3′r: 5′-GGGCACCAGATGAACGACGC or Chi3.3′r: 5′-ACTAACATACACAACGAATGCGC for CHI2 and CHI3, respectively). The matching fragment size between cDNA and respective DNA sequences shown by agarose gel electrophoresis, and the identity of genomic and cDNA sequences identified by a primer-walking strategy (data
not shown), were considered as experimental demonstration for the absence of intronic sequences within CHI2 and CHI3 genes. In silico analysis of amino acid sequences deduced from CHI2 and CHI3 Multiple matching subsegments in two protein sequences were identified with the LALIGN program http://www.ch.embnet.org/software/LALIGN_form.html implementing the algorithm of Huang & Miller [71]. The theoretical isoelectric points for the protein sequences were calculated using the Protein Isoelectric Point menu within the Sequence Manipulation Suite [72]. The presence
CP-690550 chemical structure and location of signal peptide cleavage sites in the amino acid sequences of CHI2 and CHI3 were predicted with the SignalP 3.0 Server http://www.cbs.dtu.dk/services/SignalP;”"[73]). Protein phosphorylation at serine, threonine or tyrosine residues was predicted with the NetPhos 2.0 Server [74]. AZD0156 mw Putative sites for amidation, N-myristoylation and cell attachment were identified by a protein pattern www.selleckchem.com/products/ly2835219.html search against the Prosite database http://www.expasy.org/prosite/; [75]). O-, N-, and C-glycosylated sites were predicted with EnsembleGly – a web server for prediction of O-, N-, and C-linked glycosylation sites with ensemble learning [39]. Transcript quantification by real-time reverse transcription PCR (qRT-PCR) Propagules of the strain Gb04 were grown in PG1 medium for three days, washed in fresh medium for 2 min and transferred to another portion of fresh medium (time point 0).
Twelve, 24, 36, 48 or 72 hours later the mycelium was shortly washed with distilled water, quick-frozen in liquid nitrogen and stored at -80°C. RNA was isolated from three independent samples grown per time point. For quantification of transcript mass expressed from the chitinase genes CHI2 and CHI3 as well as the endogenous about positive control NDUFV1, sense strand transcript standards were generated by in vitro transcription from a PCR product template tailed with the T7 phage promoter sequence. In more detail, for template construction a minimum sequence of 19 bases (5′-TAATACGACTCACTATAGG) required for efficient transcription was selected out of the 23 nt T7 phage promoter sequence and added to the 5′ end of the respective PCR primer. In vitro transcription was performed with the RNAMaxx™ High Yield Transcription Kit (Stratagene, Amsterdam, The Netherlands) according to the manufacturer’s instructions.