Protein deposition in developing barley endosperm

The storage proteins of barley are both qualitatively and quantitatively important in determining the nutritional quality of the grain and its technological uses. The development of the barley endosperm was followed from anthesis to grain maturity by light and electron microscopy. The deposition of storage proteins in the sub-aleurone and starchy endosperm was studied using conventional electron microscopy. Correlated studies using thick sections of zinc iodide-osmium tetroxide impregnated tissue have illustrated the three-dimensional interrelationships of the endoplasmic reticulum, Golgi apparatus and vacuoles during protein deposition. To provide a clearer understanding of protein deposition in the wild-type barley, protein body formation was also investigated in mutant barley lines where a reduced and modified synthesis of storage protein is associated with an altered protein body structure. Immunocytochemical localization of A, B, and C hordeins and the chymotrypsin inhibitors, CI-1 and CI-2, primary antibodies raised in rabbit and subsequently labelled with protein A-goId illustrated the storage protein in accumulating reserves in protein bodies of the wild-type barley, and the high-lysine mutant lines. Storage proteins were localized only in specific regions of both cytoplasmic and vacuolar protein deposits. A comparison of specimen preparation techniques including different fixation and embedding protocols indicated that for barley endosperm ,tissue post-fixed with osmium tetroxide and embedded in Spurr resin gave superior results to those embedded in LR White or Lowicryl K4M resin.In situ hybridization was used to locate mRNA for CI-1 and CI-2 chymotrypsin inhibitors in barley endosperm using a biotinylated cDNA probe. The probe was localized at an ultra structural level by incubation with avidin-peroxidase and subsequent DAB staining of the peroxidase activity. The combined approach of thin- and thick- sectioning techniques for electron microscopy, in association with the molecular techniques of immunocytochemistry and in situ hybridization, has led to the development of a new model to illustrate the course of protein body development in barley endosperm. This new model also explains those previously published results used to support apparently contradictory earlier schemes

Detectable Clonal Mosaicism from Birth to Old Age and its Relationship to Cancer

Clonal mosaicism for large chromosomal anomalies (duplications, deletions and uniparental disomy) was detected using SNP microarray data from over 50,000 subjects recruited for genome-wide association studies. This detection method requires a relatively high frequency of cells (>5–10%) with the same abnormal karyotype (presumably of clonal origin) in the presence of normal cells. The frequency of detectable clonal mosaicism in peripheral blood is low (<0.5%) from birth until 50 years of age, after which it rises rapidly to 2–3% in the elderly. Many of the mosaic anomalies are characteristic of those found in hematological cancers and identify common deleted regions that pinpoint the locations of genes previously associated with hematological cancers. Although only 3% of subjects with detectable clonal mosaicism had any record of hematological cancer prior to DNA sampling, those without a prior diagnosis have an estimated 10-fold higher risk of a subsequent hematological cancer (95% confidence interval = 6–18)

Influence of sporophore development, damage, storage, and tissue specificity on the enzymic formation of volatiles in mushrooms (Agaricus bisporus)

The enzymic oxidation of the polyunsaturated fatty acid-linoleic acid leads, in fungi, to the formation of a unique class of nonconjugated hydroperoxides, which are cleaved to form eight-carbon volatiles characteristic of mushroom and fungal flavor. However, the enzymes involved in this biosynthetic pathway, the bioavailability of the fatty acid substrate, and the occurrence of the reaction products (hydroperoxides and eight-carbon volatiles) are not fully understood. This study investigated the lipids, fatty acids, and hydroperoxide levels, as well as eight-carbon volatile variations in the fungal model Agaricus bisporus, according to four parameters: sporophore development, postharvest storage, tissue type, and damage. Eight-carbon volatiles were measured using solid phase microextraction and gas chromatography-mass spectrometry. Tissue disruption had a major impact on the volatile profile, both qualitatively and quantitatively; 3-octanone was identified as the main eight-carbon volatile in whole and sliced sporophore, an observation overlooked in previous studies due to the use of tissue disruption and solvent extraction for analysis. Fatty acid oxidation and eight-carbon volatile emissions decreased with sporophore development and storage, and differed according to tissue type. The release of 1-octen-3-of and 3-octanone by incubation of sporophore tissue homogenate with free linoleic acid was inhibited by acetylsalicylic acid, providing evidence for the involvement of a heme-dioxygenase in eight-carbon volatile production

Genome organization and transcription response to harvest of two Metallothionein-like genes in agaricus bisporus fruiting bodies

Metallothioneins are a class of small cysteine-rich proteins that have been associated with increased tolerance to metal and oxidative stresses in animals, plants, and fungi. We investigated a metallothionein-like (mt-like) gene shown previously to be upregulated in fruiting bodies of the fungus Agaricus bisporus in response to post-harvest storage. Analysis of an A. bisporus genomic DNA cosmid library identified two similar nit-like genes (men and met2) arranged as a bidirectional gene pair transcribed from the same promoter region. The promoter contained regulatory elements including 9 metal responsive elements and a CAAT box region 220 bp downstream of met1 that showed striking similarity to a feature in Coprinopsis cinerea mt-like gene promoters. Transcriptional analysis showed that both met genes are significantly and rapidly (within 3 hours) upregulated during post-harvest storage and expression is significantly greater in stipe and cap tissues compared with the gills. However, a strong directionality of the promoter was demonstrated, as transcript levels of met1 were at least two orders of magnitude greater than those of met2 in all samples teste

Hairpin-mediated down-regulation of the urea cycle enzyme argininosuccinate lyase in Agaricus bisporus

A double-stranded (ds) RNA hairpin-mediated down-regulation system was developed for the cultivated mushroom Agaricus bisporus, and the role of the urea cycle enzyme argininosuccinate lyase (asl) in mushroom post-harvest development was investigated. Hairpin expression vectors were constructed to initiate down-regulation of as] and introduced into A. bisporus by Agrobacterium tumefaciens-mediated transformation. Transcripts of asl were significantly reduced (93.1 and 99.9%) in two transformants and hairpin vector transgene sequences were maintained throughout sporophore development. Single and multiple hairpin integration events were observed in Southern analysis. Transformants with down-regulated asl exhibited reduced yield and cap expansion during post-harvest sporophore development. There were no detectable differences in urea levels between the hairpin-transformed and control strains. This is the first report of reduced gene expression resulting from the introduction of dsRNA hairpins in A. bisporus and the applications of this technology will facilitate functional studies in the mushroom. (C) 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved