PubMed Health⌕ Search

Biomedical subjects

A J Delauney

Publications and source records attributed to A J Delauney.

10 recordsLinked to original sources
PubMed2001

A cell plate-specific callose synthase and its interaction with phragmoplastin.

Callose is synthesized on the forming cell plate and several other locations in the plant. We cloned an Arabidopsis cDNA encoding a callose synthase (CalS1) catalytic subunit. The CalS1 gene comprises 42 exons with 41 introns and is transcribed into a 6.0-kb mRNA. The deduced peptide, with an approximate molecular mass of 226 kD, showed sequence homology with the yeast 1,3-beta-glucan synthases and is distinct from plant cellulose synthases. CalS1 contains 16 predicted transmembrane helices with the N-terminal region and a large central loop facing the cytoplasm. CalS1 interacts with two cell plate--associated proteins, phragmoplastin and a novel UDP-glucose transferase that copurifies with the CalS complex. That CalS1 is a cell plate--specific enzyme is demonstrated by the observations that the green fluorescent protein--CalS1 fusion protein was localized at the growing cell plate, that expression of CalS1 in transgenic tobacco cells enhanced callose synthesis on the forming cell plate, and that these cell lines exhibited higher levels of CalS activity. These data also suggest that plant CalS may form a complex with UDP-glucose transferase to facilitate the transfer of substrate for callose synthesis.

Amino Acid Motifs↗
PubMed1995

Control of de novo purine biosynthesis genes in ureide-producing legumes: induction of glutamine phosphoribosylpyrophosphate amidotransferase gene and characterization of its cDNA from soybean and Vigna.

Soybean (Glycine max) and mothbean (Vigna aconitifolia) cDNA clones encoding glutamine phosphoribosylpyrophosphate amidotransferase (PRAT), the first enzyme of the de novo purine biosynthesis pathway, have been isolated from nodule cDNA libraries. The amino acid sequence deduced from soybean clone showed > 85% homology to the PRAT sequence of mothbean and 33-47% homology to those of bacteria, yeast, chicken, rat and human. The soybean clone encodes a protein with an N-terminal sequence resembling a plastid-targeting peptide. Downstream from this peptide is a sequence similar to the 11 amino acid propeptide found in the Bacillus subtilis, chicken, rat and human PRAT proteins. The mothbean cDNA, although lacking most of the plastid presequence, encodes the putative propeptide and efficiently complements purine auxotrophy in an Escherichia coli purF mutant. Both the soybean and mothbean clones encode characteristic cysteine residues that are known to be involved in the assembly of a [Fe-S] cluster near the C-terminus of this protein. Levels of PRAT mRNA in mothbean nodules were found to increase steadily as the nodules matured from 13 days to 23 days. PRAT mRNA was not detectable in uninfected root tissue but a low level of transcript was detected in leaves. Treatment of uninfected root with L-glutamine induced the PRAT mRNA transcript suggesting that glutamine produced as a result of assimilation of fixed nitrogen is funnelled into the de novo purine biosynthesis and controls the expression of this pathway in root nodules.

Amidophosphoribosyltransferase↗
PubMed1994

Structural organization of de novo purine biosynthesis enzymes in plants: 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase cDNAs from Vigna aconitifolia.

Nodules of tropical legumes generally export symbiotically fixed nitrogen in the form of ureides that are produced by oxidation of de novo synthesized purines. To investigate the regulation of de novo purine biosynthesis in these nodules, we have isolated cDNA clones encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase from a mothbean (Vigna aconitifolia) nodule cDNA library by complementation of Escherichia coli purE and purC mutants, respectively. Sequencing of these clones revealed that the two enzymes are distinct proteins in mothbean, unlike in animals where both activities are associated with a single bifunctional polypeptide. As is the case in yeast, the mothbean AIR carboxylase has a N-terminal domain homologous to the eubacterial purK gene product. This PurK-like domain appears to facilitate the binding of CO2 and is dispensable in the presence of high CO2 concentrations. Because the expression of the mothbean PurE cDNA clone in E. coli apparently generates a truncated polypeptide lacking at least 140 N-terminal amino acids, this N-terminal region of the enzyme may not be essential for its CO2-binding activity.

Amino Acid Sequence↗
PubMed1993

Cloning of ornithine delta-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis.

Proline prototrophy was restored to an Escherichia coli proBA proline auxotroph by ornithine and a mothbean (Vigna aconitifolia) cDNA expression library. This novel strategy, "trans-complementation," allowed isolation of a cDNA encoding ornithine delta-aminotransferase (delta-OAT). This enzyme transaminates ornithine to glutamic-gamma-semialdehyde (GSA), thereby bypassing the block in GSA synthesis from glutamate in the proBA mutant. The identity of the mothbean enzyme was confirmed by its high sequence homology to mammalian and yeast delta-OATs as well as to a family of bacterial and fungal omega-aminotransferases and an absence of significant homology to various alpha-aminotransferases. The V. aconitifolia OAT cDNA encodes a polypeptide of 48.1 kDa. The native enzyme expressed in E. coli appears to be a monomer with Km of 2 mM for ornithine and 0.75 mM for alpha-ketoglutarate. Levels of mRNA in V. aconitifolia for delta 1-pyrroline-5-carboxylate synthetase (P5CS) and delta-OAT, the two key enzymes for proline synthesis, were monitored under different physiological conditions. Salt stress and nitrogen starvation induced P5CS mRNA levels and depressed OAT mRNA levels. Conversely, OAT mRNA level was elevated in plants supplied with excess nitrogen while the P5CS mRNA level was reduced. These data suggest that the glutamate pathway is the primary route for proline synthesis in plants during conditions of osmotic stress and nitrogen limitation whereas the ornithine pathway assumes prominence under high nitrogen input.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1992

A bifunctional enzyme (delta 1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants.

Many plants synthesize and accumulate proline in response to osmotic stress. Despite the importance of this pathway, however, the exact metabolic route and enzymes involved in the synthesis of proline in plants have not been unequivocally identified. We report here the isolation of a mothbean (Vigna aconitifolia) cDNA clone encoding a bifunctional enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyzes the first two steps in proline biosynthesis. The two enzymatic domains of P5CS correspond to the ProB and ProA proteins of Escherichia coli and contain a leucine zipper in each domain, which may facilitate inter- or intramolecular interaction of this protein. The Vigna P5CS enzyme activity is feedback regulated by proline but is less sensitive to end-product inhibition than is the E. coli gamma-glutamyl kinase. The P5CS gene is expressed at high levels in Vigna leaves and is inducible in roots subjected to salt stress, suggesting that P5CS plays a key role in proline biosynthesis, leading to osmoregulation in plants.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1990

A soybean gene encoding delta 1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated.

We have isolated several cDNA clones encoding delta 1-pyrroline-5-carboxylate reductase (P5CR, L-proline: NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) which catalyzes the terminal step in proline biosynthesis, by direct complementation of a proC mutation in Escherichia coli with an expression library of soybean root nodule cDNA. The library was constructed in the lambda ZapII vector, converted to a plasmid library by in vivo excision of recombinant pBluescript phagemids, and used for transformation of the E. coli mutant. Complementing plasmids contained inserts of about 1.2 kb which hybridized to a 1.3 kb RNA transcript in nodules, uninfected roots and leaves. DNA sequence analysis of one full length cDNA clone showed that it encodes a 28 586 Mr polypeptide with 39% amino acid identity to the E. coli P5CR sequence. Genomic analysis showed that there are two to three copies of the P5CR gene in the soybean genome. The steady-state level of P5CR mRNA in root nodules was twice as high as in uninfected roots and about five times higher than in leaves. Subjecting young seedlings to osmotic stress by watering with 400 mM NaCl resulted in an almost six-fold increase in the level of root P5CR mRNA, suggesting that this gene may be osmoregulated.

Amino Acid Sequence↗
PubMed1988

A stable bifunctional antisense transcript inhibiting gene expression in transgenic plants.

Tobacco plants expressing constitutive chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) activity were obtained by transformation with a chimeric CAT gene driven by the cauliflower mosaic virus 19S promoter. Plants expressing different levels of CAT activity were retransformed with vectors containing CAT sequences transcriptionally fused in the antisense orientation between the coding region of the hygromycin-resistance gene and the 3' end of the nopaline synthase gene. Several plants regenerated on high concentrations of hygromycin exhibited a loss of CAT activity, whereas plants retransformed with a vector conferring hygromycin resistance but lacking the antisense CAT sequence showed no reduction in CAT activity. RNA blot analysis revealed a strong correlation between the degree of CAT gene inactivation and the levels of stable antisense transcripts accumulated. The possibility that CAT gene inactivation was due to transferred DNA instability was discounted since a kanamycin-resistance gene contiguous with the CAT gene was expressed normally, and DNA blot analysis indicated no loss or rearrangements of the transferred DNA fragments. Thus, the imposed selection pressure enabled the selection of plants expressing high levels of stable bifunctional antisense transcripts that inhibited the activity of the targeted gene.

Journal Article↗
PubMed1983

Sequence specificity of the post-translational proteolytic cleavage of vicilin, a seed storage protein of pea (Pisum sativum L.).

Amino acid sequence data from vicilin of pea (Pisum sativum L.) were compared with predicted sequences from complementary DNA species. The sites of potential post-translational proteolytic cleavage of vicilin precursor polypeptides were located in polar regions of the polypeptide, at acidic or amide residues. Proteolysis did not take place in precursors containing a functionally distinct sequence: neutral residue-hydrophobic residue-basic residue at the cleavage site. Differences between the genomic sequences encoding vicilin thus specify proteolytic cleavage of vicilin precursor polypeptides.

Amino Acid Sequence↗
PubMed1983

The vicilin gene family of pea (Pisum sativum L.): a complete cDNA coding sequence for preprovicilin.

A cDNA plasmid bank has been constructed using mRNA from developing pea seeds and three cDNAs coding for vicilin polypeptides have been selected. These cDNAs have been sequenced and between them cover the whole of the coding sequence plus part of the 5' and 3' untranslated regions. Comparison with amino acid sequence data from the protein indicates that vicilin is synthesised as preprovicilin with subsequent removal of a signal peptide and a C-terminal peptide as well as post translational endo-proteolytic cleavage. The cDNAs represent two different classes of vicilin genes whilst amino acid data show that there are at least three major classes of vicilin polypeptide. The vicilin sequences show extensive homology with conglycinin and phaseolin except in the regions of the internal proteolytic cleavages. The evolutionary significance of this relationship is discussed.

Amino Acid Sequence↗