Computational protocol: Honey Bee Allatostatins Target Galanin/Somatostatin-Like Receptors and Modulate Learning: A Conserved Function?

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Protocol publication

[…] Protein sequence data from the improved honey bee genome assembly (Amel 4.5 []) were screened using BLAST (available at http://www.ncbi.nlm.nih.gov) for putative Apime-ASTA- and Apime-ASTC-like receptor sequences using sequences of known receptor genes for Drosophila melanogaster allatostatins A (known as DAR1, CG2872 and DAR2, CG10001) and C (Drostar1, CG7285, Drostar2 CG13702, CG14919). Two putative AST-receptor protein sequences were identified, GB43574 and GB55818 (formerly referred to as GB19021 and GB20155 respectively in the previous genome assembly, and predicted as allatostatin receptors []). We used the Splign Alignment Tool (available at http://www.ncbi.nlm.nih.gov) to detect introns in the genomic DNA sequences of these two putative AST-receptor genes, and BLAST to look for possible splice variants. We aligned the amino-acid sequences with Multiple Alignment using Fast Fourier Transform (MAFFT []), and used Phobius (http://www.phobius.sbc.su.se), tmap and toppred (both available at http://mobyle.pasteur.fr) to predict the trans-membrane domains. The consensus of the three methods was chosen and aligned to the published sequences of human opioid and somatostatin receptors (see for example []), as these receptors are the closest homologs to the Drosophila allatostatin receptors [–], and have been studied in great detail. We also used a prediction tool for intracellular coupling of the putative AST-receptor proteins to G proteins (http://bioinformatics.biol.uoa.gr/PRED-COUPLE), based on the algorithm used by []. To predict phosphorylation sites, we used NetPhosK Server []. [...] The alignment and the phylogenetic tree were done using either the Clustal Omega [] or the MUSCLE software. Both methods gave the same phylogenetic tree, and the tree shown here was built using the Phylogeny.fr package []. After alignment, ambiguous regions (i. e. containing gaps and/or poorly aligned) were removed with Gblocks (v0.91b) using the following parameters: minimum length of a block after gap cleaning = 10, no gap positions were allowed in the final alignment, all segments with contiguous non-conserved positions bigger than 8 were rejected, minimum number of sequences for a flank position = 85%. The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.0). The default substitution model was selected assuming an estimated proportion of invariant sites and 4 gamma-distributed rate categories to account for rate heterogeneity across sites. The gamma shape parameter was estimated directly from the data. Reliability for internal branch was assessed using the bootstrapping method (199 bootstrap replicates) and the bootstrap value is indicated on each branch. Urotensin-2 receptor (UR2R) was chosen as an out-group for somatostatin and galanin receptors (SSTR and GALR) based on the phylogenetic tree described in []. Phylogenetic analysis with another out-group receptor, CXCR-5, which is more distant than UR2R, did not change the results. The accession number of the proteins are: NP_444474.1 (KISS1R), NP_032108.1 (GALR1), NP_034384.3 (GALR2), NP_033242.1 (SSTR1), NP_033243.2 (SSTR2), NP_033244.2 (SSTR3), NP_033245.2 (SSTR4), NP_035555.1 (SSTR5), NP_660114.1 (MCHR1), NP_034472.1 (NPBWR1), NP_663415.1 (UR2R); fruit fly: NP_524700 (ASTAR1), NP_524544 (ASTAR2), NP_649040.2 (Drostar1), NP_649039.4 (Drostar2). […]

Pipeline specifications

Software tools Clustal Omega, MUSCLE, Phylogeny.fr, Gblocks, PhyML
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Apis mellifera
Chemicals Somatostatin