Computational protocol: Identification of multiple integrin β1 homologs in zebrafish (Danio rerio)

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Protocol publication

[…] The complete amino acid sequence of human β1 [Swiss-Prot: P05556] was used to probe the Danio rerio genome ([,]) using TBLASTN [] to identify genes with highest sequence identity to human β1. To amplify complete β1 sequences, primers were designed using putative 5' and 3' sequences (identified as described above) or from EST sequences on the NCBI database []. The following primers were used: β1–1, 5'-ATGGACCTGAAGCTACTTTTCATATC-3' and 5'-CTGATGGCCATTATTTGCCTTCG-3', β1–2, 5'-ATGGACGTAAGGCTGCTCCTG-3'and 5'-CACGTTCGTCCATTATTTGCCCTC-3', β1–3, 5'-ATGAAAATGAAGCTGCTGTTATTATC-3' and 5'-CACTTTCCCTCATATCTGGGATTC-3' β1tr-1, 5'-ATGGATATAACAGTTTTGTTATTATCAG-3' and 5'-ATGTATAACATGAGGTCATGATGTAC-3' β1tr-2 5'-ATGGATATAACAGTTTTGTTATTATCAG-3' and 5'-GTATAACATGTGTCTCAATATATGATG-3'Total RNA was prepared from 4-day old embryos using TRI reagent (Sigma), and reverse transcription was performed using Superscript II (Invitrogen) according to the manufacturer's instructions. PCR reactions were performed using Phusion (New England Biolabs). Cycling parameters were 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 60°C for 20 s and 72°C for 60 s. An additional sequence, β1tr-3, was amplified from reverse transcribed RNA from adult kidney (prepared as described below) using the same primers as for β1tr-2. The PCR reactions generated products of ~2.4 kB for β1–1, β1–2 and β1–3, and ~1.9 kB for β1tr-1, β1tr-2 and β1tr-3. EST clones ESTa and ESTb were obtained from RZPD German Resource Center for Genome Research, product numbers IRAKp961B08165Q [IMAGE:6525557] from adult kidney cDNA library and IMAGp998I1214695Q3 [IMAGE:7001749] from whole adult body cDNA library, respectively.PCR products were analysed on 1.5% agarose gels. Sequencing of PCR products or EST clones was performed using the BigDye cycle sequencing kit (Applied Biosystems). PCR products from reactions using the 5'-ATGGATATAACAGTTTTGTTATTATCAG-3' forward primer contained a mixture of sequences and therefore these products were subcloned into the Zero Blunt TOPO cloning kit (Invitrogen). Sequencing of individual clones revealed a single most abundant sequence (β1tr-1, β1tr-2 or β1tr-3). Alignment of zebrafish and human sequences was performed using CLUSTAL W []. [...] The β1 integrin sequences identified in Danio rerio were aligned with the human β1 sequence [Swiss-Prot: P05556] and the ancestral β1-like gene previously identified in Ciona intestinalis (JGI Ciona v1.0 ci0100141446) [] using CLUSTAL X []. Gap-containing sites were removed from each alignment and Maximum Likelihood trees were inferred using PROML from the PHYLIP package []. The JTT model of amino acid substitutions was used with and without global rearrangements and correction for rate heterogeneity (α value obtained from TREEPUZZLE []). The topologies of the trees were tested using three independent methods. Neighbor-Joining and Maximum Parsimony bootstrap replicates were obtained using the PHYLIP package []. Bayesian tree inference values were produced from the MrBayes programme []. […]

Pipeline specifications

Software tools TBLASTN, Clustal W, PHYLIP, MrBayes
Databases UniProt
Applications Phylogenetics, Amino acid sequence alignment
Organisms Danio rerio, Homo sapiens