Functional and evolutionary characterization of the zebrafish ryanodine receptor multigene family

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The process of gene and whole genome duplication is a fundamental mechanism in the evolution of genome and organism complexity. Genes descending from a duplication event are said to be members of a gene family. The diversity in function of the gene family members is the result of changes in amino acids in the coding region and/or changes in the regulatory domains due to random mutations in the descendant genes. We are studying the genetic regulation and molecular evolution of the intracellular calcium release channels gene family also known as ryanodine receptors (RyR) in non-mammalian vertebrates. We have chosen zebrafish as our model system for several reasons. They are hardy, disease resistant and easily reared and have become a well characterized model for studying vertebrate genetics and development. The zebrafish genome project and recent assembly of sequence data will also permit us to mine for gene sequences. Another distinct advantage of working with fish is the fact that they have discretely compartmentalized muscle fiber types. We have previously shown that fish express fiber type-specific RyR isoforms in skeletal muscle of fish. We are interested in determining the spatial and temporal expression patterns of all members of the RyR gene family in zebrafish. We will clone the regulatory regions of the zebrafish RyR gene isoforms and determine the essential regions for expression using green fluorescent tagged promoter constructs in microinjected zebrafish embryos. We will also investigate the functional role of RyR genes in development by "knocking down" expression by injecting antisense morpholinos into zebrafish embryos. To investigate the molecular evolution of the two-isoform condition in skeletal muscles of fish we will survey the genomic DNA of primitive vertebrate species including coelacanth and hagfish. Comparison of primitive and derived RyR sequences will reveal important information about both the coding and non-coding (regulatory) regions of the genes.