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.