The Application of Yeast Three-Hybrid Technology in the Mexican Axolotl (Ambystoma mexicanum) System.

The Mexican axolotl, Ambystoma mexicanum, possesses a naturally-occurring
lethal mutation, designated gene "c", for cardiac non-function. Hearts form but fail to
beat, lack organized myofibrils, and are deficient in tropomyosin. Treatment with a noncoding
RNA MIR (Myofibril-Inducing RNA) rescues this defect in organ culture.
Rescued mutant hearts have restored tropomyosin, form organized myofibrils, and beat
vigorously. Studies to elucidate the mechanism of MIR heart rescue are underway.
Current evidence suggests that MIR acts by binding with at least two proteins. The yeast
three-hybrid system is being used to screen an axolotl eDNA library for these two
proteins and other possible MIR-binding candidates. This is a method utilizing two
hybrid proteins and a hybrid RNA. An interaction between these three components will
activate the expression of reporter genes, whose activity is assayed through phenotypical
and biochemical methods. In this study, the protocol for yeast three-hybrid technology is
being established for analyzing the MIR in the Mexican axolotl, cardiac mutant animal
model.