point for the rational design of new herbicides. (C) 2002 Elsevier Science Ltd.

Oxidizing species in the mechanism of cytochrome P450

This review discusses the mechanisms of oxygen activation by cytochrome P450 enzymes, the possible catalytic roles of the various iron-oxygen species formed in the catalytic cycle, and progress in understanding the mechanisms of hydrocarbon hydroxylation, heteroatom oxidation, and olefin epoxidation. The focus of the review is on recent results, but earlier work is discussed as appropriate. The literature through to February 2002 is surveyed, and 175 referenced are cited.

Site-Directed Mutagenesis of Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus: Characterization of a Y114 → F Mutant

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to Generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K-m toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K-m was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide: acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K-d for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.

Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin-containing enzymes

Dimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum . Dimethyl sulphide dehydrogenase was shown to contain bis (molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdop-terin-containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB , ddhD and ddhC . DdhB showed sequence homology to NarH, suggesting that it contains multiple iron-sulphur clusters. Analysis of the N-terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec-dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo-trophic growth on the same substrate. Mutational analysis showed that cytochrome c (2) mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.

Catalytically active Dengue virus NS3 protease forms aggregates that are separable by size exclusion chromatography

An active form of the Dengue virus protease NS3 (CF40.Gly.NS3pro) was expressed in Escherichia coli. This construct consists of a critical 40 amino acid cofactor domain from NS2B fused to the N-terminal 184 amino acid protease domain of NS3 via a flexible, covalent linker (Gly(4)SerGly(4)). The recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the N-terminus for ease of purification using metal affinity chromatography. However, the presence of lower molecular weight impurities after affinity chromatography indicated the need for additional purification steps. The consistent appearance of these impurities suggested that they may be the products of proteolysis and/or auto-proteolysis. The latter possibility was subsequently excluded by the observation of the same impurities in a purified, catalytically inactive form of the recombinant protease (CF40.Gly.NS3pro.SA). Further analysis indicated that these impurities may represent premature translation termination products. Regardless of their origin, they were shown to form various sized aggregates with full-length CF40.Gly.NS3pro that can be separated by size exclusion chromatography, yielding fractions of active protease of sufficient purity for crystallisation trials. The ultimate goal of these studies is to obtain a crystal structure of a catalytically active form of the Dengue virus NS3 protease for structure-based drug design. (C) 2002 Elsevier Science (USA). All rights reserved.

Deep and superficial fibers of the lumbar multifidus muscle are differentially active during voluntary arm movements

AB Study Design. A cross-sectional study was conducted. Objective. To determine the activity of the deep and superficial fibers of the lumbar multifidus during voluntary movement of the arm. Summary of Background Data. The multifidus contributes to stability of the lumbar spine. Because the deep and superficial parts of the multifidus are near the center of lumbar joint rotation, the superficial fibers are well suited to control spine orientation, and the deep fibers to control intervertebral movement. However, there currently are limited in vivo data to support this distinction. Methods. Electromyographic activity was recorded in both the deep and superficial multifidus, transversus abdominis, erector spinae, and deltoid using selective intramuscular electrodes and surface electrodes during single and repetitive arm movements. The latency of electromyographic onset in each muscle during single movements and the pattern of electromyographic activity during repetitive movements were compared between muscles. Results. With single arm movements, the onset of electromyography in the erector spinae and superficial multifidus relative to the deltoid was dependent on the direction of movement, but the onset in the deep multifidus and transversus abdominis was not. With repetitive arm movements, peaks in superficial multifidus and erector spinae electromyography occurred only during flexion for most subjects, whereas peaks in deep multifidus electromyography occurred during movement in both directions. Conclusions. The deep and superficial fibers of the multifidus are differentially active during single and repetitive movements of the arm. The data from this study support the hypothesis that the superficial multifidus contributes to the control of spine orientation, and that the deep multifidus has a role in controlling intersegmental motion. (C) 2002 Lippincott Williams & Wilkins, Inc.

Ductile fabrics in the zone of active oblique convergence near the Alpine Fault, New Zealand: identifying the neotectonic overprint

The mid-crustal Alpine Schist in central Southern Alps, New Zealand has been exhumed during the past similar to3 m.y. on the hanging wall of the oblique-slip Alpine Fault. These rocks underwent ductile deformation during their passage through the similar to 150-km-wide Pacific-Australia plate boundary zone. Likely to be Cretaceous in age, peak metamorphism predates the largely Pliocene and younger oblique convergence that continues to uplift the Southern Alps today. Late Cenozoic ductile deformation constructively reinforced a pre-existing fabric that was well oriented to accommodate a dextral-transpressive overprint. Quartz microstructures below a recently exhumed brittle-ductile transition zone reflect a late Cenozoic increment of ductile strain that was distributed across deeper levels of the Pacific Plate. Deformation was transpressive, including a dextral-normal shear component that bends and rotates a delaminated panel of Pacific Plate crust onto the oblique footwall ramp of the Alpine Fault. Progressive ductile shear in mylonites at the base of the Pacific Plate overprints earlier fabrics in a dextral-reverse sense, a deformation that accompanies translation of the schists up the Alpine Fault. Ductile shear along that structure affects not only the 12-km-thick section of Alpine mylonites, but is distributed across several kilometres of overlying nonmylonitic rocks. (C) 2001 Elsevier Science Ltd. All rights reserved.