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PubMed2005

Intragenic diversity and functional conservation of the three homoeologous loci of the KN1-type homeobox gene Wknox1 in common wheat.

Common wheat represents a typical allohexaploid species and provides a good experimental system for studying genomic alterations associated with allopolyploidization. We studied three homoeologous loci of wheat Wknox1 gene, which is highly homologous to the knotted1 (kn1)-like homeobox (KNOX) genes functioning at shoot apical meristems (SAM). Wknox1 transcripts were detected in SAM, and its overexpression caused abnormal leaf morphology with occasional ectopic leaves in transgenic tobacco plants. A comparative study of the three Wknox1 genomic sequences revealed accumulation of a large number of mutations including insertions and deletions, particularly in the fourth intron and the 5'-upstream region. Some structural mutations including MITE-insertions were allocated in the evolutionary lineage of the wheat genome, suggesting that they occurred at all stages of wheat evolution. A mutation rate was the highest in the Wknox1b locus, which is consistent with the known highest degree of differentiation in the B genome. Despite the structural differentiation, all three Wknox1 homoeologs showed an identical expression pattern in wheat and their promoter regions induced the conserved expression pattern in transgenic tobacco plants. A potential of the intragenic diversity in homoeologs of essential genes as a tool for studying the genome evolution associated with allopolyploidization was discussed.

5' Flanking Region↗
PubMed2006

Transcriptional changes in powdery mildew infected wheat and Arabidopsis leaves undergoing syringolin-triggered hypersensitive cell death at infection sites.

Blumeria graminis f.sp. tritici, the causal agent of powdery mildew in wheat, is an obligate biotrophic fungus that exclusively invades epidermal cells. As previously shown, spraying of a solution of syringolin A, a circular peptide derivative secreted by the phytopathogenic bacterium Pseudomonas syringae pv. syringae, triggers hypersensitive cell death at infection sites in powdery mildew infected wheat. Thus, the fungus is essentially eradicated. Here we show that syringolin A also triggers hypersensitive cell death in Arabidopsis infected with the powdery mildew fungus Erysiphe cichoracearum. To monitor transcriptional changes associated with this effect, we cloned 307 cDNA clones representing 158 unigenes from powdery mildew infected, syringolin A sprayed wheat leaves by a suppression subtractive hybridization cloning procedure. These cDNAs were microarrayed onto glass slides together with 1088 cDNA-AFLP clones from powdery mildew-infected wheat. Microarray hybridization experiments were performed with probes derived from leaves, epidermal tissue, and mesophyll preparations of mildewed or uninfected wheat plants after syringolin A or control treatment. Similar experiments were performed in Arabidopsis using the Affymetrix ATH1 whole genome GeneChip. The results indicate a conserved mode of action of syringolin A as similar gene groups are induced in both species. Prominent groups include genes associated with the proteasomal degradation pathway, mitochondrial and other heat shock genes, genes involved in mitochondrial alternative electron pathways, and genes encoding glycolytic and fermentative enzymes. Surprisingly, in both species the observed transcriptional response to syringolin A was considerably weaker in infected plants as compared to uninfected plants. The results lead to the working hypothesis that cell death observed at infection sites may result from a parasite-induced suppression of the transcriptional response and thus to insufficient production of protective proteins necessary for the recovery of these cells from whatever insult is imposed by syringolin A.

Arabidopsis↗
PubMed1983

Regulation of hepatic receptor-dependent degradation of LDL by mevinolin in rabbits with hypercholesterolemia induced by a wheat starch-casein diet.

Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia and have a slower rate of removal of rabbit 125I-labeled low density lipoproteins (LDL) from plasma. Treating rabbits with mevinolin, a highly potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, at a daily dose of 20 mg per animal prevents the increase in plasma and LDL cholesterol. The mevinolin effect is mediated through an increased rate of removal of rabbit 125I-labeled LDL from plasma. To study the role of mevinolin on the regulation of the hepatic LDL receptor in rabbits, the binding of 125I-labeled LDL and 125I-labeled beta-VLDL (beta-migrating very-low-density lipoproteins) to liver membranes prepared from rabbits fed the wheat starch-casein diet with or without mevinolin was investigated. Liver membranes from wheat starch-casein-fed rabbits have no demonstrable EDTA-sensitive binding activity of 125I-labeled LDL and low (37 ng/mg protein) binding activity of 125I-labeled beta-VLDL. Treatment of the wheat starch-casein fed rabbits with mevinolin results in high levels of specific EDTA-sensitive binding of 125I-labeled LDL (28.7 ng/mg protein) and 125I-labeled beta-VLDL (120 ng/mg protein). To assess the functional role of the hepatic LDL receptor in response to mevinolin, the catabolism of 125I-labeled LDL by perfused rabbit livers was studied. Perfused livers from mevinolin-treated rabbits show a 3.3-fold increase in the rate of receptor-dependent catabolism of 125I-labeled LDL (4.6% X h-1) when compared with that of livers from rabbits not treated with mevinolin (1.4% X h-1). Thus, these studies demonstrate that mevinolin prevents the increase of plasma LDL cholesterol level in rabbits fed a wheat starch-casein diet by regulating the levels of hepatic LDL-binding sites and the rate of receptor-dependent catabolism of LDL by the liver.

Animals↗
PubMed1976

Analysis of the genome of plants. II. Characterization of repetitive DNA in barley (Hordeum vulgare) and wheat (Triticum aestivum).

Barley and wheat DNAs have been characterized by studying their kinetics of reassociation, melting properties and sedimentation behaviour in neutral CsCl gradients as well as in Cs2SO4 gradients containing Ag+ or Hg2+. In both species, reassociation kinetics have revealed the presence of approx. 76% redundant nucleotide sequences which have been grouped into very rapidly reassociating (Cot 0-0.01), rapidly reassociating (Cot 0.01-1.0) and slowly reassociating (Cot 1-100) fractions. The barley Cot 0-0.01 and Cot 0.01-1.0 fractions as well as the wheat Cot 0.01-1.0 fraction form narrow bands upon centrifugation in CsCl gradients. Under similar experimental conditions both Cot 0.01 and Cot 1.0-100 wheat fractions and the barley Cot 1.0-100 fraction form broad bands each having several shoulders. Thermal denaturation studies of most of the above reassociated fractions have shown a considerable degree of order in their duplexes with an average hyperchromicity of 21.5%. When native, high molecular weight barley DNA is centrifuged in Ag+/CS2SO4 density gradients (RF = 0.2), two satellites appear on the heavier side of the main band, as against one in the case of wheat. The two minor peaks, designated as satellites I and II, have buoyant densities of 1.702 and 1.698 g/cm3, respectively, in neutral CsCl gradients and together represent about 8-9% of total barley DNA. Upon centrifugation in Hg2+/CS2SO4 density gradients, one satellite is observed in both barley and wheat and it accounts for 1-2% of their genomes.

DNA↗
PubMed1989

Arabinoxylans from rye and wheat seed that interact with ice.

Arabinoxylans that interfere with growth of ice crystals have been purified from rye (Secale cereale L., Rosen) and two varieties of wheat (Triticum aestivum L., Genesee and Hillsdale) seed. The most active polysaccharide from each seed type was homogeneous in the sense that all the molecules were in the same size range, they contained the same sugar residues, and they reacted similarly in chemical characterization experiments. Structural studies showed that the polysaccharides consist of a xylan chain to which are attached side-chains that contain a single, terminal arabinose residue. The polysaccharides differ with respect to the number of arabinose residues. The xylose:arabinose ratios in the most active fractions from rye, Genesee wheat, and Hillsdale wheat are 1.26, 1.54, and 2.08, respectively. Gel-permeation column chromatography showed that the most active polysaccharide from each seed type has a molecular weight greater than 2 x 10(6) and that the rye polysaccharide is slightly larger than the Hillsdale wheat polysaccharide. The rye polysaccharide is a better inhibitor of ice-crystal growth than is the Hillsdale wheat polysaccharide.

Arabinose↗
PubMed1988

DNase I sensitivity of ribosomal RNA genes in chromatin and nucleolar dominance in wheat.

Ribosomal RNA genes at different nucleolar organizer (NOR) loci in hexaploid wheat are expressed at different levels. The degree of expression of a particular organizer depends on the genetic background, especially on the presence of other NOR loci. For example, when chromosome 1U of Aegilops umbellulata is introduced into the hexaploid wheat cultivar "Chinese Spring" the A. umbellulata NOR accounts for most of the nucleolar activity and seems to suppress the activity of the wheat NOR loci. Even in wild-type "Chinese Spring", the NOR on chromosome 1B is partially dominant to that on chromosome 6B, since the 1B locus is more active in spite of having fewer genes. We have previously shown that these and other examples of nucleolar dominance in wheat are associated with undermethylation of cytosine residues in certain regions of the dominant rDNA. Here, we show that rRNA genes at dominant loci are organized in a chromatin conformation that renders them more sensitive to DNase I digestion than other rRNA genes. In addition, we have mapped several DNase I-hypersensitive sites in the intergenic spacer region of the rDNA repeating unit. Some of these sites are located near the initiation region for the 45 S rRNA precursor, while others are associated with a series of short direct repeats 5' to the 45 S rRNA initiation site. The results are discussed in terms of a model in which repeated sequences in the wheat intergenic DNA are presumed to function as upstream promoters and transcriptional enhancers similar to those in Xenopus.

Binding Sites↗
PubMed1990

Wheat embryo DNA polymerase A reverse transcribes natural and synthetic RNA templates. Biochemical characterization and comparison with animal DNA polymerase gamma and retroviral reverse transcriptase.

Wheat DNA polymerase A has been purified from wheat germ. The previous purification procedure (Castroviejo, M. et al. (1979) Biochem. J. 181, 183-191; Tarrago-Litvak, L. et al. (1975) FEBS Lett. 59, 125-130), has been improved leading to a higher degree of purity. Several biochemical properties of the enzyme are described. Interestingly, wheat DNA polymerase A is able to copy natural poly(A)+ mRNA into cDNA, in a way that is similar to that of the human immunodeficiency virus reverse transcriptase (HIV-RT). All four dXTP and the oligo dT primer were required for cDNA synthesis. The cDNA product was completely digested in the presence of DNase I and predigestion of the mRNA template with RNase decreased dramatically the cDNA synthesis. The animal DNA polymerase gamma can not copy natural mRNA. Substances, known to alter the enzymatic activities have been used to compare enzymes properties. In the presence of glycerol, ethidium bromide or spermine, wheat DNA polymerase A, HIV-RT and DNA polymerase gamma behave similar and they differ from animal DNA polymerase alpha. Nevertheless, DNA polymerase A is more resistant than HIV-RT and DNA polymerase gamma to the chain terminator ddTTP, while the wheat enzyme is more inhibited than DNA polymerase gamma but more resistant than HIV-RT in the presence of N3-TTP.

Chromatography, Affinity↗
PubMed1992

Purification and characterization of wheat and pine small basic protein substrates for plant calcium-dependent protein kinase.

A wheat basic protein (WBP) was purified to homogeneity from wheat germ by a protocol involving extraction, centrifugation, batchwise elution from carboxymethylcellulose (CM-52), acidification with trifluoroacetic acid, neutralization and HPLC on a SP5PW cation exchange column. WBP is a 10 kDa protein and is phosphorylated on serine residues by wheat germ Ca(2+)-dependent protein kinase (CDPK). [32P]phosphoWBP exactly comigrates with WBP on SDS-PAGE. WBP does not inhibit either wheat germ CDPK or calmodulin-dependent myosin light chain kinase. Apart from histone H1, WBP is the best endogenous substrate yet found for wheat embryo CDPK. A 12 kDa pine basic protein (PBP) was purified to homogeneity from seeds of stone pine (Pinus pinea L.) by a simple procedure involving batchwise elution from carboxymethylcellulose and cation exchange HPLC. PBP is also a good substrate for CDPK and is phosphorylated on Ser residues. N-terminal sequencing of WBP and PBP revealed that these proteins are homologous to a family of small basic plant proteins having a phospholipid transfer function.

Amino Acid Sequence↗
PubMed1996

Survival of potentially pathogenic human-associated bacteria in the rhizosphere of hydroponically grown wheat.

Plants may serve as reservoirs for human-associated bacteria (H-AB) in long-term space missions containing bioregenerative life support systems. The current study examined the abilities of five human-associated potential pathogens, Pseudomonas aeruginosa, Pseudomonas cepacia, Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli, to colonize and grow in the rhizosphere of hydroponically grown wheat, a candidate crop for life support. All of these bacteria have been recovered from past NASA missions and present potential problems for future missions. The abilities of these organisms to adhere to the roots of axenic five-day-old wheat (Triticum aestivum L. cv. Yecora rojo) were evaluated by enumeration of the attached organisms after a one hour incubation of roots in a suspension (approximately 10(8) cfu ml-1) of the H-AB. Results showed that a greater percentage of P. aeruginosa cells adhered to the wheat roots than the other four H-AB. Similarly incubated seedlings were also grown under attempted axenic conditions for seven days to examine the potential of each organism to proliferate in the rhizosphere (root colonization capacity). P. cepacia and P. aerogiunosa showed considerable growth, E. coli and S. aureus showed no significant growth, and S. pyogenes died off in the wheat rhizosphere. Studies examining the effects of competition on the survival of these microorganisms indicated that P. aeruginosa was the only organism that survived in the rhizosphere of hydroponically grown wheat in the presence of different levels of microbial competition.

Antibiosis↗
PubMedDate not supplied

Characterization and quantification of low molecular weight glutenins in durum wheats.

Durum wheat proteins have been considered as a model because of the very clear-cut relationship previously evidenced between the electrophoretic type '42' or '45' of the components that are coded by the Gli-B1 chromosome locus and the intrinsic quality (gluten viscoelasticity) of cultivars. The proteins from 4 cultivars were subjected to sequential extraction and separated into five groups, respectively, in: NaCl, EtOH (gliadins-I), EtOH + mercaptoethanol (ME) (gliadins-II), AcOH + ME (glutenins-I) and SDS + ME (glutenins-II) and characterized using polyacrylamide gel electrophoresis (PAGE), SDS-PAGE and 2-dimensional (NEPHGE X SDS - PAGE) electrophoretic systems. EtOH-soluble fractions were also separated by ion-exchange chromatography, each fraction being characterized in PAGE and SDS-PAGE and its composition in major bands determined by densitometry. From the ratio of each chromatographic fraction and of each solubility group, an estimation of the major bands or electrophoretic zones was also made in respect to the whole proteins. In 'type 45' cultivars, it was shown that only 67% of the EtOH-soluble fraction (although considered as classical gliadins) had a monomeric character, giving rise to discrete bands in PAGE systems. The remainder (33%) were aggregated fractions, essentially those referred to as low molecular weight glutenins (LMWG), that migrate, upon reduction only, in SDS-PAGE systems. LMWG make up 27% of total proteins and are revealed as a strong triplet in the 44,500-51,500 MW region, in gliadin-I and especially in gliadin-II groups. In type '42' cultivars, the LMWG ratio is reduced about by half (18% of EtOH soluble fraction, 14% of total proteins). This difference, coupled with their aggregative behavior, leads to their consideration as the major functional markers of gluten quality, gliadins 42/45 being genetic markers only. Without excluding possible physicochemical differences between different LMWG allelic types, it is hypothesized that quantitative differences could explain by themselves the quality differences between the two durum wheat genetic types. Concerning the other aggregative fractions, like high molecular weight glutenin (HMWG) subunits in glutenin-I and II groups, they do not show (unlike bread wheats) quantitative or qualitative differences large enough to play a major role in explaining genetic differences in durum wheat gluten characteristics. It is recommended, especially for physicochemical studies of wheat quality, to rely on a protein classification based on monomeric or aggregative characteristics, instead of Osborne's scheme based only on fractionation by solubility.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromatography, Ion Exchange↗
PubMed1988

Sequence analysis of the wheat mitochondrial atp6 gene reveals a fused upstream reading frame and markedly divergent N termini among plant ATP6 proteins.

The nucleotide sequence of the wheat mitochondrial gene for subunit 6 (atp6) of the F1F0 ATPase complex has been determined. Unlike bacterial, chloroplast or animal/fungal mitochondrial atp6 counterparts, which encode proteins of about 230-270 amino acids, the wheat mitochondrial atp6 homologue comprises the latter part of an open reading frame (ORF) of 386 codons. The ATP6 protein may therefore by synthesized with a long N-terminal presequence. This is supported by the finding that the ORF is preceded by a conserved sequence block closely related to ones preceding several other actively transcribed wheat mitochondrial protein-coding genes. The fused upstream ORF is similar in length, but unrelated in sequence, to those preceding the maize and tobacco mitochondrial atp6 genes. In wheat, the atp6 gene is located on a recombinationally active repeated DNA element, whose length of 1.4 kb corresponds approximately to that of the atp6 mRNA. A comparison of the wheat and maize ATP6 sequences reveals unexpectedly high divergence in the region corresponding to the mature N-terminal domain and may reflect mitochondrial DNA rearrangements during atp6 gene evolution in monocotyledonous plants.

Amino Acid Sequence↗
PubMed1992

Effect of wheat bran on excretion of radioactively labeled estradiol-17 beta and estrone-glucuronide injected intravenously in male rats.

Urinary and fecal estrogen excretion were studied in male rats fed a non-fiber wheat starch diet (dietary fiber less than 1%; NF group; n = 4), a low-fiber wheat flour diet (dietary fiber 2%; LF group; n = 4) or a high-fiber wheat bran diet (dietary fiber 11.6%; HF group; n = 3). Short-term effects of the experimental diet on estrogen excretion were studied after i.v. injection of 5 microCi (0.185 MBq) of [14C]estradiol-17 beta (E2) into the tail vein of the rats fed the diets for 2 days. After 3 weeks on the experimental diets, the long-term effects were studied after injection of 5 microCi of [14C]E2 and 10 microCi of [3H]estrone-3-glucuronide (E1-gluc). The diet was found to affect estrogen excretion. The short-term effect indicated that rats fed the HF diet excreted a relatively large amount of labeled compounds in the feces during the first day after injection, while rats fed the NF or the LF diets excreted about half that amount over the same period. On the other hand, urinary excretion of labeled compounds was significantly higher in the NF and LF rats. The long-term effect resulted in steeper slopes (P less than 0.05) of the fecal excretion profiles of rats fed the HF diet as compared with rats fed the NF and LF diets, indicating an accelerated fecal excretion of labeled compounds in the HF rats. The kinetic profiles of 14C and 3H radioactivity in blood plasma indicated a fast decrease (t1/2 of less than 2 min) for both [14C]E2 and [3H]E1-gluc. It was concluded that, owing to the short-term effect of wheat bran intake, during the first 24 h after i.v. administration relatively large amounts of radioactively labeled compounds are excreted in feces of rats fed the HF diet. In contrast, excretion is lower in urine of these rats. When the microflora is adapted to the experimental diet the wheat bran diet still results in an accelerated fecal excretion of labeled compounds, which might be attributed to an interruption of the enterohepatic circulation of estrogens. This might result in lowered plasma and/or tissue estrogen levels and hence a decreased exposure of estrogen-sensitive tissue to estrogens, which might decrease risk on mammary (breast) cancer development.

Animals↗
PubMed2005

Wheat non-specific lipid transfer protein genes display a complex pattern of expression in developing seeds.

Nine cDNA clones encoding non-specific lipid transfer proteins (nsLTPs) were isolated from Triticum aestivum and Triticum durum cDNA libraries and characterized. One cDNA is predicted to encode a type 2 nsLTP (7 kDa) while others encode type 1 nsLTPs (9 kDa). All encoded proteins contain an N-terminal signal sequence and possess the characteristic features of nsLTPs. The genomic structures of the wheat nsLtp genes show that type 2 TaLtp7.1a, TaLtp7.2a and type 1 TaLtp9.2b genes lack introns while the other type 1 genes consist of one intron. Construction of a phylogenic tree of Poaceae nsLTPs shows that wheat nsLTPs can be divided into eleven distinct groups and are closely related to barley sequences. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the expression patterns of nine nsLtp genes were studied during wheat seed development and germination. We identified three different profiles of nsLtp gene transcript accumulation. Whereas TdLtp7.1a, TdLtp9.4a and TdLtp9.7a transcripts were detected during all maturation stages, TdLtp7.2a, TdLtp9.2a, TdLtp9.3a, TdLtp9.5a and TdLtp9.6a transcripts were only present in the first and TdLtp9.1a in the last stages of seed development. Moreover, these nine wheat nsLtp genes are not seed-specific and are also expressed in the coleoptile of young seedlings. The present study revealed the complexity of the wheat nsLtp gene family and showed that the expression of nsLtp genes is developmentally regulated in the seeds, suggesting a specific function for each of the corresponding proteins.

Amino Acid Sequence↗
PubMed2004

Fractionation of wheat straw by atmospheric acetic acid process.

Fractionation of wheat straw was investigated using an atmospheric acetic acid process. Under the typical conditions of 90% (v/v) aqueous AcOH, 4% H(2)SO(4) (w/w, on straw), ratio of liquor to straw (L/S) 10 (v/w), pulping temperature 105 degrees C, and pulping time 3h, wheat straw was fractionated to pulp (cellulose), lignin and monosaccharides mainly from hemicellulose with yields of approximately 50%, 15% and 35%, respectively. Acetic acid pulp from the straw had an acceptable strength for paper and could be bleached to a high brightness over 85% with a short bleaching sequence. Acetic acid pulp was also a potential feedstock for fuels and chemicals. The acetic acid process separated pentose and hexose in wheat straw to a large extent. Most of the pentose (xylan) was dissolved, whereas the hexose (glucan) remained in the pulp. Approximately 30% of carbohydrates in wheat straw were hydrolyzed to monosaccharides during acetic acid pulping, of which xylose accounted for 70% and glucose for 12%. The acetic acid lignin from wheat straw showed relatively lower molecular weight and fusibility, which made the lignin a promising raw material for many products, such as adhesive and molded products.

Acetic Acid↗
PubMed2005

Single and joint toxicity of chlorimuron-ethyl, cadmium, and copper acting on wheat Triticum aestivum.

Investigation of the toxicological effects of some agricultural pollutants on germination rate and on shoot and root elongation of wheat (Triticum aestivum) was carried out. Seeds of wheat were exposed to various concentrations of chlorimuron-ethyl with or without cadmium and copper addition. The inhibitory rates of seed germination and shoot and root elongation of wheat were calculated. Significant linear relationships between the root and shoot elongation and the concentration of chlorimuron-ethyl with or without copper or cadmium addition were observed. The interactive effects of the three pollutants on seed germination were not significant (P > 0.05), but on shoot and root elongation they were markedly significant (P < 0.01). When concentrations of added Cd2+ or Cu2+ reached the concentration under which the inhibitory rate of wheat root elongation was about 20%, cadmium (or copper) and chlorimuron-ethyl had an antagonistic effect on the inhibition of shoot and root elongation, although the interactive effects of copper were not significant (P > 0.05). However, cadmium (or copper) and chlorimuron-ethyl had significantly synergic effects on the inhibition of shoot and root elongation (P < 0.05) when concentrations of added Cd2+ and Cu2+ reached the concentrations under which the inhibitory rate of root elongation was about 70%. At the high concentration of added cadmium or copper, joint toxicity of chlorimuron-ethyl and the heavy metals was more dependent on the concentrations of the latter. The sensitivity of wheat to the toxicity of the three pollutants was in the following sequence: root elongation>shoot elongation>germination rate.

Cadmium↗
PubMed2006

Joint stress of chlorimuron-ethyl and cadmium on wheat Triticum aestivum at biochemical levels.

Biochemical responses to joint stress of chlorimuron-ethyl and cadmium (Cd) in wheat Triticum aestivum were examined. The joint action of chlorimuron-ethyl and Cd weakened the inhibition of Cd or chlorimuron-ethyl on the formation of chlorophyll. It was deduced that wheat plants had the capability to protect themselves by increasing the activity of the antioxidant enzyme peroxidase (POD) with the exposure time. The joint effect of chlorimuron-ethyl and Cd on the superoxide dismutase (SOD) activity in leaves was additive, while the joint effect on the SOD activity in roots was determined by the interaction of chlorimuron-ethyl and Cd in wheat. It was also concluded that the change of malondialdehyde (MDA) content in wheat might not be a good biomarker in the oxidative damage by chlorimuron-ethyl, while a decrease in the soluble protein content and POD activity in roots could be considered as a biomarker in the damage of wheat by chlorimuron-ethyl and Cd.

Antioxidants↗
PubMed2004

Heterologous expression of barley and wheat oxalate oxidase in an E. coli trxB gor double mutant.

Oxalate oxidase catalyses the degradation of oxalic acid to carbon dioxide and hydrogen peroxide and is of commercial importance for clinical analyses of oxalate in biological samples. Novel potential applications for oxalate oxidase include the prevention of the formation of calcium oxalate incrusts in pulp and paper manufacture and rapid determination of oxalic acid in process waters. The potential in using oxalate-degrading enzymes in industrial processes increases the interest in finding systems for heterologous expression. Oxalate oxidase from barley is a secreted multimeric glycosylated manganese-containing enzyme with several disulfide bridges, which have been found to be essential for the catalytic activity. Attempts to achieve expression of active heterologous oxalate oxidase in bacteria have up to now met little success. In this study, one oxalate-oxidase-encoding cDNA from barley and two from wheat were cloned and tested with regard to expression in Escherichia coli. The results suggest that the selection of a novel commercially available E. coli host strain, which has the ability to form disulfide bridges in heterologous proteins expressed in its cytoplasm, was important for successful expression. Although a considerable part of the heterologous protein was produced in an insoluble and inactive form, this strain, E. coli Origami B(DE3), in addition yielded soluble and active barley and wheat oxalate oxidase. One of the wheat cDNAs, Ta(M)OXO1, gave three-fold higher activity than the barley cDNA, Hv(H)OXO1, while the other wheat cDNA, Ta(M)OXO2, gave no detectable activity. This indicates that the choice of cDNA was also critical despite the high identity between the cDNAs and the encoded polypeptides (88-89% on the nucleotide level and 88-92% on the amino-acid level). Gel filtration of cell extracts containing heterologous barley and wheat oxalate oxidase resulted in an increase in the activity. This indicates that low molecular weight inhibitory compounds were present in the E. coli lysates but could be removed by the introduction of a purification step.

Amino Acid Sequence↗
PubMed2004

Detection and relative quantitation of Soil-borne cereal mosaic virus (SBCMV) and Polymyxa graminis in winter wheat using real-time PCR (TaqMan).

Soil-borne cereal mosaic virus (SBCMV) was first reported affecting wheat crops in Italy in 1960 and has spread subsequently to many other European countries, including the UK. SBCMV causes a serious disease of wheat, reducing yield by up to 70%; growing resistant varieties represents the only economical means of control. Real-time RT-PCR and PCR assays based on TaqMan chemistry were developed for the detection and quantitation of SBCMV and its vector, Polymyxa graminis. Each assay incorporated an RNA or DNA specific internal control to facilitate quantitation. Nucleic acid extracts from SBCMV-infected plants were diluted in a nucleic acid extract from a healthy plant and amplified by real-time PCR to produce a standard curve. The standard curve was used to quantify the amount of SBCMV and P. graminis in plant samples. The sensitivity of the real-time assays were compared to established serological quantitation and conventional PCR methods by testing a range of SBCMV-infected wheat varieties. The results indicate that real-time assays were a 1000 times more sensitive than ELISA for the quantitation of SBCMV, and a 100 times more sensitive than conventional PCR for the quantitation of P. graminis. Real-time assays enabled sensitive, reproducible and specific detection of both virus and vector in wheat tissues. The real-time assays are potentially useful tools for determining variations in virus and vector concentrations in plant tissue from wheat varieties differing in resistance to SBCMV.

Enzyme-Linked Immunosorbent Assay↗