The AP1 and AP2 primers supplied by the manufacturer The touchdo

The AP1 and AP2 primers supplied by the manufacturer. The touchdown and nested PCR parameters

Veliparib used were those described previously [60]. DNA sequencing and analysis All sequencing reactions for the ssg-2 gene were conducted using the ABI PRISM™ 377 automated DNA sequencer (Applied Biosystems) and the Thermo Sequenase II Dye terminator Cycle Sequencing Premix Kit (Amersham Biosciences) as described previously [19]. Sequencing of the sspla 2 gene products was done commercially using the SeqWright sequencing service (Fisher Scientific, Houston, TX, USA) Bioinformatics Sequence Analysis The theoretical molecular weights were calculated using the on-line ExPASy tool http://​www.​expasy.​ch/​tools/​. On-line Prosite Scan (Proscan Search) search was used to identify potential motifs RGFP966 present in SSG-2 and Entospletinib research buy SSPLA2 http://​npsa-pbil.​ibcp.​fr/​cgi-bin/​npsa_​automat.​pl?​page=​npsa_​prosite.​html[45].

The protein classification was performed using the PANTHER Gene and Protein Classification System http://​www.​pantherdb.​org[40] and on-line Blocks Analysis Server http://​blocks.​fhcrc.​org/​blocks/​blocks_​search.​html[37]. The calmodulin-binding domain was identified using the on line Calmodulin Target Database http://​calcium.​uhnres.​utoronto.​ca/​ctdb/​ctdb/​sequence.​html[44]. On-line database searches and comparisons for SSG-2 were performed using the Integrated Protein Classification (iProClass) database [61] and its BLAST algorithm implementation with a cutoff of 10-7, a low complexity filter and the Blosum 62 matrix. The iProClass/UniProt accession numbers of the sequences used for the multiple sequence alignment of G protein subunits were: S. schenckii (SSG-2), Q8TF91; M. grisea (MAGA), O13314; C. parasitica (CPG2), Q00581; N. crassa (GNA3) Q9HFW7; R. necatrix (WGA1/RGA1), Q9HFA3; E. nidulans (GANB), Q9UVK8, and S. schenckii (SSG-1), O74259. On-line database searches and comparisons for SSPLA2 were performed with the BLAST Rho algorithm http://​www.​ncbi.​nlm.​nih.​gov/​BLAST/​

with a cutoff of 10-7, a low complexity filter and the BLOSUM 62 matrix [39]. The Pfam analysis was done on-line using the using the Wellcome Trust Sanger Institute server http://​pfam.​sanger.​ac.​uk/​[42]. The GenBank accession numbers for the multiple sequence alignment of phospholipases were: A. nidulans (PLA2), XP_663815; S. schenckii (SSPLA2), ACJ04517.1; M. grisea (hypothetical protein), XP_363597; N. crassa (PLA2), XP_962511; C. globosum (hypothetical protein) XP_001223932; P. anserina (hypothetical protein) XP_001909265, and G. zeae (PLA2), XP_382145. Multiple sequence alignments were built using MCOFFEE http://​www.​igs.​cnrs-mrs.​fr/​Tcoffee/​tcoffee_​cgi/​index.

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