With MAFFT (v. 6) configured for highest accuracy (MAFFT with E-INS-i …

With MAFFT (v. 6) configured for highest accuracy (MAFFT with E-INS-i option). Prior to the phylogenetic analysis, signal peptide sequences and other N and C terminal extentions peculiar to individual taxa were excluded. In total 337 and 249 characters were used for GHF5 endoglucanase and pectate lyase respectively for phylogenetic anlaysis. The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.0 aLRT). The JTT substitution model was selected assuming an estimated proportion of invariant sites (of 0.018) and Staurosporine 4 gamma-distributed rate categories to account for rate heterogeneity across sites. The gamma shape parameter was estimated directly from the data (gamma = 1.603). Reliability for internal branch was assessed using the aLRT test (SH-Like). Graphical representation and edition of the phylogenetic tree were performed with TreeDyn (v198.3). In situ hybridisation In situ hybridisation was performed as described previously[75]. PCR products were generated from the plasmid stocks of the cDNA clones of Aa-eng-1 and Aa-pel-1 using gene specific primers (Table 5). Sense or antisense stra nds were labelled with digoxigenin by asymmetric PCR and hybridised to fixed, permeabilised fragments of mixed stage A. avenae. After washing to remove unbound probe, specifically hybridising probe was detected using Alkaline-phosphatase conjugated Anti-Digoxigenin antibody and NBT/BCIP stock solution (Roche Diagnostics). Specimens were examined with differential interference microscopy (Nikon).Cm s gDm g sFigure 9 gland cells ofandavenae bytranscripts in the esophageal Localization of Aa-eng-1 A. Aa-pel-1 in situ hybridisation Localization of Aa-eng-1 and Aa-pel-1 transcripts in the esophageal gland cells of A. avenae by in situ hybridisation. Nematode sections were hybridized with antisense (A) and sense (B) Aa-eng-1 digoxigenin-labeled cDNA probes. Hybridisation was also carried out with digoxigenin-labeled antisense (C) and sense (D) Aa-pel-1 cDNA probes. G, esophageal glands; S, stylet; M, metacorpus. The bar indicates a length of 20 m.defined as the representation of a single gene by multiple non-overlapping clusters, was estimated by comparing A. avenae cluster sequences with C. elegans [39].Gene ontology (GO) Cluster sequences were classified into Gene Ontology functional categories [54] based on BLAST similarities to known genes in the NCBI Swiss-Prot protein sequence (Swiss-Prot) and using the BLAST2GO annotation tool [55,56] with an E-value cut-off of 1e-05 and summarized according to their biological processes, molecular functions and cellular components. To obtain the complete GO mapping, a node sequence filter in the GO graph was used 50 for biological process, 20 for molecular function, and 20 for the cellular component. The multi-level pie charts were generated using sequence cut-offs of 140, 50 and 40 for biological process, molecular function and cellular components respectively. Identification of genes encoding cell-wall-degrading enzymes and sequence analyses BLASTX searches against the GenBank database were used to identify A. avenae clusters encoding potential cell-walldegrading enzymes. To obtain full PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 length sequences of genes, the plasmid clones from which each of these sequences were obtained were identified and resequenced both directions using designed primers (Table 5) in order to obtain the full-length cDNA sequences. The genomic coding region of each cDNA clone was obta.