The Molecular Evolution of Primitive Skeletal Ryanodine Receptor Isoforms. Alfonso Go & Francisco Valles
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In cloning and characterizing skeletal ryanodine receptor isoform (RyR1) in primitive vertebrates, we have cloned and sequenced cDNA fragments using degenerate primers on the Xenopus laevis, African Clawed frog. In our investigation, skeletal muscles from the Xenopus were taken and its RNA was isolated. From these RNA, a cDNA library was created. Using PCR, we probed the library with degenerate primers that were based on known RyR1 sequence from fish. Degenerate primers differ from "normal" primers in that they contain variable bases. This means that certain bases in its sequence vary. We were able to isolate and sequence a 90 amino acid sequence of the RyR receptor. Our results indicate that there could be different RyR1 isoforms in the skeletal muscle of the Xenopus. Three of the five clones show resemblance to the RyR3 isoform, one show resemblance to RyR2 and another resembles a non-RyR sequence. In addition to our investigations with the Xenopus, we also began to investigate another primitive vertebrate, the Amphioxus (lancelets). Using the same degenerate primers that were used for the Xenopus, we were successful in detecting a cDNA band that is possibly from a RyR receptor. This DNA fragment is in the process of being cloned and sequenced.