Genetic and biochemical analysis of common wheat cultivars lacking puroindoline a.

Puroindoline a (Pin-a) and puroindoline b (Pin-b), two basic isoforms encoded by the Pina-D1 and Pinb-D1 loci respectively, involved in controlling grain texture in wheat, were isolated from starch granules of soft wheat cultivars using three different extraction procedures, and fractionated by acidic polyacrylamide gel electrophoresis (A-PAGE). Tris buffer containing 1% Triton X-114 extracted Pin-a and small amounts of Pin-b, whereas 1% SDS preferably extracted Pin-b. Large amounts of both puroindolines were isolated by a solution containing 50% propan-2-ol and 50 mM NaCl. This solution extracted reduced amounts of Pin-b and no traces of Pin-a from starch granules of 20 hard common wheats containing the null allele Pina-D1b. The absence of Pin-a was confirmed by immunostaining with an anti-Pin-a antiserum. With the exception of two cultivars, null Pin-a cultivars gave no PCR fragment with three primer pairs specific to either the coding region or the promoter region of Pina-D1a, suggesting that major changes had occurred at the Pina-D1 locus in these genotypes. Cultivars Fortuna and Glenman were unique in giving size-specific PCR fragments with all primer pairs for the allele Pina-D1a and showed a cytosine deletion at position 267 in the coding region of the Pin-a gene, which resulted in a TGA stop codon at position 361. However, there was no evidence of a mutated protein in the A-PAGE or SDS-PAGE patterns of Fortuna and Glenman. The novel gene, provisionally named Pina-D1c, is the first null allele due to a point mutation that has been identified at the Pina-D1 locus.

An attempt of specific desensitising treatment with gliadin in celiac disease.

Gluten-free diet is the current treatment of celiac disease. We decided to verify the occurrence of histological and serological modification and/or clinical manifestations during a gradual and progressive introduction of gliadin in the diet and if it may induce a tolerance to food, as it occurs in allergic patients. We studied the case of a celiac woman with complete clinical and histological remittance after 10 years of gluten free diet. She took increasing daily doses of gliadin, reaching the final dose of 9 g of gliadin (15 g of gluten) in 6 months. Then she started a free dietary regimen. During the 15-month follow-up period esophago-gastro-duodenoscopy showed normal Kerckring folds and villi. Anti-gliadin, anti-endomysium and anti-tissue-transglutaminase antibodies, as well as the haematological and biochemical parameters remained normal. Our results represent a new approach in research concerning celiac disease, and could provide a future line of study for its management.

Genetic improvement of plant for coeliac disease.

Wheat, rye and barley are toxic for patients with coeliac disease. Toxicity has been found to result, respectively, from proteins such as gliadins, secalins and hordeins. Agglutination of in vitro cultured human myelogenous leukaemia K 562 (S) cells proved to be a suitable model for detection of toxic components of proteins. Five toxic peptides derived from an A-gliadin protein have been found to agglutinate the K 565 (S) cells. Triticum monococcum is a diploid wheat species widely grown during the Bronze Age. Proteins from monococcum are unable to agglutinate the K 562 (S) cells.

Agglutinating activity of alcohol-soluble proteins from quinoa seed flour in celiac disease.

The edible seeds of the quinoa plant contain small quantities of alcohol-soluble protein which, after peptic-tryptic digestion, are unable to agglutinate K562(s) cells. When separated by affinity chromatography on sepharose-6B coupled with mannan, peptic-tryptic digest separated in two fractions. Fraction B peptides (about 1% of total protein) were shown to agglutinate K562(s) cells at a very low concentration, whereas peptides in fraction A and in the mixed fraction A+B were inactive, suggesting that fraction A contains protective peptides that interfere with the agglutinating activity of toxic peptides in fraction B.

In vitro assessment of acetic-acid-soluble proteins (glutenin) toxicity in celiac disease.

Acetic-acid-soluble storage proteins from gluten of the bread wheat cv. Sprint 3 were fractionated by adsorption chromatography on 2000 A controlled-pore glass (CPG) beads, and glutenin polymers with molecular mass higher than 10(7) Da and free from monomeric gliadins were recovered. The glutenin polymers were found to consist of high-molecular-weight (HMW) and low-molecular-weight (LMW) glutenin subunits. Peptic-tryptic (PT) digests of glutenins were examined for their agglutination activity on human myelogenous leukemia K 562(S) cells, agglutination being strongly correlated with toxicity for the celiac intestine. The peptide fraction at a concentration of 1 g/L of culture medium was able to agglutinate 30% of K 562(S) cells, suggesting a moderate toxic effect. This toxicity may be accounted for by homologies in amino acid sequences between glutenin subunits and alpha/beta- and gamma-gliadins.

In vitro toxicity testing of alcohol-soluble proteins from diploid wheat Triticum monococcum in celiac disease.

Peptic-tryptic digests of alcohol-soluble proteins from flours of 10 accessions of Trificum monococcum with contrasting storage protein compositions and bread-making characteristics were found unable to agglutinate K562(S) cells even at a peptide concentration as high as 14 g/L, agglutination being strongly correlated with toxicity in celiac disease. When fractionated by affinity chromatography on Sepharose-6B coupled with mannan, peptic-tryptic digests separated into three fractions. Fraction C peptides were shown to agglutinate K562(S) cells, whereas peptides in fractions A and B and in the mixed fraction B + C were inactive, suggesting that fraction B contains "protective" peptides that interfere with toxic peptides in fraction C in their agglutinating activity. These results offer an opportunity to study the biochemical and genetic bases of wheat toxicity at the diploid level. Moreover, the reduced toxicity, if any, of Triticum monococcum in the celiac disease, along with the good grain characteristics of some "monococcum" accessions, greatly increases the economical prospects of this wheat species.

Production and genetic characterization of near-isogenic lines in the bread-wheat cultivar Alpe.

Two biotypes of the bread-wheat cultivar Alpe were shown to possess contrasting alleles at each of the glutenin (Glu-B1, Glu-D1, Glu-B3 and Glu-D3) and gliadin (Gli-B1 and Gli-D1) loci on chromosomes 1B and 1D. Fourteen near-isogenic lines (NILs) were produced by crossing these biotypes and used to determine the genetic control of both low-molecular-weight (LMW) glutenin subunits and gliadins by means of one-dimensional or two-dimensional electrophoresis. Genes coding for the B, C and D groups of EMW subunits were found to be inherited in clusters tightly linked with those controlling gliadins. Southern-blot analysis of total genomic DNAs hybridized to a γ-gliadin-specific cDNA clone revealed that seven NILs lack both the Gli-D1 and Glu-D3 loci on chromosome 1D. Segregation data indicated that these "null" alleles are normally inherited. Comparison of the "null" NILs with those possessing allele b at the Glu-D3 locus showed one B subunit, seven C subunits and two D subunits, as fractionated by two-dimensional A-PAGExSDS-PAGE, to be encoded by this allele. Alleles b and k at Glu-B3 were found to code for two C subunits plus eight and six B subunits respectively, whereas alleles b and k at Gli-B1 each controlled the synthesis of two ß-gliadins, one γ and two ω-gliadins. The novel Gli-B5 locus coding for two ω-gliadins was shown to recombine with the Gli-B1 locus on chromosome 1B. The two-dimensional map of glutenin subunits showed α-gliadins encoded at the Gli-A2 locus on chromosome 6A. The use of Alpe NILs in the study of the individual and combined effects of glutenin subunits on dough properties is discussed.

Gliadin alleles in Canadian western red spring wheat cultivars: use of two different procedures of acid polyacrylamide gel electrophoresis for gliadin separation.

Gliadin allele compositions of 21 Canadian spring common wheat cultivars, most of which belong to the Canada western red spring (CWRS) class, were studied and great similarity in their genotypes was confirmed. It was found that alleles frequent in the set of Canadian wheats (such as Gli-B1d, Gli-D1j, Gli-A2m, and Gli-D2h) are very rare or absent in common wheat cultivars from other regions and countries studied earlier, indicating that germplasm of CWRS cultivars is rather unique. It may be suggested that alleles frequent in Canadian cultivars relate to important technological characteristics of these wheats and may possibly serve as marker genes during selection for quality traits. Similarity of gliadin electrophoregrams obtained by two different acid polyacryl-amide gel electrophoretic procedures for the same genotype was established, and the component composition of allelic variants of blocks of gliadin components found in the set of Canadian cultivars and in standard cultivars Chinese Spring and Bezostaya 1 are described.

Recombination mapping of Gli-5, a new gliadin-coding locus on chromosomes 1A and 1B in common wheat.

Inheritance studies of gliadin loci on chromosomes 1A and 1B were carried out in the progeny from crosses between cv "Salmone" and six other common wheat varieties. The map distance between the Rg-1 locus for glume colour and the gliadin locus Gli-B1 on the satellite of chromosome 1B was calculated as 2.0±0.6 cM. An additional gliadin locus, Gli-B5, was mapped between Gli-B1 and Rg-1, 1.4 cM from the former. A genetic distance of 1.8±0.4 cM was obtained between the Hg-1 locus for hairy glumes and a gliadin locus that seems to be remote from Gli-A1 and homoeologous to Gli-B5. Statistically significant differences in recombination values were found in the six crosses, indicating the influence of genotype on the frequency of recombination. The similarity in chromosomal location of seed storage protein genes in wheat, barley and rye is discussed.

Genetics of gliadins coded by the group 1 chromosomes in the high-quality bread wheat cultivar Neepawa.

The inheritance and biochemical properties of gliadins controlled by the group 1 chromosomes of the high-quality bread wheat cultivar Neepawa were studied in the progeny of the cross Neepawa x Costantino by six different electrophoretic procedures. Chromosome 1B of Neepawa contains two gliadin loci, one (Gli-B1) coding for at least six ω- or γ-gliadins, the other (Gli-B3) controlling the synthesis of gliadin N6 only. The map distance between these loci was calculated as 22.1 cM. Amongst the chromosome 1A gliadins, three proteins are encoded at the Gli-A1 locus whereas polypeptides N14-N15-N16 are controlled by a remote locus which recombines with Gli-A1. Six other gliadins are controlled by a gene cluster at Gli-D1 on chromosome 1D. Canadian wheat cultivars sharing the Gli-B1 allele of Neepawa were found to differ in the presence or absence of gliadin N6. The electrophoretic mobilities of proteins N6 and N14-N15-N16 were unaffected by the addition of a reducing agent during two-dimensional sodium dodecyl sulphate polyacrylamid-gel electrophoresis, suggesting the absence of intra-chain disulphide bonds in their structure.

Preparative isoelectric focusing of reduced wheat gluten proteins.

Proteins extracted from gluten of the bread wheat cultivar Fiorello 2 in the presence of 2-mercaptoethanol or dithiothreitol were separated by isoelectric focusing in a free solution in a pH 3-10 gradient containing 50% v/v 1-propanol or urea. The collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels (high and medium molecular weight glutenin subunits) and 16% gels (low molecular weight gliadins). The isoelectric focusing pattern of gluten polypeptides in 50% v/v 1-propanol was comparable to that obtained on two-dimensional gel electrophoresis, based on isoelectric focusing and polyacrylamide gel electrophoresis or nonequilibrium pH gradient electrophoresis and polyacrylamide gel electrophoresis. A similar isoelectric focusing pattern was also observed when 3M urea was used as solvent. New gluten polypeptides, similar in mobility to the high molecular weight subunits of glutenin were detected at acidic pH.

Early and late heat shock proteins in wheats and other cereal species.

Coleoptiles and roots of 3-day-old seedlings from five cereal species (Triticum aestivum L., T. durum Desf., Hordeum vulgare L., Secale cereale L., and Triticale) respond to heat shock at 40 degrees C by synthesizing a new set of 13 strong bands (as revealed by one-dimensional sodium dodecyl sulfate gel electrophoresis) as well as some 20 degrees C proteins. Heat shock proteins (HSPs) belong to three different size groups: high molecular mass HSPs in the 103 to 70 kilodalton range, intermediate molecular mass HSPs in the 62 to 32 kilodalton range, and low molecular mass HSPs about 17 to 16 kilodalton in size. At the beginning of the heat shock coleoptiles show a reduced ability to synthesize intermediate molecular mass HSPs but after 4 hours at 40 degrees C they exhibit fully developed HSP patterns identical to that found in roots. Synthesis of early HSPs declines after 7 hours of treatment followed by the appearance of a new set of 12 protein bands (late HSPs) in the ranges 99 to 83, 69 to 35, and 15 to 14 kilodaltons. After 12 hours at 40 degrees C, three other late HSPs of 89, 45, and 38 kilodalton are induced. The induction of late HSPs after 7 hours at 40 degrees C appears to be associated with an enhancement of radioactive methionine incorporation into proteins.

Distribution and clustering of two highly repeated sequences in the A and B chromosomes of maize.

Clones from a family of highly repeated sequences present in a heterochromatin rich maize line have been characterized by sequencing and chromosome location. The repeats differ from each other in length and degree of sequence homology, and show areas rich in purine and pyrimidine. In "situ" hybridization experiments indicate that the repeats are mainly located in the knob heterochromatin of the A chromosomes and the centromeric heterochromatin of the B chromosome. However, in addition to previously published data, some copies are also distributed in euchromatic regions of the A chromosomes and in the distal heterochromatic block of the B chromosome. The results are discussed in relation to the centromeric activity of maize heterochromatin.

Multiplicity and diversity of cloned zein cDNA sequences and their chromosomal localisation.

We have constructed and screened cDNA libraries from total maize endosperm poly(A) RNA or from a mRNA fraction enriched in zein sequences. From these libraries we have isolated clones representative of the major classes of zein cDNA sequences and have characterised them by crosshybridisation, by hybrid-selected translation, by in situ hybridisation to maize chromosomes, and hybridisation to genomic Southern blots. We conclude that at least four types of non cross-hybridising zein sequences are present, two coding for light chains and two for heavy chains. At least in the case of the light zeins, there is considerable sequence diversity among the clones which hybridise to each type. Similar results are obtained by translation of the mRNAs selected by each clone. In situ hybridisation shows that the light chain zein genes are located on chromosomes 4, 7, and 10, whilst genes coding for some of the heavy chain zeins are confined to the distal part of the long arm of chromosome 4.