Isolation and nucleotide sequence analysis of the of Rhinella arenarum ß-catenin: an mRNA and protein expression study during the larval stages of the digestive tract development.

ABSTRACT

ß-catenin interacts with several proteins mediating key biological processes, such as cadherin-mediated cell-cell adhesion as well as signal transduction. This work was done to establish the molecular basis and regulation of the formation pattern of cadherin/ß-catenin-mediated adherens junctions, using an animal model of unknown gene sequence, the toad Rhinella arenarum. A Rhinella arenarum ß-catenin homolog was isolated from larval tissue, their sequence compared and analyzed with those of eight other vertebrates using bioinformatics tools. The mRNA and protein expression levels of ß-catenin were determined during the development of Rhinella arenarum digestive tract both by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and immunohistochemistry-morphometry respectively. Using Xenopus laevis frog specific primers, a fragment 539 bp of Rhinella arenarum toad ß-catenin cDNA was obtained and sequenced. The resulting putative sequence of 177 amino acids showed high similarity at the amino acid level (97%) when compared to other six vertebrates (Xenopus laevis, Xenopus tropicalis, Mus musculus, Rattus norvegicus, Bos taurus and Homo sapiens), with sequences and structural domains characteristic of catenins. Subsequently, using primers specifically designed for Rhinella arenarum nucleotide sequence, ß-catenin-mRNA increasing levels were found during the Rhinella arenarum metamorphosis. Finally, increasing ß-catenin protein expression during development has confirmed the specificity the detection of Rhinella arenarum ß-catenin. Summarizing, we have isolated and sequenced a ß-catenin-homologue sequence from the Rhinella arenarum toad, which is highly conserved between species, and following we have detected ß-catenin mRNA and protein levels during their digestive tract development.