Syntheses of haptens containing dioxaphosphorinan methoxyacetic acid linker arms for the production of antibodies to organophosphate pesticides.

Four generic heterobifunctional reagents, namely 2-(2-chloro-5-methyl-1,3,2-dioxaphosphorinan-5-yl)methoxyacetic acid methyl ester, p-sulfide, 2-(2-chloro-5-methyl-1,3,2-dioxaphosphorinan-5-yl)-methoxyacetic acid methyl ester, p-oxide, 2-(2-mercapto-5-methyl-1,3,2-dioxaphosphorinan-5-yl)-methoxyacetic acid bispotassium salt, p-sulfide-, and (2-methoxy-5-methyl-1,3,2-dioxaphosphorinan-5-yl)methoxyacetic acid, methyl ester, have been synthesized and used to prepare organophosphate, thiophosphate, and dithiophosphate haptens containing a functional carboxyl group which can be used to conjugate the haptens to proteins. These hapten-protein conjugates have been used as antigens for preparing polyclonal sera against all classes of organophosphate pesticides. The eight examples used protein-hapten conjugates of chlorpyrifos, parathion, diazinon, paraoxon, azinphos, dimethoate, demeton, and dichlorvos. These were all immunogenic and resulted in sera containing antibodies that recognized the corresponding parent pesticide with high specificity.

A system for tissue-specific copper-controllable gene expression in transgenic plants: nodule-specific antisense of aspartate aminotransferase-P2.

A vector system, based on copper controllable gene expression, has been developed to give control over place as well as time of expression of an introduced gene. This system consists of two elements: (1) the yeast ace1 gene encoding a metallo-regulatory transcription factor, ACE1, under control of either an organ-specific or a constitutive promoter; and (2) a gene of interest under control of a chimaeric promoter consisting of the 46 bp TATA fragment of the CaMV 35S RNA promoter linked to four repeats of the ACE1 binding site. The functioning of the system in an organ-specific manner was tested in nodulated Lotus corniculatus plants which consisted of non-transformed shoots plus transformed hairy root tissue 'wild-type tops/transgenic roots'. After addition of copper ions to the plant nutrient solution, beta-glucuronidase (GUS) expression was visualized either specifically in nodules or in both roots and nodules when the ace1 gene was placed under control of the nod45 promoter or the CaMV 35S RNA promoter, respectively. The nodule-specific system was used to express antisense constructs of aspartate aminotransferase-P2 in transgenic Lotus corniculatus plants. When expression was induced by the addition of copper ions to the plant nutrient solution aspartate aminotransferase-P2 activity declined dramatically, and a decrease of up to 90% was observed in nodule asparagine concentration.

Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum.

We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum. An open reading frame coding for 1,016 amino acids was identified. The B. japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium. Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains. Within the proline dehydrogenation domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA. Within the P5C dehydrogenase domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Bacillus subtilis ipa76d, and S. cerevisiae put2. A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain. This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C. Unlike the gene isolated from E. coli, S. typhimurium, and K. pneumoniae, the B. japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP). Additionally, the B. japonicum gene lacks the putative C-terminal regulatory domain present in the E. coli and S. typhimurium genes. The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome. Symbiotically active mutant strains that were devoid of putA activity were isolated. With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B. japonicum bacteroids is transcriptionally regulated by drought and other stresses.

Evolutionary analysis of aspartate aminotransferases.

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family.

Analysis of the lupin Nodulin-45 promoter: conserved regulatory sequences are important for promoter activity.

The promoter from the Lupinus angustifolius late nodulin gene, Nodulin-45, has been analysed to identify cis-elements and trans-acting factors. Various regions of the Nodulin-45 promoter, fused to the luciferase reporter gene, were introduced into Lotus roots using an Agrobacterium rhizogenes, transformation procedure. The transgenic roots were then nodulated. The promoter region A (-172 to +13, relative to the transcription start site) was capable of directing low-level expression of the reporter gene and in a nodule-enhanced manner when compared to roots. The addition of region C (-676 to -345) resulted in a significant increase in the expression within the nodule, whilst a low level of root expression was maintained. The C region, which confers this high-level nodule expression, contains the nodule consensus motifs AAAGAT and CTCTT. When region C was ligated to a minimal promoter element from the unrelated asparaginase gene rather than the Nodulin-45 A region, nodule-enhanced expression was still apparent, but at a much lower level. Mutation of the AAAGAT element in this construct resulted in a further significant decrease of expression. Gel retardation assays revealed that a factor from lupin nodule nuclear extracts interacted with two sequences of the C region. The binding of the factor to both of these regions could be removed by the addition of an oligonucleotide containing the AT-rich binding site for the soybean factor NAT2. This suggests that the lupin factor identified here is a NAT2 homologue. No factor binding was observed to the AAAGAT or CTCTT elements present in the C region.

Repression of the L-asparaginase gene during nodule development in Lupinus angustifolius.

Upon the establishment of an effective nitrogen-fixing symbiosis in amide-transporting plants the enzymatic activity and transcript levels of L-asparaginase are dramatically decreased. This decrease in L-asparaginase activity is essential for the correct functioning of the Rhizobium-legume symbiosis in lupin in which asparagine, synthesized from recently fixed nitrogen, is exported to aerial parts of the plant for use in growth and development. Concomitant with this decrease in L-asparaginase transcript a DNA-binding protein was detected in the nodules. This binding protein was not detectable in ineffective nodules, in nodules treated with nitrate, or in root tips, mature roots, developing flowers or developing seeds. The DNA-binding activity was shown to interact with a 59 bp sequence proximal to the transcription start site. Within this sequence a CTAAAAT direct repeat and a ACTGT/TGTCA incomplete inverted repeat were implicated in the binding of protein to the DNA by DNase I protection experiments. Competitive binding studies with synthesized binding sites were consistent with the CTAAAAT/TGTCA sequence pair proximal to the transcription start site having the highest affinity for the DNA-binding protein. We postulate that this DNA-binding protein is associated with repression of L-asparaginase gene expression in mature lupin root nodules.

Cloning and characterization of a cDNA encoding aspartate aminotransferase-P1 from Lupinus angustifolius root tips.

A root tip cDNA library, constructed in the lambda Zap II expression vector, was immunoscreened with a monoclonal antibody raised against aspartate aminotransferase-P1 from Lupinus angustifolius L. var Uniharvest. One 1452-base pair clone was isolated. The encoded protein sequence had high homology to both plant and animal aspartate aminotransferase sequences. The clone was converted to the phagemid form and expressed in Escherichia coli. The expressed protein was enzymically active and could be immunocomplexed with aspartate aminotransferase-P1-specific antibodies. The complete aspartate aminotransferase-P1 cDNA was cloned into the yeast expression vector pEMBL-yex4 and transformed into Saccharomyces cerevisiae strain BRSCS6, which possesses a mutated aspartate aminotransferase gene (the asp5 mutation). Complementation of the mutation was achieved using this construct.

Copper-controllable gene expression system for whole plants.

We describe a system for gene expression in plants based on the regulation mechanism of the yeast metallothionein (MT) gene. The system consists of two elements: (i) the yeast ace1 (activating copper-MT expression) gene encoding a transcription factor under control of the cauliflower mosaic virus (CaMV) 35S RNA promoter, and (ii) a gene of interest under control of a chimeric promoter consisting of the 90-base-pair domain A of the CaMV 35S RNA promoter linked to the ACE1 transcription factor-binding site. At elevated copper ion concentrations, the ACE1 protein changes conformation, binds to, and activates transcription from the chimeric promoter. To test the functioning of the system in plants, a construct containing the beta-glucuronidase (GUS) reporter gene under control of the chimeric promoter was prepared, and transgenic tobacco plants were produced. It was shown that GUS activity in the leaves of transgenic plants increased up to 50-fold, either after addition of 50 microM CuSO4 to the nutrient solution or after application of 0.5 microM CuSO4 to the plants in a foliar spray. This GUS expression was repressed after the removal of copper ions. The results show that the activity of the described chimeric promoter directly depends on copper ion concentration and that this system can be used in experiments that demand precise timing of expression.

Molecular cloning of the gene encoding developing seed L-asparaginase from Lupinus angustifolius.

A genomic sequence encoding Lupinus angustifolius L-asparaginase has been obtained, and is the first report of this gene from a plant source. The 3.2 kb of DNA sequenced contains a 1136 bp 5' flanking sequence, four exons and three introns. Intron-exon borders were mapped by comparing the genomic sequence with that of a L. arboreus cDNA. Primer extension analysis revealed transcription start sites 16 bp and 13 bp 5' of the initiating ATG for L. angustifolius and L. arboreus, respectively. The 5' flanking region contained sequences associated with seed-specific expression.

Molecular cloning of a cDNA encoding aspartate aminotransferase-P2 from lupin root nodules.

Two isoenzymic forms of aspartate aminotransferase are present in the plant fraction of developing lupin root nodules. One of these forms, aspartate aminotransferase-P2 (AAT-P2), increases dramatically with the onset of biological nitrogen fixation and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. A day 18 lupin nodule cDNA library in the lambda ZapII vector was immunoscreened with a monoclonal antibody specific for AAT-P2 and yielded two near-full-length 1700 bp clones. These clones were sequenced. Amino acid sequences from three peptides derived from immunopurified AAT-P2 were aligned, and showed 100% homology with the amino acid sequence deduced from the cDNA clones. The DNA sequence showed 50% homology with AAT sequences from a range of animal sources. Conversion of the clones to the phagemid form allowed their expression in Escherichia coli where both exhibited enzyme activity that could be immunoprecipitated with AAT-P2-specific monoclonal antibodies. Western blot analysis revealed protein moieties with molecular masses of 39, 43, 45 and 55 kDa. The 5' end of the clones coded for a hydrophobic leader sequence of about 50 amino acids indicative of a targeting sequence and consistent with the plastid localisation of nodule AAT-P2.

The isolation and characterisation of a cDNA clone encoding L-asparaginase from developing seeds of lupin (Lupinus arboreus).

An L-asparaginase cDNA clone, BR4, was isolated from a Lupinus arboreus Sims developing seed expression library by screening with polyclonal antibodies to the seed asparaginase. The cDNA hybridised with an oligonucleotide probe designed from amino acid sequence data and was found on sequencing to be 947 bp in length. Six polypeptide sequences obtained previously could be placed along the longest open reading frame. Computer-aided codon use analysis revealed that the cDNA sequence was consistent with other plant genes in terms of codon use. The cDNA insert was used to analyse asparaginase transcription in various tissues by northern blot analysis. A transcript size of approximately 1.2 kb was detected in L. arboreus seed total and poly(A)+ RNA. The level of this transcript declined from 30 days after anthesis to an undetectable level by day 55. Furthermore, under the high stringency conditions used, the seed asparaginase cDNA did not hybridise with total or poly(A)+ RNA isolated from root tips, suggesting that the asparaginase known to be present in this tissue may be the product of a different gene. Southern analysis suggested the seed asparaginase is a single-copy gene. The plant asparaginase amino acid sequence did not have any significant homology with microbial asparaginases but was 23% identical and 66% similar (allowing for conservative substitutions) to a human glycosylasparaginase.

L-asparaginase from developing seeds of Lupinus arboreus.

Asparaginase (EC 3.5.1.1) activity reached a maximum 40 days post anthesis in developing seeds of Lupinus arboreus and this correlated with the appearance of other ammonia assimilatory enzymes. Asparaginase, purified from these developing seeds, was resolved into three isoforms, designated asparaginases A, B and C. A major protein species in asparaginase A preparations co-focussed with enzyme activity on an isoelectric focussing gel. When analysed by SDS-PAGE, asparaginase isoforms A and B each yielded several polypeptides with M(r)s in the 14,000 to 19,000 ranged. These peptides are fragmentation products of an M(r) 36,000 asparaginase subunit. Polyclonal antibodies raised against asparaginase isoforms A and B precipitated asparaginase activity from a partially purified L. arboreus seed extract. Immunoaffinity chromatography recovered polypeptides with M(r)s between 14,000 and 19,000. Partial protein sequences were obtained for these asparaginase polypeptides.

Localization of sucrose synthase in soybean root nodules.

Immunogold labelling studies using polyclonal antibodies to sucrose synthase suggest that this enzyme is exclusively located in the cytoplasm of soybean nodule cells. A significantly greater intensity of labelling was found in the cytosol of uninfected interstitial cells of the central nodule region compared with the cytosol of the infected cells. Labelling was low in the bulk of the cortical cells but was found to be high in the vascular endodermis and in the cortical cells between the bundle and the infected region. The results are discussed in relation to carbohydrate supply and metabolism and in the context of nodule structure.

Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P(2) from Lupin Root Nodules.

Twenty-one monoclonal antibodies were raised against the aspartate aminotransferase-P(2) isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. Induction of this isoenzyme is positively correlated with the onset of N(2) fixation in effective root nodules and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. The monoclonal antibodies produced were all of the IgG class, recognized five different epitopes on the protein, and represented greater than 90% of the available epitopes. These epitopes were not unique to lupin nodule aspartate aminotransferase-P(2) but were shown to be present on the enzyme from tobacco leaves and potato. Four of the epitopes were conformational with a fifth epitope recognized by the appropriate monoclonals in both its native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium Iupini NZP2257 extracts. Antibodies against two epitopes showed some cross-reaction with the constitutive aspartate aminotransferase-P(1) isoenzyme also found in lupin root nodules. However, affinity of these monoclonals for AAT-P(1) was three orders of magnitude lower than for AAT-P(2). Monoclonals against the other epitopes appeared to be specific for aspartate aminotransferase-P(2).

Distribution of N within Pea, Lupin, and Soybean Nodules.

The (15)N abundance of some, but not all, legume root nodules is significantly elevated compared to that of the whole plant. It seems probable that differences in (15)N enrichment reflect differences in the assimilatory pathway of fixed N. In that context, we have determined the distribution of naturally occurring (15)N in structural fractions of nodules from soybean (Glycine max L. Merr.), yellow lupin (Lupinus luteus), and pea (Pisum sativum) nodules and in chemical components from soybean nodules and to a lesser extent, pea and lupin nodules. None of the fractions of pea nodules (cortex, bacteriod, or host plant cytoplasm) was enriched in (15)N. The differences among bacteriods, cortex, and plant cytoplasm were smaller in lupin than in soybean nodules, but in both, bacteriods had the highest (15)N enrichment. In soybean nodules, the (15)N abundance of bacteriods and cortex was higher than plant cytoplasm, but all three fractions were more enriched in (15)N than the entire plant. Plant cytoplasm from soybean nodules was fractionated into protein-rich material, nonprotein alcohol precipitable material (NA), and a low molecular weight fraction. The N of the latter was further separated into N of ureides, nucleotides and free amino acids. Most of these components were either similar to or lower in (15)N abundance than the plant cytoplasm as a whole, but the NA fraction showed unusual (15)N enrichment. However, the percentage of nodule N in this fraction was small. NA fractions from yellow lupin and pea nodules and from soybean leaves were not enriched in (15)N. Nor was the NA fraction in ruptured bacteriods and cortical tissue of soybean nodules. Variation among soybean nodule fractions in the preponderance in protein of different amino acids was not large enough to explain the differences in (15)N abundances among them. A hypothesis, consistent with all known data, concerning the mechanism leading to the observed excess (15)N of lupin and soybean bacteriods is offered.

Serine hydroxymethyltransferase from soybean root nodules : purification and kinetic properties.

Serine hydroxymethyltransferase has been purified 1,550-fold from the plant fraction of soybean (Glycine max [L]. Merr. cv Williams) nodules. The pH optimum for the enzyme was at 8.5. The native molecular weight was 230,000 with a subunit molecular weight of 55,000 which suggested a tetramer of identical subunits. The enzyme kinetics for the enzyme were Michaelis-Menten; there was no evidence for cooperativity in the binding of either substrates or product inhibitors. There were two K(m) values for serine at 1.5 and 40 millimolar. The K(m) for l-tetrahydrofolate was 0.25 millimolar. l-Methyl-, l-methenyl-, and l-methylene-tetrahydrofolates were all noncompetitive inhibitors with l-tetrahydrofolate with K(i) values of 1.8, 3.0, and 2.9 millimolar, respectively. Glycine was a competitive inhibitor with serine with a K(i) value of 3.0 millimolar. The intersecting nature of the double reciprocal plots together with the product inhibition data suggested an ordered mechanism with serine the first substrate to bind and glycine the last product released. The enzyme was insensitive to a wide range of metabolites which have previously been reported to affect its activity. These results are discussed with respect to the roles of serine hydroxymethyltransferase and the one-carbon metabolite pool in control of the carbon flow to the purine biosynthetic pathway in ureide biogenesis.

Phosphoserine aminotransferase in soybean root nodules : demonstration and localization.

Phosphoserine aminotransferase activity was detected in the plant and bacteroid fractions from soybean (Glycine max) root nodules. Both total and specific activities increased in the plant fraction during nodule development. Serine-pyruvate aminotransferase activity was not detectable in the plant or bacteroid fractions of these nodules. Sucrose density gradient fractionation indicated a proplastid localization for phosphoserine aminotransferase. The data presented support a role for this enzyme in carbon supply to purine biosynthesis in the pathway of ureide biogenesis in soybean nodules.

Comparison of Cellulolytic Activities in Clostridium thermocellum and Three Thermophilic, Cellulolytic Anaerobes.

Avicelase, carboxymethyl cellulase (CMCase), and beta-glucosidase activities have been compared between Clostridium thermocellum and three extremely thermophilic, cellulolytic anaerobes, isolates TP8, TP11, and KT8. The three isolates were all small, gram-negative staining, oval-ended rods which occurred singly and, at exponential phase, in long chains. They were nonflagellated and no spores were visible. The KT8 and TP11 isolates caused clumping of the cellulose during growth. In all four organisms the CMCase activity paralleled cell growth; however, in C. thermocellum and TP8 the avicelase activity did not increase until early stationary phase. Total CMCase activity in C. thermocellum was significantly higher than in the three isolates; however, avicelase activities were much more comparable among the four organisms. C. thermocellum produced higher levels of ethanol, and all four organisms produced similar concentrations of acetate. The amounts of free and bound CMCase and avicelase activities were investigated. In C. thermocellum and TP8 most of the CMCase and avicelase activities were bound to the cellulose in the medium. In contrast, most of the CMCase activity in TP11 and KT8 was free in the culture supernatant; a significant percentage of avicelase activity was also free. The TP8 isolate was also grown on a defined medium with urea as sole nitrogen source and cellulose serving as the carbon source. Under these conditions the pattern of enzyme production was the same as that in the enriched medium, although the level of that production was considerably reduced.