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List of 290 FLNC reads showing cross-contig mapping. (XLS 40 kb

Phylogenetic tree of Triticeae γ-type prolamins compared to a <i>T. monococcum</i> cDNA.

<p>Derived amino acid sequences, minus repetitive domains (see text), were aligned with Clustal V. The root of the tree was using an α-gliadin as an outgroup. Genbank accession numbers identify each sequence. Classifications of related genes are shown with brackets and names on the right of the figure.</p

A New Class of Wheat Gliadin Genes and Proteins

<div><p>The utility of mining DNA sequence data to understand the structure and expression of cereal prolamin genes is demonstrated by the identification of a new class of wheat prolamins. This previously unrecognized wheat prolamin class, given the name δ-gliadins, is the most direct ortholog of barley γ3-hordeins. Phylogenetic analysis shows that the orthologous δ-gliadins and γ3-hordeins form a distinct prolamin branch that existed separate from the γ-gliadins and γ-hordeins in an ancestral Triticeae prior to the branching of wheat and barley. The expressed δ-gliadins are encoded by a single gene in each of the hexaploid wheat genomes. This single δ-gliadin/γ3-hordein ortholog may be a general feature of the Triticeae tribe since examination of ESTs from three barley cultivars also confirms a single γ3-hordein gene. Analysis of ESTs and cDNAs shows that the genes are expressed in at least five hexaploid wheat cultivars in addition to diploids <em>Triticum monococcum</em> and <em>Aegilops tauschii</em>. The latter two sequences also allow assignment of the δ-gliadin genes to the A and D genomes, respectively, with the third sequence type assumed to be from the B genome. Two wheat cultivars for which there are sufficient ESTs show different patterns of expression, i.e., with cv Chinese Spring expressing the genes from the A and B genomes, while cv Recital has ESTs from the A and D genomes. Genomic sequences of Chinese Spring show that the D genome gene is inactivated by tandem premature stop codons. A fourth δ-gliadin sequence occurs in the D genome of both Chinese Spring and <em>Ae. tauschii</em>, but no ESTs match this sequence and limited genomic sequences indicates a pseudogene containing frame shifts and premature stop codons. Sequencing of BACs covering a 3 Mb region from <em>Ae. tauschii</em> locates the δ-gliadin gene to the complex <em>Gli-1</em> plus <em>Glu-3</em> region on chromosome 1.</p> </div

Prolamin repetitive domains.

<p>The repetitive domains of several Triticeae prolamins are shown with repeats arrayed vertically. The sequences from which the repetitive domains originate are identified by Genbank accession numbers and membership in different prolamin classes. Lines connecting repeat motifs in δD and δA indicate conserved repeats. Underlined repeats in δD are repeat differences with δA. Cysteine residues are boxed. The arrow indicates the repeat motif that is FPQQM in wheat hexaploid cv Recital, but FP.M in cv Chinese Spring.</p

Alignment of δ-gliadins with γ-type prolamins.

<p>The derived amino acid sequences for δ-gliadins and barley γ3-hordeins are aligned with wheat, barley, and rye γ-prolamins, but without the repetitive domains (position of the repetitive domain indicated by the arrowhead). The vertical order is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052139#pone-0052139-g003" target="_blank">Figure 3</a>. Residue positions common to all aligned sequences are shaded with yellow. Residue positions shared only by the upper or lower initial branches of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052139#pone-0052139-g003" target="_blank">Figure 3</a> are shaded with blue. Horizontal lines separate sequences from the two initial branches of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052139#pone-0052139-g003" target="_blank">Figure 3</a>. Accession numbers of previously reported sequences are given on the right, and classifications by prolamin type is given to the left. Cysteine residues are shaded in green and conserved cysteine positions are indicated by asterisks below the alignments. Domains similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052139#pone-0052139-g001" target="_blank">Figure 1</a> are indicated below the alignments.</p

General Structure of the wheat prolamins.

<p>The general structure of the wheat prolamin classes is diagrammed showing the main sequence domains, conserved cysteine residues (S), and intramolecular disulfide bonds (lines connecting Ss). The signal peptides (SIG) are shaded. The mature polypeptide sequence of the α- and γ-gliadins and LMW-glutenins are composed of five sections: (I) a short non-repetitive peptide, (II) the repetitive domain composed of variations of short motifs, (III) a non-repetitive region containing most of the cysteine residues, (IV) a glutamine-rich domain, and (V) the C-terminal non-repetitive domain containing at least one cysteine residue. The ω-gliadins usually have no cysteines and therefore no disulfide bonds. The disulfide bonds are taken from references: α-gliadins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052139#pone.0052139-Mller1" target="_blank">[15]</a>, γ-gliadins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052139#pone.0052139-Mller2" target="_blank">[16]</a>, and the HMW- and LMW-glutenins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052139#pone.0052139-Keck1" target="_blank">[17]</a>.</p

Phylogenetic tree of γ-type Triticeae prolamins and δ-gliadins.

<p>Derived amino acid sequences are aligned with Clustal V as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052139#pone-0052139-g001" target="_blank">Figure 1</a>. Genbank accession numbers identify each sequence. Classifications of related genes are shown with brackets and names on the right of the figure. The root of the tree was using an α-gliadin sequence as an outgroup. Branches containing sequences from only one Triticeae species are shown by capital letters on or near those branches: wheat (W), barley (B), rye (R).</p

δ-gliadin gene chromosome location.

<p>A 3.1 Mb region of the <i>Ae. tauschii</i> 1D chromosome covered by 28 overlapping BACs in two contigs was sequenced as indicated by the horizontal line. Prolamin and closely related AAI (alpha-amylase-inhibitor) genes are indicated above the line with colors identifying prolamin types. Longer colored lines indicate apparently intake reading frames while shorter colored lines indicate pseudogenes or gene fragments. Annotated non-prolamin genes are indicated by black vertical lines below the region sequence. Longer black vertical lines indicate syntenic genes among the known grass genomes.</p

Image_1_Proteomic characteristics of saliva in patients with different subgroups of IgG4-RD.jpeg

BackgroundImmunoglobulin G4-related disease (IgG4-RD) is a newly defined disease entity, with great heterogeneity among IgG4-RD subgroups with different organ involvement patterns. Identification of the proteomic characteristics of IgG4-RD subgroups will be critical for the understanding of the pathogenic mechanisms of IgG4-RD.MethodIn this study, we performed proteomic analysis using Tandem Mass Tags (TMT) technology with “high field” mass analyzer with improved resolution and sequencing speed to investigate the proteomic profile of saliva and plasma samples from ten untreated IgG4-RD patients and five healthy controls (HCs). Differentially expressed proteins (DEPs) were identified by “t test” function in R package. Functional enrichment analysis was used to investigate pathways enriched in IgG4-RD samples.ResultsMost salivary DEPs identified in IgG4-RD patients compared with HCs were mainly enriched in neutrophil mediated GO bioprocess. Within the comparisons between four IgG4-RD subgroups, more DEPs were identified in the comparison of Mikulicz group and Head and neck group. Among four subgroups of IgG4-RD, Head and neck group showed the most distinctive proteomic expression pattern when compared with HCs. Moreover, “Neutrophil mediated process” related GO bioprocess was commonly identified between comparisons of Mikulicz group and Head and neck group, Head and neck group and Retroperitoneal aorta group, Head and neck group and HCs, IgG4-RD patients with saliva gland involvement and those without saliva gland involvement. Key DEPs that involved in this GO bioprocess were identified. Besides, we performed proteomic analysis for plasma samples between ten IgG4-RD and five HCs and there were several DEPs identified overlapped in saliva and plasma.ConclusionWe identified multiple processes/factors and several signaling pathways in saliva that may be involved in the IgG4-RD pathogenesis.</p

Table_4_Proteomic characteristics of saliva in patients with different subgroups of IgG4-RD.xlsx

BackgroundImmunoglobulin G4-related disease (IgG4-RD) is a newly defined disease entity, with great heterogeneity among IgG4-RD subgroups with different organ involvement patterns. Identification of the proteomic characteristics of IgG4-RD subgroups will be critical for the understanding of the pathogenic mechanisms of IgG4-RD.MethodIn this study, we performed proteomic analysis using Tandem Mass Tags (TMT) technology with “high field” mass analyzer with improved resolution and sequencing speed to investigate the proteomic profile of saliva and plasma samples from ten untreated IgG4-RD patients and five healthy controls (HCs). Differentially expressed proteins (DEPs) were identified by “t test” function in R package. Functional enrichment analysis was used to investigate pathways enriched in IgG4-RD samples.ResultsMost salivary DEPs identified in IgG4-RD patients compared with HCs were mainly enriched in neutrophil mediated GO bioprocess. Within the comparisons between four IgG4-RD subgroups, more DEPs were identified in the comparison of Mikulicz group and Head and neck group. Among four subgroups of IgG4-RD, Head and neck group showed the most distinctive proteomic expression pattern when compared with HCs. Moreover, “Neutrophil mediated process” related GO bioprocess was commonly identified between comparisons of Mikulicz group and Head and neck group, Head and neck group and Retroperitoneal aorta group, Head and neck group and HCs, IgG4-RD patients with saliva gland involvement and those without saliva gland involvement. Key DEPs that involved in this GO bioprocess were identified. Besides, we performed proteomic analysis for plasma samples between ten IgG4-RD and five HCs and there were several DEPs identified overlapped in saliva and plasma.ConclusionWe identified multiple processes/factors and several signaling pathways in saliva that may be involved in the IgG4-RD pathogenesis.</p