Computational protocol: New Data on the Presence of Hemocyanin in Plecoptera: Recomposing a Puzzle

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Protocol publication

[…] Two different multiple alignments of the proteins belonging to the hemocyanin superfamily (HcSF) were performed: the first one only for Plecoptera sequences, and the second for sequences of Plecoptera and other groups of arthropods.Multiple alignment: Plecoptera. From our cDNA and from the Genbank database, sequences were deduced in 29 stonefly species of 14 hemocyanins (6 of the subunit 1 (hc1) and 8 of the subunit 2 (hc2)) and 27 hexamerins. lists the sequences used for the alignment. Six Myriapoda hemocyanin sequences (i.e., Scutigera coleoptrata AJ344359, AJ344360, AJ431378, AJ431379, AJ512793 and Spirostreptus sp. AJ297738) were used in the alignment, since Myriapoda are in an ancestral position with respect to Plecoptera (). The final alignment included 48 sequences, 520 nucleotides, and 154 amino acids positions.Multiple alignment: Arthropod HcSF. The alignment of Plecoptera sequences was completed with others sequences of the arthropod hemocyanin superfamily, retrieved from the GenBank database. The alignment was composed of crustacean prophenoloxidases (PPO), insect prophenoloxidases (PPO), crustacean cryptocyanins (CC) or pseudohemocyanins (Phc), crustacean hemocyanins (hc), Myriapoda hemocyanins (hc), insect hemocyanins (hc), and insect hexamerins (hx).The hexamerin receptors were ignored in this study because only a small part of the sequences aligned well with the hemocyanin conserved region we analyzed. A list of sequences for the non—Plecopteran taxa used in this study is provided in . The final alignment comprises 102 sequences, 785 nucleotides, and 161 amino acid positions.Sequence alignment and phylogenetic inference. Multiple alignment of nucleotides and amino acid sequences was constructed with the MAFFT online version () matrix BLOSUM62. Long gap regions, as well as some highly divergent regions, were removed from the final data set, and in order to optimize the results of the phylogenetic analysis, we operated a selection of conserved blocks from multiple alignments with Gblocks server (). The appropriate phylogenetic model for nucletidic sequences was selected with MrModeltest2 (). The amino acid sequence evolution model was chosen by ProtTest () using the Akaike information criterion. Nucleotidic tree constructions were performed by Bayesian analysis with MrBayes 3.1–2 (GTR model). The reliability of the trees was tested by bootstrap analysis () with 1000 replications. Amino acid trees were inferred by Maximum Likelihood (ML) methods. The phylogenetic analyses were performed with PhyML ( () with100 replications. Distances between pairs of protein sequences were calculated according to the LG model () assuming a gamma distribution of substitution rate. […]

Pipeline specifications

Software tools MAFFT, Gblocks, MrModelTest, ProtTest, MrBayes, PhyML
Application Phylogenetics
Chemicals Hemocyanin