Computational protocol: G Protein Activation without a GEF in the Plant Kingdom

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Protocol publication

[…] The sequences of G protein signaling components were found using BLASTp (E value<0.1) against protein database and the translated BLAST (tBLASTn, E value<0.1) against genomic DNA sequences registered in Phytozome v7.0 (released on Apr/8/2011; by using A. thaliana genes as queries. Full-length or partial DNA sequences of Gα, Gβ, Gγ, and 7TM-RGS for Triticum aestivum, Hordeum vulgare, P. taeda, Picea glauca, and M.polymorpha were identified with tBLASTn in the nucleotide collection (nr/nt) database or the expressed sequence tags (EST) database at National Center for Biotechnology Information (NCBI) or the species-specific EST database (E value<10). The partial DNA sequences were combined to determine the full cDNA sequences. G protein components of P. dactylifera were found using assembled-gene sequences downloaded from Weill Cornell Medical College in Qatar ( 7TM-RGS gene of P. dactylifera was assembled manually. Gα genes of P. taeda and P. glauca were cloned from the cDNA libraries and their sequences were determined, because information from the databases was insufficient to define the full length sequence. To screen all the possible RGS-like genes, P. dactylifera RGS and S. italica RGS and RGS domain sequences from H. sapiens RGS4, G protein-coupled receptor kinase,, LARG and sorting nexin 13 were also used as query sequences (E value<10). [...] Phylogenetic trees were constructed with MEGA5.0 . Full length Gα, Gβ and 7TM-RGS protein sequences were aligned with ClustalW using the following parameters; Gap opening penalty and gap extension penalty for initial pairwise alignment, 10 and 0.1; Gap opening penalty and gap extension penalty for multiple alignment, 10 and 0.2; Gonnet protein weight matrix; Residue-specific penalties, ON; Hydrophilic penalties, ON; Gap separation distance, 4; End gap separation, OFF; Use negative matrix, OFF. The maximum likelihood (ML) trees were made using the Complete-Deletion option of gaps and the JTT (Jones-Taylor-Thornton) substitution model with gamma distributed rate variation, which was estimated as the best-fitting substitution model using MEGA5.0. The consensus phylogenetic trees were shown with the bootstrap values from 1000 repetitions. Homo sapiens Gαi1, Gαq, Gβ1 and Gβ5 were included as out groups. […]

Pipeline specifications

Software tools BLASTP, TBLASTN, MEGA, Clustal W
Applications Phylogenetics, Amino acid sequence alignment
Organisms Arabidopsis thaliana
Chemicals Amino Acids, Guanine Nucleotides, Guanosine Triphosphate