Computational protocol: An evolutionary analysis of cAMP-specific Phosphodiesterase 4 alternative splicing

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Protocol publication

[…] PDE4 translated amino acid sequences were aligned using the Clustal function of Mega v4, according to the default settings. PDE4 splice variant relationships based on the catalytic domain were determined using the Neighbor Joining algorithm of Mega v4. The tree shown represents three independent runs, each with 1000 bootstrap replicates using the PAM matrix of amino acid evolution. One long form splice variant nucleotide sequence from each clade was selected for each species of interest (representative of species individual PDE4 gene isoforms, highlighted in Additional File ), and translated in the clustal function of MEGA. Mr Bayes and PhyML were used to construct a vertebrate PDE4 gene family phylogeny using the amino acid sequences common to all PDE4 long forms (UCR-1, UCR-2, and catalytic domains). The tree represents three and four independent runs of 1000 bootstrap replicates and four million generations, in PhyML and Mr Bayes, respectively. Mr Bayes was allowed to estimate the best fixed rate model of protein evolution during the run by setting the prior for amino acid model to mixed. The best fixed rate model was Jones-Taylor Thornton (JTT) model (gamma = 0.946). PhyML analyses were run under this model as well. [...] Multi PipMaker http://bio.cse.psu.edu[,] was used for PDE4 gene comparisons across vertebrate taxa with respect to exon presence/conservation. In each case, the human PDE4 gene of interest (PDE4A, PDE4B, PDE4C, or PDE4D) was used as the reference gene, in the forward orientation. Multi PIP maker offers an additional function in which user defined exon positions are displayed. Exon positions (coordinates) within each human PDE4 gene were determined using the human gene sequence and conserved splice variant amino termini, facultative (not present in all forms) and constitutive exons (present in all forms) within the two sequence BLAST (bl2seq) function from NCBI http://blast.ncbi.nlm.nih.gov/Blast.cgi and then formatted into an exon file for use in Multi PipMaker. […]

Pipeline specifications

Software tools MEGA-V, PhyML, PipMaker
Applications Phylogenetics, GWAS, Genome data visualization
Organisms Homo sapiens