Analysis of the genome of plants. II. Characterization of repetitive DNA in barley (Hordeum vulgare) and wheat (Triticum aestivum).

Barley and wheat DNAs have been characterized by studying their kinetics of reassociation, melting properties and sedimentation behaviour in neutral CsCl gradients as well as in Cs2SO4 gradients containing Ag+ or Hg2+. In both species, reassociation kinetics have revealed the presence of approx. 76% redundant nucleotide sequences which have been grouped into very rapidly reassociating (Cot 0-0.01), rapidly reassociating (Cot 0.01-1.0) and slowly reassociating (Cot 1-100) fractions. The barley Cot 0-0.01 and Cot 0.01-1.0 fractions as well as the wheat Cot 0.01-1.0 fraction form narrow bands upon centrifugation in CsCl gradients. Under similar experimental conditions both Cot 0.01 and Cot 1.0-100 wheat fractions and the barley Cot 1.0-100 fraction form broad bands each having several shoulders. Thermal denaturation studies of most of the above reassociated fractions have shown a considerable degree of order in their duplexes with an average hyperchromicity of 21.5%. When native, high molecular weight barley DNA is centrifuged in Ag+/CS2SO4 density gradients (RF = 0.2), two satellites appear on the heavier side of the main band, as against one in the case of wheat. The two minor peaks, designated as satellites I and II, have buoyant densities of 1.702 and 1.698 g/cm3, respectively, in neutral CsCl gradients and together represent about 8-9% of total barley DNA. Upon centrifugation in Hg2+/CS2SO4 density gradients, one satellite is observed in both barley and wheat and it accounts for 1-2% of their genomes.

Comparison of the EPR properties of photosystem I iron-sulphur centres A and B in spinach and barley.

The properties of Photosystem I iron-sulphur centres A and B from spinach and barley chloroplasts were investigated by electron paramagnetic resonance spectroscopy (EPR). Barley chloroplasts were shown to photoreduce significant amounts of centre B at cryogenic temperatures unlike those from spinach which only photoreduced centre A. Centre B in barley chloroplasts was also reduced by dithionite before centre A and the EPR spectrum of reduced centre B was obtained. Illumination of barley chloroplasts at 15 K where centre B was chemically reduced resulted in the reduction of centre A and the appearance of spectral features indicating interaction between the two reduced centres. The variation of behaviours of iron-sulphur centres A and B between species favours a scheme of electron flow for Photosystem I where either centre A or centre B act as parallel electron acceptors from the earlier acceptor X.

P-700 content and polypeptide profile of chlorophyll-protein complexes of spinach and barley thylakoids.

Of the six chlorophyll-protein complexes of spinach and barley resolved by mild gel electrophoresis, two were chlorophyll a-protein complexes of PS I, namely CP1a and CP1, which accounted for up to 30% of the total chlorophyll. Both of these complexes had one P-700 per 120 chlorophyll a molecules. Since spinach and barley thylakoids have some 400 chlorophyll molecules per P-700, these complexes may not have lost any of the chlorophyll associated with them in vivo. This may account for CP1a and CP1 having the characteristic low-temperature fluorescence normally associated with PS I in vivo, which is not found in complexes with low chlorophyll/P-700 ratios. Two-dimensional electrophoresis showed that all of the chlorophyll a and P-700 of CP1 was bound to 70 kilodalton polypeptides. The PS I reaction centre complex of lowest mobility, CP1a, contained CP1 and four additional low molecular weight polypeptides. The three light-harvesting complexes resolved had major 25 and 23 kilodalton polypeptides. The presumed reaction centre complex of PS II contained major 50 and 47 kilodalton polypeptides.

Isolation and partial characterization of two antifungal proteins from barley.

We have developed a simple assay for detecting antifungal compounds utilizing impregnated paper discs on agar to inhibit mycelial spread of an indicator organism, Trichoderma reesei. Using this assay we have isolated and purified to apparent homogeneity two antifungal proteins from dehusked barley grain. Both proteins are present at high concentrations: over 10 mg of each protein can be isolated per 100 g of grain. The first protein has a molecular weight of 30 000 and is identical to the 30 kDa ribosome-inactivating protein previously isolated from barley. This protein very effectively inactivates fungal ribosomes and this may explain its antifungal activity and biological role. The second antifungal protein has a molecular weight of 28 000 and is 20-fold more potent than the 30 kDa protein in inhibiting growth of Trichoderma. In addition to Trichoderma, the 28 kDa protein also efficiently inhibits growth of Phycomyces blakesleeanus, Alternaria alternaria and a protoplast-forming mutant of Neurospora crassa. The 28 kDa protein does not inactivate fungal ribosomes and we are currently investigating other possible enzymatic activities of this protein.

Functional reconstitution of the malate carrier of barley mesophyll vacuoles in liposomes.

The malate carrier of barley (Hordeum vulgare L.) mesophyll vacuoles was highly purified by chromatography on hydroxyapatite followed by affinity-chromatography using 5-amino-1,2,3-benzenetricarboxylic acid as ligand. The carrier, reconstituted in asolectin liposomes, had properties similar to those described previously for the carrier in intact vacuoles (Martinoia, E., Flügge, U.I., Kaiser, G., Heber, U. and Heldt, H.W. (1985) Biochim. Biophys. Acta 806, 311-319). The apparent Km for malate uptake was 2-3 mM, and the uptake was inhibited by other carboxylic acids (preferentially tricarboxylic). The sulfhydryl reagent, p-chloromercuribenzenesulfonate, as well as the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, also inhibited malate uptake. The transport was dependent on the membrane potential with an optimum at about 35 mV.

Accurate measurements of coupling constants from two-dimensional nuclear magnetic resonance spectra of proteins and determination of phi-angles.

A new and simple method to measure 3JHNH alpha coupling constants of proteins by adding and subtracting traces from corresponding two-dimensional nuclear Overhauser enhanced spectroscopy and two-dimensional correlated spectroscopy cross peaks after scaling is proposed. The optimal scaling for the addition and the subtraction of the two traces is obtained by minimizing an error function. The method was proven to give accurate and precise measurements of coupling constants when tested with a series of simulated spectra. The accuracy of the method was better than 0.1 Hz for all test cases including the limiting case of J = 2.0 Hz and line-width = 11.0 Hz. The accuracy of the method was better than 0.1 Hz for all test cases including The 3JHNH alpha coupling constants were measured in two-dimensional nuclear magnetic resonance spectra of the two proteins barley serine proteinase inhibitor (CI-2) and the bacterial ribonuclease (barnase) of Bacillus amyloliquefaciens. The experimentally measured coupling constants were used to calculate the constants in a Karplus equation to be: 3JHNH alpha = 6.7 cos2(phi-60) -1.3 cos(phi-60) +1.5. These constants are in good accordance with those obtained for basic pancreatic trypsin inhibitor (BPTI). In addition, special emphasis is given to the measurements of positive phi-angles, and to the contribution of molecular dynamics on the apparent coupling constants.

Correlation of some published amino acid sequences for photosystem I polypeptides to a 17 kDa LHCI pigment-protein and to subunits III and IV of the core complex.

Photosystem I (PSI) in barley consists of at least 11 polypeptides of which three have apparent sizes of 15-19 kDa. Two of these polypeptides (subunits III and IV) are constituents of the core complex (CCI), the third is a component of the light-harvesting complex (LHCI). After fractionation of PSI into its CCI and LHCI components, each of the polypeptides has been isolated and its N-terminal region sequenced. We conclude that the gene sequence published for subunit IV of spinach [(1988) FEBS Lett. 237, 108-112] is not that of subunit IV but rather that of the 17 kDA LHCIc pigment protein. We confirm that the published sequence for subunit III [(1988) Curr. Genet. 14, 511-518] is indeed that of subunit III; seemingly conflicting identifications, based on apparent sizes on SDS-PAGE, of which polypeptides are subunits III and IV are probably explained by subunit III's electrophoretic migration rate being dependent on the solvent.

A barley flour inhibitor of insect alpha-amylase is a major allergen associated with baker's asthma disease.

A barley salt-soluble protein of 14.5 kDa, which inhibits the alpha-amylase from the insect Tenebrio molitor, has been identified as a major IgE-binding component of sera from baker's asthma patients. The N-terminal amino acid sequence of this protein indicates that it is a member of a previously described family of alpha-amylase/trypsin inhibitors.

Extreme variations in the ratios of non-synonymous to synonymous nucleotide substitution rates in signal peptide evolution.

Nucleotide sequences encoding signal peptides from the precursors of alpha-amylase/trypsin inhibitors from cereals are homologous to those corresponding to the precursors of thaumatin II and of plastocyanins. Non-synonymous (KA) and synonymous (KS) rates of nucleotide substitutions have been calculated for all possible binary combinations. Extreme variation in KA/KS ratios has been observed; from the 0.167 average found within the plastocyanin family to an average of 1.90 calculated for the inhibitors/thaumatin II transition. A similar calculation has been carried out for the signal peptide sequences of thionins, which are unrelated to those of the alpha-amylase/trypsin inhibitor family, and an average KA/KS of 0.12 has been obtained. This variation can be largely explained in terms of an empirical index of stability related to amino acid composition and seems to be independent of functional constraints.

[Nature and fractionation of barley proteins extracted by ethanol, isopropanol and n-propanol in different proportions]. / Nature et fractionnement des protéines de l'Orge extraites par l'éthanol, l'isopropanol et le n-propanol à des titres différents-19h

Barley alcoholsoluble protein extractabilities by aqueous ethanol, isopropanol and n-propanol were measured at room temperature. The quantities extracted by each alsohol strongly depend on the concentration of the alcohol. The most efficient concentration for the three alcohols were by increasing order: 45 per cent ethanol, 40 per cent isopropanol, 35 per cent (w/w) n-propanol. Hrodein preparations extracted by these three alcoholic solutions and by 75 per cent (w/w) ethanol were compared by means of the flour nitrogen percentage they contain and by electrophoresis, amino-acid analysis and Sephadex G 100 gel filtration. The preparations studied do not differ markedly in their amino-acid composition or electrophoretic pattern which shows at least 17 different bands. On the other hand, Sephadex G 100 gel filtration separates two main groups of proteins, The first one is present at the same level in all preparations studied and consists of electrophoretically typical hordeins (already described hordeins). The other group represents a fraction of the preparation, the more abundant as the solvent is more effective. This second group is excluded on Sephadex G 100 chromatography and does not give well defined bands by starch gel electrophoresis. Consequently it is related to some glutelins. Nevertheless its amino-acid composition is very close to the mean hordein composition. Electrophoretic comparison with glutelins extracted by acetic acid and with hordeins, all reduced and alkylated, discloses a great similitude between this fraction, the glutelins and some hordein fast components alpha, beta and gamma.

Isolation and some properties of a subtilisin inhibitor from barley.

An inhibitor affecting subtilisin [EC 3.4.21.14] was isolated from barley (Hordeum vulgare L cv. Kikaihadaka) by extraction with 1% sodium chloride, fractionation with ammonium sulfate, chromatography on CM- and DEAE-cellulose columns, and gel filtration on Sephadex G-100. The final preparation appeared to be homogeneous on the basis of polyacrylamide gel electrophoresis; the inhibitory activity against subtilisin was increased about 140-fold during purification. This inhibitor was protein having a molecular weight of about 20,000, and containing 177 amino acid residues. Both the amino- and carboxyl-terminal residues were alaine. The inhibitor inactivated subtilisin, probably for formation of an enzyme-inhibitor complex in a molar ratio of 1: 1, but had little or no effect on the activities of other enzymes tested. The dissociation constant of the subtilisin-inhibitor complex was 1.5 X 10(-10) M. The inhibitor appears to be distinct from the barley microbial proteinase isoinhibitors reported by Mikola and Suolinna, in respect of most of its physiochemical and inhibitory properties.

Studies on trypsin inhibitor in barley. I. Purification and some properties.

To clarify the properties and functions of a trypsin inhibitor from Japanese barley in comparison with the inhibitor from Pirkka barley, an inhibitor was isolated from the barley Hordeum distichum L var. emend Lamark by extraction with 1% NaCl, ammonium sulfate fractionation and repeated chromatography on DEAE-cellulose and CM-cellulose. The final purified preparation of the inhibitor was found to be homogeneous by both chromatographic and electrophoretic analysis. The inhibitor was thermostable and was stable over the broad pH range from 2 to 11. No inhibition was observed by heavy metal ions and many reagents at 10(-2) M, except that p-chloromercuribenzoate caused a 69% loss of activity. The inhibitor was subjected to isoelectric focusing at pH 7.51 and its molecular weight was calculated to be 14,200+/-900 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent dissociation constant for the complex between the inhibitor and trypsin[EC 3.4.21.4] was 1.64 X 10(-7)M with casein as a substrate. One microgram of purified inhibitor inhibited 1.5 mug of pure trypsin in the hydrolysis of alpha-N-benzoyl-DL-arginine-p-nitroanilide. By chemical modification of arginyl residues in the inhibitor with 1,2-cyclohexanedione, the inhibitor was shown to be an arginine inhibitor. The inhibitor contained relatively many basic amino acids and few half cystines as compared with Pirkka barley trypsin inhibitor.

Leucine transfer RNA from barley. Characterization and purification of isoaccepting species.

Barley embryo leucine tRNA separated on reversed-phase chromatography-5 (RPC-5) into 5 fractions, whereas tRNA isolated from barley seedlings grown both in the light and in the dark, contained 4 species of tRNALeu. Species 2 and 3 were predominant; their relative ratio changed depending upon the growth conditions of the seedlings. Fractionation of crude barley tRNA successively on BD-cellulose, RPC-5 and Sepharose 4B enabled preparative isolation and purification of four leucine isoaccepting tRNAs. The species isolated differed in their main nucleotide composition, melting profiles and MgCl2 titration curves.

The amino acid sequence of ferredoxin from Hordeum vulgare.

Ferredoxin from barley consists of a single polypeptide chain of 97 amino acids, four of which are cysteine. These residues, which bind the iron atoms of the active centre, are in identical positions to those of other ferredoxins. The primary structure shows considerable similarity with other plant ferredoxins.

Examination of Chinese and U.S.S.R. cereals for the Fusarium mycotoxins, nivalenol, deoxynivalenol and zearalenone.

Cereals, foods and feeds sampled in Taiwan, China and the U.S.S.R. were contaminated with nivalenol, deoxynivalenol and zearalenone. The frequencies and levels of contamination are similar to those observed in the cereals of Japan and Korea. This is the first report on the natural occurrence of nivalenol, deoxynivalenol and zearalenone in Chinese and U.S.S.R. cereals, foods and feeds.

Comprehensive evaluation of fatty acids in foods. VI. Cereal products.

Information on total lipid and fatty acid composition or cereal grains and their products used for food has been collated in a comprehensive search of world literature published since 1960. Data considered most suitable for use for representing contents of total lipids and fatty acids have been tabulated and are presented. In developing these data, attention was given to stability and other chemical properties of the lipids, lipid composition of the different parts of the grain, and to such factors as genetics, production, processing, and analytical methods used in extracting and determining the fat and fatty acids in cereal products.

The sequence statistics and solution conformation of a barley (1----3, 1----4)-beta-D-glucan.

The sequence statistics and aqueous solution conformation of the 40 degrees water-soluble (1----3,1----4)-beta-D-glucan isolated from barley (Hordeum vulgare) have been modeled realistically using the known sequence-distributions of (1----3) and (1----4) linkages, theoretical conformational analysis, and the statistical mechanical theory of polymer-chain conformation. This barley beta-glucan fraction consists of (1----4)-beta-glucooligosaccharides, predominantly of d.p. 4 or less, joined by single beta-(1----3) linkages. Approximate treatments of the sequence statistics which do not take into account the small mole fraction (approximately 2%) of (1----4)-beta-glucooligosaccharides of d.p. approximately 10 significantly underestimate the chain extension in solution. A correct prediction of the observed chain extension is achieved when these longer, highly extended (1----4)-beta-glucooligosaccharide blocks are included in a model which randomly incorporates all (1----4)-beta-glucooligosaccharide segments in the proportions observed experimentally. Chain flexibility in the 40 degrees water-soluble beta-glucan fraction is shown to arise principally from the isolated beta-(1----3) linkages; blocks of two or more contiguous beta-(1----3) linkages provide a source of additional flexibility which may influence the properties of barley beta-glucan fractions containing a significant proportion of such sequences.

The regional distribution of selenium in Greek cereals.

The selenium content of hard and soft wheat, barley, oats, rye and corn grown in approximately 100 different locations of Greece has been determined fluorimetrically. The mean values +/- SD for these cereal types were 0.29 +/- 0.19, 0.21 +/- 0.12, 0.16 +/- 0.10, 0.14 +/- 0.10, 0.19 +/- 0.10 and 0.12 +/- 0.08 ppm Se (dry weight basis), respectively. Based on data for selenium in corn from 96 different locations, a geobotanic map of Greece for the selenium in soil available for uptake by plants was prepared. Macedonia, West Epirus, south-east Thessaly, north-east Sterea Hellas and the Aegean Islands produce corn deficient or low in selenium, but only sporadic selenium-deficiency diseases in animals have been observed in many of these areas, probably because the farm animals are given mixed food or they are free to graze. No areas with toxic levels of selenium in soil were found.

Soil to plant transfer of 239 + 240Pu, 238Pu, 241Am, 137Cs and 90Sr from global fallout in flour and bran from wheat, rye, barley and oats, as obtained by field measurements.

Crops of wheat, rye, barley and oats were grown on fields where the contamination of the soil with radionuclides resulted exclusively from global fallout debris. After machine harvesting and milling, the concentrations of 239 + 240Pu, 241Am, 137Cs and 90Sr were determined separately in bran and flour, as well as in the soils. The concentrations of 239 + 240Pu in wheat, rye and barley flour were between 59 and 180 microBq kg-1, and in oat flour 1100 microBq kg-1. The range of concentrations of the other radionuclides for the four flours were: 241Am, 5-70 microBq kg-1, 137Cs, 260-380 mBq kg-1; 90Sr, 310-1300 mBq kg-1. The corresponding concentrations of the radionuclides were always considerably higher in the bran than in the flour, with the exception of oats. The range of this factor depends on the cereal: for 239 + 240Pu, 19-25; 241Am, 10-38; 137Cs, 4-6; and for 90Sr, 4-7. Similar differences between flour and bran were observed for stable elements, for example K, Ca and Fe. The soil-to-plant transfer factors of 239 + 240Pu for wheat, rye and barley flour were between 0.00026 and 0.00078 (oats 0.017) and for bran between 0.0048 and 0.02 (oats 0.014). For 241Am the corresponding values were somewhat lower. For the wheat, rye and barley flour the transfer factors of 137Cs were between 0.013 and 0.018 (oats 0.069) and, for the bran, between 0.08 and 0.10 (oats 0.16). The corresponding values for 90Sr were significantly higher: for flour between 0.08 and 0.14 (oats 1.2) and for bran between 0.62 and 0.93 (oats 1.5).

Some properties of large and small granules of waxy barley (Hordeum vulgare L.) endosperm starch.

Large and small starch granules were isolated and characterized from mature barley kernels with waxy endosperms. The large granules of any given waxy cultivar contained more amylose than the small granules of the same cultivar. It was also found that large granules contained a greater amount of long amylopectin B chains and had a lower fraction III:fraction II ratio, one of the structural characteristics of amylopectin, than the small granules in the same cultivar. Small granules showed a wider range of gelatinization and smaller heat of gelatinization by differential scanning calorimetry.