Frank C. Greene

The nucleotide and deduced amino acid sequences of an HMW glutenin subunit gene from chromosome 1B of bread wheat (Triticum aestivum L.) and comparison with those of genes from chromosomes 1A and 1D

SummaryThe nucleotide and deduced amino acid sequences of a high molecular weight glutenin subunit gene derived from chromosome 1B of bread wheat (Triticum aestivum L.) are reported. The encoded protein corresponds to the y-type subunit 1B9. Comparison of the 5′ upstream untranslated regions of this gene and a previously reported silent y-type gene derived from chromosome 1A showed a deletion of 85 bp in the latter. A sequence present in this region of the 1By 9 gene shows homology with part of the “-300 element” which is conserved in the 5′ upstream regions of other prolamin genes from barley, wheat and maize (Forde BG et al. 1985). It is suggested that the absence of this element is responsible for the lack of expression of the 1Ay gene. Comparison of the derived amino acid sequence with those reported previously for the silent 1Ay gene and the expressed x-type (1Dx2) and y-type (1Dy12) genes derived from chromosome 1D showed that the three y-type proteins are closely related. In contrast the x-type subunit (1Dx2) shows clear differences in the N-terminal region and in the number, type and organisation of repeats in the central repetitive domain.

Allergy to green coffee: Failure of patients allergic to green coffee to react to chlorogenic acid, roasted coffee, or orange

Abstract Clinical skin tests upon twenty-nine French patients and allergic human serum transfer tests in monkeys indicate that primary allergy to green coffee does not involve chlorogenic acid or its known isomers. Allergy to green coffee appears to be a typical example of atopic hypersensitivity to proteins specific, in this allergy, to the green coffee bean. There were no cross-reactions to castor beans or oranges, nor were there any reaginic reactions to roasted coffee or to coffee beverage.

Purification and characterization of wheat α-gliadin synthesized in the yeast, Saccharomyces cerevisiae

Abstract The development of efficient methods for production and purification of plant seed storage proteins in heterologous microbial hosts would facilitate structure-function studies of these proteins. This report describes such methods applied to the production and isolation of wheat α-gliadin, a prolamine-type seed storage protein, from Saccharomyces cerevisiae . Beginning with the vector, growth conditions, and extraction methods of Neill et al. [Gene 55 (1987) 303–317], we implemented several improvements to increase the yields of α-gliadin per volume of yeast cell culture. The CYC1::Gli-A2 -Y transcriptional fusion vector, pAY31 (Neill et al., 1987), was modified by replacing the ARS1 region of replication with that of the 2 μ plasmid of yeast. We formulated a new medium, a derivative of synthetic defined (SD) medium supplemented with several nitrogen sources, that allows both selection for maintenance of plasmids and growth to high cell densities. Stationary phase cultures of cells bearing the modified expression vector, and grown in this medium with glycerol and lactate as carbon sources, contain significantly higher levels of α-gliadin than log-phase cultures grown in SD glucose. Sonication in 80% ethanol selectively and efficiently extracts the α-gliadin from cell pellets of small- or large-scale cultures, allowing the purification of several hundred μg of the wheat protein per liter injust a few high-yield steps. The α-gliadin isolated from yeast elutes at the same position in HPLC as the A-gliadin fraction purified from wheat flour. N-terminal amino acid (aa) sequencing reveals that the signal peptide is removed from the gliadin precursor in yeast cells. About two-thirds of the molecules have the same N terminus as A-gliadin made in wheat endosperm cells. The remaining third of the α-gliadin is apparently cleaved at a similar processing site three aa closer to the N terminus of the prepeptide.

Interactions of anionic and cationic fluorescent probes with proteins: The effect of charge

A family of fluorescent probes, consisting of 2-p-toluidinylnapththalene-6-sulfonate (TNS) and neutral and cationic sulfonamido derivatives has been utilized to study the influence of electrostatic forces in protein-amphiphile interactions. 2-p-Toluidinylnaphthalene-6-[N-β-ethylammonium chloride] sulfonamide (III) binds to a lower number of discrete sites in bovine serum albumin and sperm whale apomyoglobin than does TNS, and is also bound less efficiently by β-lactoglobulin. The fluorescence characteristics of the bound probes indicate that their environments are hydrophobic, but thatpH and ionic strength influence the binding. The initial binding of III to discrete sites on both apomyoglobin and bovine serum albumin induces the cooperative binding of additional probe molecules. TNS, but not III, fluoresces in α-chymotrypsin solutions. An [N-β-ethyltrimethyl ammonium] sulfonamido derivative (IV), but not TNS, fluoresces in bovine trypsin solutions. Fluorescence-enhancing interactions were detected between TNS, III, and polyvinylpyrrolidone, but not between these probes and ribonuclease A, α-chymotrypsinogen, lysozyme, or γ-globulins. The wheat prolamin A-gliadin binds more TNS than III. The accessibility of all binding sites of gliadin is lower atpH 5.0 than atpH 3.1. It is suggested that, in general, charge effects are more likely to enhance the binding of anionic than cationic amphibiles by proteins.

Use of monkeys to demonstrate allergic cross-reactions among the euphorbiaceae: Ricinus communis, poinsettia pulcherrima, euphorbia esula

Abstract Individuals who are sensitized by or allergic to one species of Euphorbiaceae may be allergic to other species also. Guinea pigs passively sensitized with sera from rabbits that had been sensitized to Ricinus communis (castor bean) exhibited passive cutaneous anaphylaxis with extravasation of Evans blue dye when challenged with antigens extracted from the blossoms and seeds of Poinsettia pulcherrima , or Euphorbia esula. Macaca irus monkeys passively sensitized with blood sera from castor bean-allergic human beings exhibited PCA reactions when challenged with blossom antigens from either of these euphorbias.

PASSIVE TRANSFER OF HUMAN ALLERGIES TO PROSIMIANS: SKIN-REACTIONS IN THE LEMUROID, NYCTICEBUS COUCANG (SLOW LORIS).

Summary Human serum transfer tests were conducted upon the lemuroid prosimian, Nycticebus coucang (slow loris). Intradermal injections of allergic sera passively sensitized the skin of the slow loris. Challenge with the allergens affecting the human patients caused erythema, piloerection, blanching, whealing, and extravasation of intravenously injected colloidal dye in the passively sensitized sites. The human allergic serum transfer test may be an aid in ordinal and subordinal classification of species suspected of being related to the Anthropoidea. Mr. Leonard Harrison, animal caretaker, rendered invaluable aid in maintaining the animals in healthy condition. Mr. Wm. G. Murray filmed the procedures and performed the photographic services. Dr. Raphael Panzani of Marseille, France, supplied the sera from castor bean allergic patients. Dr. C. W. Denko, Ohio State University, supplied the sera from ragweed atopic patients. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Dept. of Agriculture to the exclusion of others that may be suitable.

Neutralization of Specific Reagins in Monkeys Passively Sensitized by Cross-Reactive Allergy Sera

Summary Blood sera from 60 allergy patients in Marseille, France, were examined by the reagin passive transfer test in macaque monkeys. Twenty-six of the sera were highly reactive with partially purified castor bean seed protein. Six of the reactive sera were also highly reactive with castor pollen extract. By utilization of the phenomenon of reagin neutralization (specific desensitization) with castor pollen extract, sites passively sensitized with these 6 sera were rendered non-reactive to the pollen allergens: the pollen-desensitized sites were still highly reactive to the seed protein. The results appear to support the conclusion that allergy to castor bean pomace may be hypersensitivity to one or more of several components of the seed, pollen, or female blossoms.

Progress Towards Genetic Engineering of Wheat with Improved Quality

Progress is reported on the goal of our laboratory to use biotechnological approaches to improve wheat quality. The wheat gene family we have chosen for our initial efforts is that of the high-molecular-weight (HMW) glutenins. These polypeptides play a critical role in the formation of the disulfide cross-linked protein matrix correlated with dough functionality. The development of sufficiently reliable wheat transformation methodology will allow direct testing of the effect of native and modified HMW-glutenin genes on wheat quality. Our laboratory now has the capability of transforming wheat reliably enough to carry out such studies. We have used the highly regenerative hard white spring cultivar Bobwhite. DNA is delivered via bombarding immature embryos with gold particles coated with the bar gene (which confers resistance to the herbicides bialaphos and Basta). Over a nine month period we produced 9 lines of transformed wheat. The criteria for successful transformation was herbicide resistance, transgene encoded enzyme activity, integration of transforming DNA into high-molecular-weight wheat DNA, and co-segregation of the DNA and phenotype in the T1 and T2 generations. Wheat transformation is a key component in a schema proposing a complete set of approaches to apply biotechnology to questions of wheat quality via manipulation of the HMW-glutenin genes. A second important component is the heterologous expression of HMW-glutenin genes, and the use of the resulting proteins in the study of glutenin physical chemistry and dough supplementation experiments. While the techniques are still in the process of fine-tuning, we believe all the necessary tools are now available to attempt modification of the physical properties of dough.