Cloning and nucleotide sequence of a cDNA encoding the precursor of the barley toxin alpha-hordothionin.

A cDNA library, prepared from developing barley endosperm, was screened for thionin recombinants. Clone pTH1 was that with the largest insert out of three identified. The longest reading frame in the 610-base-pair insert codes for a protein of 127 amino acids that includes an internal sequence of 45 amino acids, which is identical to that obtained for the alpha-hordothionin by direct protein sequencing. The deduced thionin sequence is preceded by a leader sequence of 18 residues and followed by a sequence that corresponds to an acidic protein of 64 amino acids. This structure supports previous evidence indicating that thionin is synthesized as a much larger precursor, which undergoes two processing steps: the cotranslational cleavage of a leader sequence and the post-translational one of a larger peptide. The size of the mRNA was estimated to be about 950 bases by Northern analysis. Thionin concentration in mature endosperm of barley cv. Bomi was about twice that of its high-lysine mutant Risø 1508. The same difference was observed in thionin mRNA in the corresponding developing endosperms, indicating that gene expression is partially blocked in the mutant at a pretranslational level.

Characterization of cDNA clones of the family of trypsin/α-amylase inhibitors (CM-proteins) in barley (Hordeum vulgare L.).

Recombinants encoding members of the trypsin/α-amylase inhibitors family (also designated CM-proteins) were selected from a cDNA library prepared from developing barley endosperm. Inserts in two of the clones, pUP-13 and pUP-38, were sequenced and found to encode proteins which clearly belong to this family, as judged from the extensive homology of the deduced sequences with that of the barley trypsin inhibitor CMe, the only member of the group for which a complete amino acid sequence has been obtained by direct protein sequencing. These results, together with previously obtained N-terminal sequences of purified CM-proteins, imply that there are at least six different members of this dispersed gene family in barley. The relationship of this protein family to the B-3 hordein and to reserve prolamins from related species is discussed in terms of their genome structure and evolution.

Synthesis and processing of thionin precursors in developing endosperm from barley (Hordeum vulgare L.).

Thionin is a lysine-rich polypeptide (mol. wt. 5000) which is synthesized in developing barley endosperm from 8 days to 30 days after anthesis. Two thionin precursors (THP1 and THP2) have been identified using monospecific antibodies (A-TH) prepared against the mature protein. THP1, which is the only polypeptide recognized in vitro by A-TH, is encoded by a 7.5S mRNA obtained from membrane-bound polysomes, and its alkylated derivative has an apparent mol. wt. of 17 800. THP2, which is selected together with mature thionin by A-TH among labelled proteins in vivo, differs from THP1 in apparent mol. wt. (17 400 alkylated) and in electrophoretic mobility at pH 3.2. Both THP1 and THP2 are competed out of the antigen-antibody complex by purified thionin. The conversion of THP2 into thionin, which has been demonstrated in a pulse-chase experiment in vivo, is a post-translational process. As it has not been possible to detect THP1 in vivo it is assumed that it is converted co-translationally into THP2. Final deposition of thionin as an extrinsic membrane protein, possibly associated with the endoplasmic reticulum, has been tentatively established on the basis of subcellular fractionation experiments.

In vivo and in vitro synthesis of CM-proteins (A-hordeins) from barley (Hordeum vulgare L.).

CM-proteins from barley endosperm (CMa, CMb, CMc, CMd), which are the main components of the A-hordein fraction, are synthesized most actively 10 to 30 d after anthesis (maximum at 15-20 d). They are synthesized by membranebound polysomes as precursors of higher apparent molecular weight (13,000-21,000) than the mature proteins (12,000-16,000). The largest in vitro product (21,000) is the putative precursor of protein CMd (16,000), as it is selected with anti-CMd monospecific IgG's, and is coded by an mRNA of greater sedimentation coefficient (9 S) than those encoding the other three proteins (7.5 S). CM-proteins always appear in the soluble fraction, following different homogenization and subcellular fractionation procedures, indicating that these proteins are transferred to the soluble fraction after processing.

Thionins: plant peptides that modify membrane permeability in cultured mammalian cells.

Thionins, which are high-sulphur polypeptides present in the endosperm of wheat and related species, have ben found to prevent growth and to inhibit macromolecular synthesis in cultured mammalian cells. Baby hamster kidney (BHK) cells were markedly more sensitive to thionins than the other cell lines tested (monkey CV1, mouse L, human HeLa). A thionin concentration of 5 microgram/ml (1 microM) completely blocked translation in BHK cells. It was later found that omission of both calcium and magnesium ions from the medium strongly enhanced the inhibitory effects of thionins (BHK cells, 80% inhibition, 0.5 microgram/ml). Several lines of evidence indicate that thionins might act at the membrane level. Indeed, both the 86Rb+ content and the nucleotide pool of BHK cells were drastically decreased at thionin concentrations that inhibited translation. In addition, thionin concentrations that did not affect macromolecular synthesis in these cells, allowed inhibition of translation by antibiotics, such as hygromycin B, that are not able to cross the cell plasma membrane by themselves. Our results suggest that the inhibition of protein, RNA and DNA synthesis in BHK cells might be a consequence of membrane leakiness induced by thionin treatment. In this respect, particularly striking was the parallelism found between 86Rb+ leakage and inhibition of protein synthesis by treatment with different genetic variants of thionins (alpha 1 purothionin, alpha 2 purothionin, beta purothionin from wheat; hordothionin from barley), as well as with the viscotoxins, which are homologous polypeptides from the European mistletoe.

Homoeologous chromosomal location of the genes encoding thionins in wheat and rye.

Thionins are high sulphur basic polypeptides present in the endosperm of Gramineae. In wheat there are three thionins encoded by genes located in the long arms of chromosomes 1A, 1B and 1D. Rye has one thionin encoded by a gene which has been assigned to chromosome 1R after analysis of the Imperial-Chinese Spring rye-wheat disomic addition lines. Commercial varieties and experimental stocks with a 1B/1R substitution carry the thionin from rye (ß R) instead of the α B thionin from wheat. The ß R thionin gene is not located in the large chromosomal segment representing most of the short arm of chromosome 1R.

Control of galactosyl diglycerides in wheat endosperm by group 5 chromosomes.

Lower levels of monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) have been found in tetraploid wheats as compared with those in hexaploid wheats. The same difference has been found between hexaploid cultivars and tetraploid lines derived from them by D genome extraction. A lower level of MGDG and DGDG is also present in Triticum carthlicum (AABB) as compared with Aegilops squarrosa (DD) or with the synthetic T. spelta (AABBDD) obtained from them. Analysis of the appropriate nullitetrasomic and ditelosomic lines indicates that a gene or genes located in the short arm of chromosome 5D are responsible for the observed difference and that group 5 chromosomes can be ranked as to their influence on the MGDG and DGDG levels in the order 5B > 5D > 5A and 5D > 5B > 5A, respectively. These results further support our previous identification of DGDG as the lipid factor responsible for petroleum ether solubility of lipopurothionins. Since DGDG contributes to baking quality by improving the retention of fermentation gases, the present observations imply that the difference in bread-making quality between the two types of wheat is not due only to proteins contributed by the D genome.

Gene expression in alloploids: genetic control of lipopurothionins in wheat.

Lipopurothionins are complexes of basic polypeptides and polar lipids found in petroleum ether extracts of wheat endosperm. Location of the structural genes for the protein moiety and of genes probably controlling the lipid moiety has been achieved by analysis of compensated nulli-tetrasomic and ditelosomic lines of Triticum aestivum L. cv. Chinese Spring, as well as of other genetic stocks. There are two electrophoretic variants of the apoprotein designated alpha and beta purothionins. Structural genes for alpha purothionins are located in the long arm of chromosomes 1B and 1D, and for the beta variant in the long arm of 1A. These genes have been tentatively designated Pur-A1, Pur-B1, and Pur-D1. The aminoacid composition of purified alpha and beta purothionins from Triticum aestivum (genomes AABBDD) and T. durum (AABB), and of beta purothionin from T. monococcum (AA) is also consistent with this conclusion and suggests that the alpha purothionin encoded by gene Pur-B1 probably differs from that encoded by gene Pur-D1 in at least three positions of the aminoacid sequence. A gene (or genes) located in the short arm of chromosome 5D markedly affects the level of lipopurothionin but does not affect apoprotein synthesis. It is concluded that they control the lipid moiety which is required for solubility in petroleum ether.

Inhibition of brewer's yeasts by wheat purothionins.

Purothionins are basic polypeptides with antimicrobial properties that are present in the endosperm of wheat and other Gramineae. Susceptibility to crude and electrophoretically purified purothionins among brewing starters has been investigated. Seven yeast strains of Saccharomyces uvarum (syn. carlsbergensis), four strains of Saccharomyces cerevisiae, and four wild strains (Saccharomyces spp.) have been tested in three culture media. All the strains were susceptible to the crude preparation in a yeast extract-glucose medium. Determinations of minimal inhibitory and biocidal concentrations yielded double end points in about half of the assays. The highest sensitivity to purothionins was obtained in malt extract medium. Sensitivity to electrophoretically purified purothionins was of the same order or smaller than to the crude preparation. Possible explanations for this unexpected result are presented.