hin the Agouti 871700-17-3 supplier peptide evolution, the scenario is very improbable. We find that more likely, Agouti-like peptides, like most vertebrate gene families, were formed through classical subsequent gene duplications where the AgRP is likely to be the most ancestral, first splitting from a common ancestor to ASIP and A2 and then later the A2 split from ASIP followed by a split resulting in ASIP2 and AgRP2. The finding of a single copy of AgRP2 in spotted gar and double copies of A2 in European eel appear consistent with a 3R origin, but the position of the AgRP2 and ASIP2 genes outside linear synteny blocks on their respective TSGD-duplicated chromosomes in Medaka could suggest a random copying event into the TSGD chromosomal context. using the PHI pattern C-x-C-x-C-C-x-C-x-C-x-C-xC-x-C-x-C, and reporting sequences with the pattern at position 75 and E-value WORSE than the threshold. This is to allow for length variability in the last inter-cysteine segment, which has the length 8 in chicken, and the length 9 in human or mouse. Furthermore, we compared the 1,357 spider toxin sequences found in the ��Protein��database, with Atlantic cod ASIP2. 5. Phylogenetic Analysis of A1, A2, and Agouti-like Sequences A multiple sequence alignment was generated for the final set of AgRP and ASIP like sequences using MAFFT version 6 with the EINS_I version having default parameters. The alignments were inspected and edited using Jalview. The phylogenetic PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22202440 analysis was performed using a Bayesian approach as implemented in MrBayes version 3.1.2. Markov Chain Monte Carlo analysis was used to approximate the posterior probabilities of the trees. Analysis was run using a gamma shaped model for the variation of evolutionary rates across sites and the mixed option was used to estimate the best amino acid substitution model. Each analysis was set to run for 3,000,000 generations and every hundredth tree was sampled. A stop rule was 
applied to determine when to terminate the MCMC generations as recommended in the MrBayes manual. If the MCMC analysis does not hit the stop value within the default number of generations, additional generations were run for it to reach the minimum split frequencies. The first 25% of the sampled trees were discarded to reassure a good sample from the posterior probability distribution. A consensus tree was built from the remaining 75% of the sampled trees with the MrBayes sumt command using the 50% majority rule method. The sump command was used to control so that an adequate sample of the posterior probability distribution was reached during the MCMC procedure. The phylogenetic tree was drawn in FigTree 1.3.1. To root the tree, consensus sequences from arthropods used in the phylogenetic analysis were generated using HMMEMIT from HMMER3 package. First, the sequences that belong to the arthropod sequences identified in UniProt search and the spider sequences were aligned separately and separate HMM profiles were built from those alignments. Each HMM profiles serves as an input for the HMMEMIT program and a consensus sequence. were obtained using option ��2C��as implemented in the HMMER3 package. The consensus sequence is formed using a plurality rule that selects the maximum probability residue at each match state from the HMM profiles. Materials and Methods 1. Database Annotation of A1 and A2 Sequences Please refer to the online appendix, for details. 2. Experimental Determination of European Sea Bass AgRP1, AgRP2, ASIP1; Turbot ASIP1; Solea A
