Computational protocol: The Red Imported Fire Ant (Solenopsis invicta Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor

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Protocol publication

[…] The cDNA sequence obtained was analyzed with the Lasergene software (DNASTAR, Madison, WI, USA) and the prepropeptide open reading frame (ORF) was predicted by the “ORF finder” tool in the same. In addition, the initiation site (AUG) was also confirmed through analysis using ATGpr (atgpr.dbcls.jp) that identified the Kozak consensus sequence. Detection of poly (A) signal sequences by the poly(A) signal miner http://dnafsminer.bic.nus.edu.sg/PolyA.html . The BLAST search algorithms at NCBI were used to identify the sNPF prepropeptide sequences from other insect species and alignment of the S. invicta sNPF prepropeptide to those was with ClustalW included in Megaline (Lasergene, DNASTAR). The sNPF prepropeptide sequence was analyzed with the prediction server (cbs.dtu.dk/services/SignalIP/) to determine the signal peptide sequence. Subsequently, to determine the first cleavage site for the active peptide sequence, a sequence of two basic amino acids (RK) was localized by eye-gazing in the propeptide because these are the common cleavage sites in insect neuroendocrine peptides . However, Southey et al. have also identified ‘R’ in addition to ‘RK’ as a potential cleavage site. The sNPF sequence has an ‘R’ residue downstream of RK, which could also be a first cleavage site. To determine the C-terminal sequence the alignment to other peptides and the orthologous neuropeptide of Apis mellifera sequence were also compared to the S. invicta sNPF sequence. Based on these comparative analyses and the amidation signal at the C-terminus, the putative active peptides were predicted as SLRSALAAGHLRYa (sNPF1) or SALAAGHLRYa (sNPF2) (). The evolutionary relationships of the insect sNPF prepropeptide amino acid sequences were analyzed by MEGA (version 5.05) and neighbor-joining method with 1000 bootstrap replicates. The resulting tree was exported to Newick and then to FigTree 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) for creating the unrooted phylogenetic tree figure. […]

Pipeline specifications

Software tools Open Reading Frame Finder, ATGpr, DNAFSMiner, Clustal W, MEGA, FigTree
Applications Phylogenetics, Transcription analysis
Organisms Solenopsis invicta, Homo sapiens, Apis mellifera