Isolation and Characterization of Upstream Regulatory Region for Yellowfin Tuna RyR1 Isoforms

Abstract

Muscle contractions depend on the release of calcium from the sarcoplasmic reticulum (SR) of muscle cells. Calcium release channels on the SR, called ryanodine receptors (RyR), act as calcium gates and regulate calcium concentration in the cytoplasm. Previous research has found several isoforms of RyR, including RyR1 in skeletal muscle, RyR2 in cardiac muscle, and RyR3 in both. Two fiber-specific RyR1 isoforms, have been identified as fast and slow twitch. To characterize the transcriptional regulation responsible for this differential expression, we began by isolating the extreme 5? region for RyR1 slow twitch in Yellowfin Tuna ( Thunnus albacares ) using the Polymerase Chain Reaction (PCR). Primers for this region were based on a sequence from the Marlin RyR1 slow sequence. This region will be sequenced in order to identify Yellowfin Tuna specific primers. Using the Marlin primers, the non-coding 5? upstream regulatory region has been successfully amplified with the hope of identifying a putative transcriptional regulatory region. Further research will ultimately provide insight into the molecular basis for the dynamics of the differential expression of these isoforms.