Nigrosine staining of wheat endosperm proteolipid patterns on starch gels

We have previously characterized a group of proteolipids from wheat endosperm, designated CM proteins, which are soluble in chloroformmethanol (2:1, v/v) and have a molecular weight lower than 25,000 daltons (1-3). These have been also studied by Redman and Ewart (4). The CM proteins are suitably fractionated into several components by starch gel electrophoresis at pH 3.2 (1). A sensitive staining procedure was required in connection with genetic studies of these proteins because phenotypes had to be ascertained in small endosperm fractions dissected without impairing normal germination and plant development. We report here on Nigrosine staining conditions for CM proteins under which high sensitivity and selectivity are achieve

Heterogeneity of wheat endosperm proteolipids (CM proteins)

Proteins extracted with CHCl3-MeOH from wheat endosperm have been fractionated by Sephadex G-100 and the 15 000–20 000 MW range fraction, designated CM protein, has been examined by combined electrofocusing (pH range 5–8) and electrophoresis (pH 3.2) and the heterogeneity of the electrophoretic components has been ascertained. It has been shown by joint mapping and by sequential extraction that CM proteins are extracted by 70% EtOH but not by H2O, although they can be made water-soluble after dialysis against an acid buffer, pH 3.2, 3 M urea, without losing their solubility in CHCl3-MeOH mixtures. It is concluded that CM proteins fit the definition of a Folch—Lees proteolipid. The Triticum aestivum (genomes ABD) map can be reconstructed by mixing T. durum (AB) and Aegilops squarrosa (D). The low intragenomic variability of CM protein is confirmed

Loss of redundant gene expression after polyploidization in plants

Based on chromosomal location data of genes encoding 28 biochemical systems in allohexaploid wheat,Triticum aestivum L. (genomes AABBDD), it is concluded that the proportions of systems controlled by triplicate, duplicate, and single loci are 57%, 25%, and 18% respectively

Genetics of CM-proteins (A-hordeins) in barley

The CM-proteins, which are the main components of the A-hordeins, include four previously described proteins (CMa-1, CMb-1, CMc-1, CMd-1), plus a new one, CMe-1, which has been tentatively included in this group on the basis of its solubility properties and electrophoretic mobility. The variability of the five proteins has been investigated among 38 Hordeum vulgare cultivars and 17 H. spontaneum accessions. Proteins CMa-1, CMc-1 and CMd-1 were invariant within the cultivated species; CMd was also invariant in the wild one. The inheritance of variants CMb-1/CMb-2 and CMe-1/CMe-2,2 was studied in a cross H. spontaneum x H. vulgare. The first two proteins were inherited as codominantly expressed allelic variations of a single mendelian gene. Components CMe-2,2 were jointly inherited and codominantly expressed with respect to CMe-1. Gene CMb and gene(s) CMe were found to be unlinked. The chromosomal locations of genes encoding CM-proteins were investigated using wheat-barley addition lines. Genes CMa and CMc were associated with chromosome 1, and genes CMb and CMd with chromosome 4. These gene locations further support the proposed homoeology of chromosomes 1 and 4 of barley with chromosomes groups 7 and 4 of wheat, respectively. Gene(s) CMe has been assigned to chromosome 3 of barley. The accumulation of protein CMe-1 is totally blocked in the high lysine mutant Riso 1508 and partially so in the high lysine barley Hiproly

HETEROGENEITY OF WHEAT ENDOSPERM PROTEOLIPIDS (CM PROTEINS)

Abstract-Proteins extracted with CHCl 3 -MeOH from wheat endosperm have been fractionated by Sephadex G-100 and the 15 000-20 000 MW range fraction, designated CM protein, has been examined by combined electrofocusing (pH range 5-8) and electrophoresis (pH 3-2) and the heterogeneity of the electrophoretic components has been ascertained. It has been shown by joint mapping and by sequential extraction that CM proteins are extracted by 70% EtOH but not by H 2 0, although they can be made water-soluble after dialysis against an acid buffer, pH 3-2, 3 M urea, without losing their solubility in CHCl 3 -MeOH mixtures. It isconcluded that CM proteins fit the definition of a Folch-Lees proteolipid. The Triticum aestivum (genomes ABD) map can be reconstructed by mixing T. durum (AB) and Aegilops squarrosa (D). The low intragenomic variability of CM protein is confirmed

Chromosomal control of non-gliadin proteins from the 70% ethanol extract of wheat endosperm

The non-gliadin fraction of the 70% ethanol extracts of compensated nulli-tetrasomics and ditelosomics of Triticum aestivum cv. Chinese Spring has been analyzed by combined electrofocusing and electrophoresis. Seventeen of the 21 protein map components of the euploid have been ascribed to eight chromosomes: 4A, 3BS, 6BS, 7BS, 3D, 4D, 5D and 7DS. The relationship of the different map components with other proteins previously associated with the same chromosomes is discusse

Evaluation of monocytes as carriers for armed oncolytic adenoviruses in murine and Syrian hamster models of cancer

Replication-competent (oncolytic) adenoviruses (OAV) can be adapted as vectors for the delivery of therapeutic genes, with the aim of extending the antitumor effect beyond direct cytolysis. Transgene expression using these vectors is usually intense but short-lived, and repeated administrations are hampered by the rapid appearance of neutralizing antibodies (NAbs). We have studied the performance of monocytes as cell carriers to improve transgene expression in cancer models established in athymic mice and immunocompetent Syrian hamsters. Human and hamster monocytic cell lines (MonoMac6 and HM-1, respectively) were loaded with replication-competent adenovirus-expressing luciferase. Intravenous administration of these cells caused a modest increase in transgene expression in tumor xenografts, but this effect was virtually lost in hamsters. In contrast, intratumoral administration of HM-1 cells allowed repeated cycles of expression and achieved partial protection from NAbs in preimmunized hamsters bearing pancreatic tumors. To explore the therapeutic potential of this approach, HM-1 cells were loaded with a hypoxia-inducible OAV expressing the immunostimulatory cytokine interleukin-12 (IL-12). Three cycles of treatment achieved a significant antitumor effect in the hamster model, and transgene expression was detected following each administration, in contrast with the rapid neutralization of the free virus. We propose monocytes as carriers for multiple intratumoral administrations of armed OAVs

Biochemical markers associated with two Mv chromosomes from Aegilops ventricosa in wheat-Aegilops addition lines

The distribution of three biochemical markers, U-1, CM-4 and Aphv-a, -b, among wheat-Aegilops addition lines carrying Mv chromosomes from Aegilops ventricosa (genomes DvMv) has been investigated. Addition lines which had been previously grouped together on the basis of common non-biochemical characters carried marker U-1, a protein component from the 2M urea extract. The added chromosome, in the appropriate genetic background, seems to confer a high level of resistance to the eyespot disease, caused by the fungus Cercosporella herpotrichoides. The other two markers were concomitantly associated with another similarly formed group of addition lines. Both CM-4, a protein component from the chloroform:methanol extract, and Aphv-a, -b, alkaline phosphate isozymes, have been previously shown to be associated with homoeologous chromosome group 4, which suggests that the added chromosome in the second group of addition lines is 4Mv

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/-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