GESA--a two-dimensional processing system using knowledge base techniques.

The successful analysis of two-dimensional (2-D) polyacrylamide electrophoresis gels demands considerable experience and understanding of the protein system under investigation as well as knowledge of the separation technique itself. The present work concerns the development of a computer system for analysing 2-D electrophoretic separations which incorporates concepts derived from artificial intelligence research such that non-experts can use the technique as a diagnostic or identification tool. Automatic analysis of 2-D gel separations has proved to be extremely difficult using statistical methods. Non-reproducibility of gel separations is also difficult to overcome using automatic systems. However, the human eye is extremely good at recognising patterns in images, and human intervention in semi-automatic computer systems can reduce the computational complexities of fully automatic systems. Moreover, the expertise and understanding of an "expert" is invaluable in reducing system complexity if it can be encapsulated satisfactorily in an expert system. The combination of user-intervention in the computer system together with the encapsulation of expert knowledge characterises the present system. The domain within which the system has been developed is that of wheat grain storage proteins (gliadins) which exhibit polymorphism to such an extent that cultivars can be uniquely identified by their gliadin patterns. The system can be adapted to other domains where a range of polymorpic protein sub-units exist. In its generalised form, the system can also be used for comparing more complex 2-D gel electrophoretic separations.

Chromosomal location of genes coding for endosperm proteins of Hordeum chilense, determined by two-dimensional electrophoresis of wheat-H. chilense chromosome addition lines.

The proteins of Hordeum chilense grain were resolved into 25 major components by two-dimensional electrophoresis. Their solubilities in aqueous alcohol solutions were determined to distinguish prolamin storage proteins from metabolic and structural proteins. The prolamins were divided into two groups, based on the presence or absence of intermolecular disulfide bonds determined by gel-filtration chromatography. Using an incomplete set of Chinese Spring wheat-H. chilense disomic addition lines, the structural genes of 21 of the 26 most dominant seed proteins were assigned to chromosomes. The great majority of the prolamin genes, including those coding for a high molecular weight (HMW) prolamin subunit, was present on chromosome 1 Hch. However, a small number of prolamin genes also occurred on chromosomes 5 Hch and 7 Hch. A minor protein, probably belonging to the nonstorage group of proteins, is coded by genes on 5 Hch. Various ditelosomic addition lines and ditelosomic and disomic substitution lines for chromosome 7 Hch were also analyzed by electrophoresis. This technique revealed that the genes for three major prolamins occur on the beta arm of chromosome 7 Hch and that a gene for a minor protein, also thought to be a prolamin, occurs on the alpha arm. These results are discussed in relation to the evolution of prolamin genes in the Triticeae.

Wheat storage proteins: diversity of HMW glutenin subunits in wild emmer from Israel : 1. Geographical patterns and ecological predictability.

The diversity of HMW glutenin subunits in the tetraploid wild progenitor of wheat, Triticum turgidum var. dicoccoides was studied electrophoretically in 231 individuals representing 11 populations of wild emmer from Israel. The results show that (a) The two HMW glutenin loci, Glu-A1 and Glu-B1, are rich in variation, having 11 and 15 alleles, respectively, (b) Genetic variation in HMW glutenin subunits is often severely restricted in individual populations, supporting an island population genetic model, (c) Significant correlations were found between glutenin diversity and the frequencies of specific glutenin alleles and physical (climate and soil) and biotic (vegetation) variables. Our results suggest that: (a) at least part of the glutenin polymorphisms in wild emmer can be accounted for by environmental factors and (b) the endosperm of wild emmer contains many allelic variants of glutenin storage proteins that are not present in bread wheat and could be utilized in breeding varieties with improved bread-making qualities.

Electrophoretic analysis of the high-molecular-weight glutenin subunits of Triticum monococcum, T. urartu, and the A genome of bread wheat (T. aestivum).

The high molecular weight (HMW) subunit composition of glutenin was analysed by sodium dodecyl sulphate, polyacrylamide gel electrophoresis (SDS-PAGE) in the A genome of 497 diploid wheats and in 851 landraces of bread wheat. The material comprised 209 accessions of wild Triticum monococcum ssp. boeoticum from Greece, Turkey, Lebanon, Armenia, Iraq, and Iran; 132 accessions of the primitive domesticate T. monococcum ssp. monococcum from many different germplasm collections; one accession of free-threshing T. monococcum ssp. sinskajae; 155 accessions of wild T. urartu from Lebanon, Turkey, Armenia, Iraq, and Iran; and landraces of T. aestivum, mainly from the Mediterranean area and countries bordering on the Himalayan Mountains. Four novel HMW glutenin sub-units were discovered in the landraces of bread wheat, and the alleles that control them were designated Glu-Ald through Glu-Alg, respectively. The HMW subunits of T. monococcum ssp. boeoticum have a major, "x" subunit of slow mobility and several, less prominent, "y" subunits of greater mobility, all of which fall within the mobility range of HMW subunits reported for bread wheat. In T. monococcum ssp. monococcum the range of the banding patterns for HMW subunits was similar to that of ssp. boeoticum. However, two accessions, while containing "y" subunits were null for "x" subunits. The single accession of Triticum monococcum ssp. sinskajae had a banding pattern similar to that of most ssp. boeoticum and ssp. monococcum accessions. The HMW subunit banding patterns of T. urartu accessions were distinct from those of T. monococcum. All of them contained one major "x" and most contained one major "y" subunit. In the other accessions a "y" subunit was not expressed. The active genes for "y" subunits, if transferred to bread wheat, may be useful in improving bread-making quality.

Intrachromosomal mapping of the nucleolar organiser region relative to three marker loci on chromosome 1B of wheat (Triticum aestivum).

Restriction enzyme digestion of the ribosomal RNA genes of the nucleolar organisers of wheat has revealed fragment length polymorphisms for the nucleolar organiser on chromosome 1B and the nucleolar organiser on 6B. Variation between genotypes for these regions has also been demonstrated. This variation has been exploited to determine the recombination frequency between the physically defined nucleolar organiser on 1B (designatedNor1) and other markers; two loci,Glu-B1 andGli-B1 which code for endosperm storage proteins andRf3, a locus restoring fertility to male sterility conditioned byT. timopheevi cytoplasm.Gli-B1 andRf3 were located on the short-arm satellite but recombine with the nucleolar organiser giving a gene order ofNor1 - Rf3 - Gli-B1. Glu-B1 is located on the long arm of 1B but shows relatively little recombination withNor1, which is, in physical distance, distal on the short arm. This illustrates the discrepancy between map distance and physical distance on wheat chromosomes due to the distal localisation of chiasmata. The recombination betweenNor1 andRf3 indicates that, contrary to previous suggestions, fertility restoration is not a property of the nucleolar organiser but of a separate locus.

Development and characterisation of a line of bread wheat, Triticum aestivum, which lacks the short-arm satellite of chromosome 1B and the Gli-B1 locus.

About 360 offspring of a tri-parental cross were screened by gel electrophoresis and unexpectedly one of them did not contain chromosome 1B ω-gliadins derived from either of the primary parents. A line disomic for the ω-gliadin null was developed from the surviving embryo half of the unique grain. Two dimensional electrophoresis revealed that all the storage protein genes at Gli-B1, coding for γ-gliadins, ß-gliadins and low-molecular-weight subunits of glutenin as well as the ω-gliadin, were not expressed. The nuclei of dividing root-tip cells were shown by light microscopy to lack the normal short-arm satellites of chromosome 1B, indicating that the genes for the missing storage proteins had been lost through a terminal deletion. Using a radioactive ribosomal RNA probe, the deficient 1B chromosomes were shown to contain ribosomal RNA genes demonstrating that at least two-thirds of the short arm was still present. Examination of serial sections of chromosome 1B at metaphase by low-power electron microscopy showed that the point of scission of this chromosome was within the secondary constriction where the ribosomal RNA genes are located. The Gli-B1 locus must therefore be carried on the short-arm satellite. Transmission of the deficient chromosome from female gametes to progeny was normal (i.e., about 50%) but from pollen it was poor (8.8%). Recombination mapping indicated that the distance from the ribosomal RNA genes (Nor1) to Glu-B1 was 22 cM, equivalent to 13 cM from Nor1 to the centromere.

Genetic linkage between endosperm storage protein genes on each of the short arms of chromosomes 1A and 1B in wheat.

The storage proteins of the endosperm of wheat grain which are known to be controlled by genes on the short arms of the homoeologous group 1 chromosomes are (1) the ω-gliadins, (2) most of the γ-gliadins, (3) a few ß-gliadins and (4) the major lowmolecular-weight subunits of glutenin. Several crosses were made between varieties or genetic lines which had contrasting allelic variants for some of these proteins and which were coded by genes on chromosomes 1A or 1B. The progeny were analysed by one or more of several electrophoretic procedures. The results of all the analyses are consistent with the hypothesis that chromosomes 1A and 1B each contain just one, complex locus, named Gli-A 1 and Gli-B 1 respectively, which contain the genes for the ω-, γ- and ß-gliadins and the low-molecular-weight subunits of glutenin.

Control of lectins in Triticum aestivum and Aegilops umbellulata by homoeologous group 1 chromosomes.

Each of the three genomes in hexaploid wheat controls the expression of a specific lectin in the embryo. The chromosomes which control their synthesis were determined using nullisomic-tetrasomic and inter-varietal chromosome substitution lines of 'Chinese Spring'. All three wheat lectins were shown to be controlled by the homoeologous group 1 chromosomes. Using ditelosomic lines of 'Chinese Spring' the lectin genes could be localized on the long arms of chromosomes 1A and 1D. Inter-specific addition and substitution lines of Aegilops umbellulata chromosomes to 'Chinese Spring' indicated that chromosome 1U, which is homoeologous to the group 1 chromosomes of wheat, controls lectin synthesis.

The use of irradiated pollen for differential gene transfer in wheat (Triticum aestivum).

The use of irradiated pollen to bring about limited gene transfer in wheat has been investigated. Doses of X-rays of 2Kr, 3Kr and 5Kr were used to generate M1 progeny between maternal and paternal genotypes differing in quantitative and major gene characters. Cytological studies of M1 plants revealed hybrids with widespread aneuploidy and structural rearrangements in the paternal genome. These effects resulted in phenotypic variation between M1 progeny and complex multivalent formation at meiosis. All M1 plants at the 5Kr and 3Kr doses were sterile and all but 2 plants at the 2Kr dose.Studies of the two M2 families from these plants revealed disturbances in genotype frequencies for some of the marker loci with an excess of maternal homozygotes and a deficit of paternal homozygotes. This was also reflected in a more maternal appearance for quantitative characters. These results are interpreted as showing that irradiation damage to the paternal genome in M1 plants results in the differential transmission of maternal alleles.

Characterisation of high molecular weight gliadin and low-molecular-weight glutenin subunits of wheat endosperm by two-dimensional electrophoresis and the chromosomal localisation of their controlling genes.

Gliadins, here defined as those proteins of defatted wheat endosperm which dissolve in 70% (v/v) ethanol at room temperature, were fractionated by gel filtration using Sephadex G-100. The protein which eluted with the void volume of the column, often described as high-molecular-weight (HMW) gliadin, was fractionated by the two different, two dimensional gel electrophoresis procedures of O'Farrell (1975) and O'Farrell et al. (1977). The next two fractions to elute from the gel column, ω-gliadin and α-, ß-, γ-gliadin, were analysed similarly. The subunits of HMW gliadin and the classical (i.e. non-aggregated) gliadins map at distinctive positions on the electrophoregrams, the majority of the HMW gliadin subunits being more basic and having a slightly slower electrophoretic mobility than the α-, ß-, γ-gliadins. These experiments demonstrate that those gliadins which aggregate to form HMW gliadin are distinct molecular entities and thus coded by different genes to those gliadins which do not aggregate. Glutenin, here prepared by a modification of the pH 6.4 precipitation procedure of Orth and Bushuk (1973), was also analysed by two-dimensional electrophoresis. The low-molecular-weight subunits were found to correspond exactly with the HMW gliadin subunits. Using the nullisomic-tetrasomic lines and the ditelocentric lines of 'Chinese Spring', the genes controlling the synthesis of all the major HMW gliadin subunits were shown to be located on the short arms of chromosomes 1A, 1B and 1D, as are the genes coding for the ω-gliadins and the majority of the γ-gliadins.

Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin : Part 3. Telocentric mapping of the subunit genes on the long arms of the homoeologous group 1 chromosomes.

The genes controlling the synthesis of the high-molecular-weight subunits of glutenin on the long arms of chromosomes 1A and IB were mapped to the ω-gliadin genes on the short arms by analysing the progeny of three test crosses by sodium dodecyl sulphate, polyacrylamide-gel electrophoresis. Only very weak linkages were detected: the percentage recombination ranged from 39% to 47% and as the values did not significantly differ from each other, the data was pooled. A mean recombination of 43% was obtained and the map distance between glutenin and gliadin genes was calculated to be 66 cM. The analysis of three crosses involving telocentric lines revealed that the glutenin subunit genes on chromosomes 1A, IB and ID are tightly linked to the centromere, the mean map distance being 9.0 cM.

Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin : Part 1: Allelic variation in subunits amongst varieties of wheat (Triticum aestivum).

The high-molecular-weight (HMW) subunits of glutenin from about 185 varieties were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). About 20 different, major subunits were distinguished by this technique although each variety contained, with only a few exceptions, between 3 and 5 subunits. Further inter-varietal substitution lines to those already described (Payne et al. 1980) were analysed and the results indicate that all the HMW subunits are controlled by the homoeologous group 1 chromosomes. All hexaploid varieties studied except 'NapHal' contained two major subunits controlled by chromosome 1D. Their genes were shown to be tightly linked genetically for only four different types of banding patterns were observed. The nominal molecular weights determined after fractionation in 10% polyacrylamide gels were between 110,000 and 115,000 for the larger of the two subunits and between 82,000 and 84,000 for the smaller. One quarter of the varieties contained only one major HMW subunit controlled by chromosome 1B whereas the rest had two. The chromosome 1B subunits were the most varied and nine different banding patterns were detected. All the subunits had mobilities which were intermediate between those of the two chromosome 1D-controlled subunits. Only two types of HMW subunit controlled by chromosome 1A were detected in all the varieties examined; a single variety never contained both of these subunits and 40% of varieties contained neither. The chromosome 1A-controlled subunits had slightly slower mobilities in 10% gels than the largest HMW subunit controlled by chromosome 1D. About 100 single grains were analysed from each of five different crosses of the type (F1 of variety A × variety B) × variety C. The results indicate that the genes on chromosome 1B which control the synthesis of subunits 6, 7, 13, 14 and 17 are allelic, as are the genes of the chromosome 1A-controlled subunits, 1 and 2.

Control by homoeologous group 1 chromosomes of the high-molecular-weight subunits of glutenin, a major protein of wheat endosperm.

The electrophoretic mobilities of the high-molecular-weight (HMW) subunits of glutenin from 7 varieties were compared by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate (SDS). In total, 12 subunits were clearly resolved and they had nominal molecular weights of between 95,000 and 140,000. The chromosomes which control their synthesis were determined using monosomic lines and inter-varietal substitution lines. All subunits were shown to be controlled by the homoeologous group 1 chromosomes. Each variety contains between 3 and 5 HMW subunits; two are under the control of the 1D chromosome, 1 or 2 are controlled by chromosome 1B and 0 or 1 by chromosome 1A. The segregation of two 1D-controlled subunits of similar electrophoretic mobilities were analysed in the F2 progeny of crosses between 'Chinese Spring' and 'Holdfast'. The results suggest that the genes which code for the two proteins are allelic.

Identification of a high-molecular-weight subunit of glutenin whose presence correlates with bread-making quality in wheats of related pedigree.

The subunit composition of glutenin was analysed by SDS-polyacrylamide-gel electrophoresis using two varieties of contrasting pedigrees. 'Maris Widgeon', a variety of good bread-making quality, was shown to contain 2 glutenin subunits not present in 'Maris Ranger', a much higher yielding variety that is unsuitable for making bread. A third subunit was only found in 'Maris Ranger' glutenin. To determine if any of these subunits are directly related to bread-making quality, 60 randomly-derived F2 progeny from a 'Maris Widgeon' x 'Maris Ranger' cross were analysed for bread-making quality and for glutenin subunit composition. A strong correlation was demonstrated between the presence of one of the two subunits inherited from 'Maris Widgeon', and quality. This subunit (termed subunit 1 glutenin) had an approx. mol. wt. of 145,000. It was also found in 'Maris Freeman', a bread-making variety selected from the same cross previously made in 1962. In further crosses involving 'Maris Widgeon' or its descendants, more bread-making varieties have been produced in the last decade at the Plant Breeding Institute, Cambridge and all but one have inherited glutenin subunit 1. The subunit has been traced back through 'Holdfast' to 'White Fife', a Canadian hard spring wheat of excellent breadmaking quality. Some 67 varieties were screened for the presence of glutenin subunit 1 and it was found in 31% of them. Several unrelated varieties of good bread-making quality did not contain subunit 1 glutenin.

Subunit composition of wheat glutenin proteins, isolated by gel filtration in a dissociating medium.

Proteins were extracted from wheat meal or flour in 0.1 M acetic acid, 3 M urea and 0.01 M CTAB and fractionated in columns of cross-linked Sepharose in the same solvent. An heterogeneous fraction of high molecular weight eluted from the column which, when reduced and subjected to SDS-polyacrylamide-gel electrophoresis, separated into 12 components. Their molecular weights ranged from about 31,500 to 136,000. The unreduced protein was insoluble in salt solutions and aqueous ethanol but soluble in 0.1 M acetic acid and was therefore defined as glutenin. Glutenins of different molecular weight were made up from the same subunits but in different proportions. The ethanol-soluble proteins (gliadins) of the flour were fractionated in Sephadex G-100. The protein component that was excluded from the Sephadex gel, often described as high-molecular-weight gliadin, was shown to contain 8 distinguishable subunits and they had identical mobilities to 8 of the 12 subunits of glutenin.

Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.).

Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products have identical mobilities to the vicilin and legumin subunits by SDS-PAGE. Certain products were specifically immunoprecipitated with antisera prepared against purified vicilin and legumin fractions. Various RNA fractions including poly A-rich RNA have also been isolated from polyribosomes and shown to direct the synthesis of polyripeptides whose properties are similar to the storage protein subunits. The results are discussed in relationship to other investigations with seed storage protein biosynthesis in vitro.

Evidence for the nucleotide sequence of 5-S rRNA from the flowering plant Secale cereale (Rye).

Evidence for the sequence of rye 5-S rRNA was derived from the analysis of partial and complete enzymic digests of the 32P-labelled molecule. The probable sequence of 5-S rRNA from four other flowering plants was deduced by aligning, with the homologous sequences in the rye 5-S rRNA, oligonucleotides produced by T1 and pancreatic A ribonuclease digestion. The most dissimilar differed in only seven positions. From a comparison of the sequence of rye 5-S rRNA with those known for other types of organism, it was possible to distinguish some structural features of the molecule which are common to all of them. Also, information was obtained about the possible phylogenetic relationship of the flowering plants to other organisms whose 5-S rRNA has been sequenced.

In vitro translation of the long-lived messenger ribonucleic acid of dry seeds.

The total RNA extracted from rye embryos (Secale cereale) and seed of the broad bean (Vicia faba), pea (Pisum sativum) and oil-seed rape (Brassica napus) exhibits low levels of template activity when incubated in a wheat-germ cell-free protein-synthesising system. The RNA from pea, rapeseed and rye embryos was fractionated by chromatography on oligo dT-cellulose columns. Most of the template-active RNA bound to the column at high ionic strength, indicating that it is polyadenylated. The remainder would not bind, even when passed through the column several times. The proteins synthesised in vitro from the template-active RNA migrated as numerous bands in polyacrylamide gels and ranged in molecular weight from 10,000 to 70,000. The banding patterns obtained were quite different for the three species of seed tested. It is concluded that dry seeds contain a store of intact, long-lived mRNA.

Early ribonucleic acid synthesis during the germination of rye (Secale cereale) embryos and the relationship to early protein synthesis.

Incorporation studies with radioactive precursors showed that synthesis of protein and RNA is initiated in germinating embryos of rye within the first hour of imbibition of water. By polyacrylamide-gel fractionations of radioactive nucleic acid components, the appearance of products of transcription of the genome was shown to follow the sequence: heterogeneous (ribonuclease-sensitive) RNA, 4S and 5S RNA by 20min, 31S and 25S rRNA by 40min, and 18S RNA by 60min. "Fingerprint' analysis of T1-ribonuclease digests show that all the large oligonucleotides present in 25S and 18S RNA are present in the 31S species, indicating that 31S RNA is the precursor rRNA molecule to both 25S and 18S RNA. The importance of these early RNA syntheses and in particular the possible template function of the heterogeneous RNA is discussed in relation to the concept of long-lived mRNA and the coding for protein synthesis in the first hours of germination.