Temporal and tissue localization of a cowpea (Vigna unguiculata) cystatin.

A cowpea (Vigna unguiculata L. Walpers cv. Pitiúba) cystatin was analysed to determine its localization during development and germination of cowpea seeds, using western blotting with a specific antiserum.

Genetic modification and plant food allergens: risks and benefits.

Plant genetic engineering has the potential to both introduce new allergenic proteins into foods and remove established allergens. A number of allergenic plant proteins have been characterized, showing that many are related to proteins which have potentially valuable properties for use in nutritional enhancement, food processing and crop protection. It is therefore important to monitor the allergenic potential of proteins used for plant genetic engineering and major biotechnology companies have established systems for this.

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein.

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E.

Endosperm-specific activity of a storage protein gene promoter in transgenic wheat seed.

The characterization of the promoter of a wheat (Triticum aestivum) cv. Cheyenne high molecular weight glutenin subunit (HMW subunit) gene, Glu-1D-1 is reported. The nucleotide sequence of the promoter from position -1191 to -650 with respect to the transcription start site was determined, to add to that already determined.

Production of C20 polyunsaturated fatty acids (PUFAs) by pathway engineering: identification of a PUFA elongase component from Caenorhabditis elegans.

Using a combination of database-mining and functional characterization, we have identified a component of the polyunsaturated fatty acid (PUFA) elongase. Co-expression of this elongating activity with fatty acid desaturases has allowed us to heterologously reconstitute the PUFA biosynthetic pathway. Both these enzymes (desaturases and elongase components) have undergone gene-duplication events which provide a paradigm for the diverged nature of PUFA biosynthetic activities.

Mutagenesis of the borage Delta(6) fatty acid desaturase.

The consensus sequence of the third histidine box of a range of Delta(5), Delta(6), Delta(8) and sphingolipid desaturases differs from that of the membrane-bound non-fusion Delta(12) and Delta(15) desaturases in the presence of glutamine instead of histidine. We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b(5) fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Delta(6) desaturase.

Prolamin aggregation, gluten viscoelasticity, and mixing properties of transgenic wheat lines expressing 1Ax and 1Dx high molecular weight glutenin subunit transgenes.

The composition of high molecular weight (HMW) subunits of glutenin determines the gluten strength and influences the baking quality of bread wheat. Here, the effect of transgenes coding for subunits 1Ax1 and 1Dx5 was studied in two near-isogenic wheat lines differing in their HMW subunit compositions and mixing properties. The subunits encoded by the transgenes were overexpressed in the transformed lines and accounted for 50-70% of HMW subunits.

Chimeras of Delta6-fatty acid and Delta8-sphingolipid desaturases.

The Borago officinalis Delta6 fatty acid desaturase (Boofd6) shares 58% identity in its amino acid sequence with Boofd8, a Delta8 sphingolipid desaturase from the same plant species. In order to localise the distinct catalytic properties of Boofd6 and Boofd8 to individual regions within them, a set of chimeras of these two enzymes were constructed and expressed in yeast. Chimera 2 is different from the other chimeras and Boofd6 in that it did not have any detectable desaturase activity on 18 carbon fatty acids.

Overexpression, purification, and in vitro refolding of the 11S globulin from amaranth seed in Escherichia coli.

An amarantin 11S globulin cDNA encoding one of the most important storage proteins of amaranth seeds, with a high content of essential amino acids, was expressed in Escherichia coli. A good level of expression of recombinant amarantin with a molecular weight of 59 kDa was obtained. The recombinant protein was extracted by ammonium sulfate precipitation and purified to homogeneity using ion-exchange chromatography and reversed phase high-performance liquid chromatography.

Elastomeric proteins: biological roles, structures and mechanisms.

Elastomeric proteins are able to withstand significant deformations without rupture before returning to their original state when the stress is removed. Although elastomeric proteins differ considerably in their amino acid sequence, they all have a complex domain structure and share two common properties. Namely, they contain elastomeric domains, comprised of repeated sequences, and additional domains that form intermolecular crosslinks.

A high resolution (1)H magic angle spinning NMR study of a high-M(r) subunit of wheat glutenin.

This work describes the application of (1)H magic angle spinning (MAS) nmr to the study of hydrated 1Dx5 wheat high-M(r) subunit. 1Dx5 is a water-insoluble 88 kDa protein, associated with good baking performance, and whose structure in the solid and low-hydration states is not known. High-resolution MAS (HR-MAS) results in a threefold resolution improvement of the (1)H spectra of the hydrated wheat protein, compared to standard MAS.

Expression and characterisation of a highly repetitive peptide derived from a wheat seed storage protein.

The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens. They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences. The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli.

Genetically modified crops: methodology, benefits, regulation and public concerns.

The genetic modification of crop plants from the methodology involved in their production through to the current debate on their use in agriculture are reviewed. Techniques for plant transformation by Agrobacterium tumefaciens and particle bombardment, and for the selection of transgenic plants using marker genes are described. The benefits of currently available genetically modified (GM) crops in reducing waste and agrochemical use in agriculture, and the potential of the technology for further crop improvement in the future are discussed.

Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway.

A Caenorhabditis elegans ORF encoding the presumptive condensing enzyme activity of a fatty acid elongase has been characterized functionally by heterologous expression in yeast. This ORF (F56H11. 4) shows low similarity to Saccharomyces cerevisiae genes involved in fatty acid elongation.

Characterization of a monoclonal antibody specific for HMW subunits of glutenin and its use to investigate glutenin polymers.

A monoclonal antibody, IFRN 1602, has been developed to a synthetic peptide based on the sequence (94)GSVTCPQQV(101) of HMW subunit 1Dx5. The antibody bound strongly to the synthetic peptide based on the cognate sequence of HMW subunit 1Dx2 which contains a serine instead of a cysteine residue. However, it recognized the immunizing peptide by enzyme-linked immunosorbent assay (ELISA) only poorly, probably because the peptide exists as a disulfide-bonded dimer under the assay conditions.

Structural characterization of a methionine-rich, emulsifying protein from sunflower seed.

The 2 S seed storage protein, sunflower albumin 8, contains an unusually high proportion of hydrophobic residues including 16 methionines in a mature protein of 103 amino acids. A structural model, based on the known structure of a related protein, has been constructed as a four-helix bundle cross-linked by four disulphide bonds. This model structure is consistent with data from circular dichroism and nuclear magnetic resonance experiments.

Extraction, separation, and purification of wheat gluten proteins and related proteins of barley, rye, and oats.

The wheat proteins, which are active in celiac disease and other glutenrelated conditions, are defined as prolamins, in that they are soluble as individual subunits in alcohol-water mixtures, such as 50% (v/v) aqueous propan-1-ol or 60-70% (v/v) aqueous ethanol. However, in wheat grain and flour, about half of these subunits are present in polymers that are not soluble in alcohol-water mixtures unless disulfide bonds between the component subunits are reduced using an agent such as 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). These alcohol-insoluble polymers are traditionally called glutenins and the related alcohol-soluble monomers are called gliadins; the two groups of proteins together form the major part of the gluten fraction.

Identification of microphases in mixed alpha- and omega-gliadin protein films investigated by atomic force microscopy.

Pure and mixed films of alpha- and omega-gliadins were studied by tapping mode atomic force microscopy (AFM). The technique was sensitive to the chemistry of the surface properties of the films, allowing imaging of the mixed gliadin phases at different ratios. In addition to the study of the phases at the micrometer level, higher resolution images allowed visualization of the protein films at the molecular level.

Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.

Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase.

Transgenic Arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug Deroceras reticulatum (Müller).

Transgenic Arabidopsis thaliana has been developed which expresses the oryzacystatin mutant OC-I delta 86, which is an inhibitor of the major proteinase present in the digestive gland of the slug, Deroceras reticulatum. When fed on leaf tissue from plants expressing this inhibitor the growth of juvenile slugs was significantly reduced by 31% compared with those feeding on control leaf tissue. Furthermore, while surviving slugs did not individually consume less when feeding on leaf tissue expressing OC-I delta 86, the total amount of leaf tissue eaten was 50% less, due to reduced survival of slugs.

Direct kinetic evidence for folding via a highly compact, misfolded state.

The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.

The localization and expression of the class II starch synthases of wheat.

The starch granules of hexaploid wheat (Triticum aestivum) contain a group of three proteins known as SGP-1 (starch granule protein-1) proteins, which have apparent molecular masses of 100, 108, and 115 kD. The nature and role of these proteins has not been defined previously. We demonstrate that these polypeptides are starch synthases that are present in both the starch granule and the soluble fraction at the early stages of wheat endosperm development, but that are exclusively granule bound at mid and late endosperm development.

Molecular cloning and characterisation of a maize cDNA for a homologue of the large subunit of the eukaryotic initiation factor 3 (eIF3).

In order to identify genes that are specifically expressed in distinct cell populations of the maize root apex, we have constructed PCR-directed cDNA libraries from microdissected populations of cells, and screened them by differential hybridisation. A meristem-specific cDNA was isolated and characterised. This cDNA, termed ZmeIF3A, encodes a protein homologous to the large subunit of the eukaryotic translation Initiation Factor 3 (eIF3), which is an essential multi-protein complex for the initiation of protein synthesis.

High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds.

Proteinaceous serine proteinase inhibitors are widespread throughout the plant kingdom where they play an important role in protection against pests and pathogens. Here, we describe the isolation and characterisation of a novel 14 amino acid residue cyclic peptide from sunflower seeds, which is a potent inhibitor of trypsin (Ki=100 pM). The crystal structure of this peptide in complex with bovine beta-trypsin shows both sequence and conformational similarity with the trypsin-reactive loop of the Bowman-Birk family of serine proteinase inhibitors.

Small angle X-ray scattering of wheat seed-storage proteins: alpha-, gamma- and omega-gliadins and the high molecular weight (HMW) subunits of glutenin.

Small angle X-ray scattering in solution was performed on seed-storage proteins from wheat. Three different groups of gliadins (alpha-, gamma- and omega-) and a high molecular weight (HMW) subunit of glutenin (1Bx20) were studied to determine molecular size parameters. All the gliadins could be modelled as prolate ellipsoids with extended conformations.