Computational protocol: Enter the Dragon: The Dynamic and Multifunctional Evolution of Anguimorpha Lizard Venoms

Similar protocols

Protocol publication

[…] The rat ileum muscle preparations were isolated from adult male rats. The rats were euthanised by CO2 asphyxiation. The isolated preparations were individually mounted in 15 mL parallel organ baths containing a Krebs solution with the following constituents (mM): NaCl, 118.4; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25 and glucose, 11.1. The Krebs solution was continuously bubbled with carbogen (95% O2 and 5% CO2) to maintain a pH between 7.2–7.4 at a temperature of 32–34 °C. A resting tension between 1 and 3 g was found to be the optimal starting baseline. Stimulation was performed with 50 μg/mL of crude venom; deionised H2O (170 μL) was used as a control. The venom was left in the organ bath with the preparation for approximately 30 min or until the twitch response was completely abolished. Studies were conducted under University of Queensland Animal Ethics Committee approval # SBMS/310/12 (30 March 2012). [...] A phylogeny was assembled using previous genetic studies [,,] and was used for all further analyses conducted in R version 3.2.5 [] using the ape package [] for general handling of phylogenetic and trait data. Ancestral states were estimated and reconstructed over the tree in order to investigate the evolutionary history of the traits and consequently their relation to one another over time. The continuous functional traits were reconstructed by maximum likelihood in the contMap function in phytools []. We then fit PGLS models [] in caper [] to test for relationships. [...] Kallikrein, lysosomal acid lipase and chitinase datasets were analysed using Bayesian inference implemented on MrBayes, version 3.2.1 using lset rates = invgamma with prset aamodelpr = mixed, which enables the program to optimize between nine different amino acid substitution matrices. The analysis was performed by running a minimum of 10 million generations in four chains, and saving every 100th tree. The log-likelihood score of each saved tree was plotted against the number of generations to establish the point at which the log likelihood scores reached their asymptote, and the posterior probabilities for clades established by constructing a majority-rule consensus tree for all trees generated after completion of the burn-in phase. [...] Publicly available kallikrein sequences were retrieved from GenBank by using Varanus komodoensis kallikrein sequence as a query for a BLAST search within Anguimorpha [,,]. Sequences with less than 70% coverage were discarded. Sequences were edited to only include the codons for the mature protein using AliView and aligned by using AliView’s “Realign everything as translated amino acids” to translate the codons, align the resulting amino acids using MUSCLE, and reverse translating them [,]. MrBayes version 3.2 was used to create a phylogenetic tree of the sequences for performing the later selection analyses []. Using the closest sequence similarity reptile venom structure (GU441485) as input, the Phyre2 webserver generated a custom protein structure based on the published structure (PDB ID: 3S9C) []. This structure has a resolution of 1.8 Å and was 41% identical with the query sequence and with conservation of the cysteine residues. Protein models were rendered in UCSF Chimera version 1.10.2 []. Conservation scores were calculated using the UCSF Chimera implementation of AL2CO under the default settings [,]. Tests for selection were performed using HyPhy version 2.220150316: overall dN/dS value was calculated using the AnalyzeCodonData method, persistent site-by-site selection was analyzed with the FUBAR method, and episodic site-by-site selection was analyzed with the MEME method [,,]. MEME is used for identifying sites that experience episodic selection pressures, where as FUBAR is improvement on site-wide selection analysis []. […]

Pipeline specifications