Functional and evolutionary characterization of the zebrafish ryanodine receptor multigene family

基因和全基因组复制的过程是基因组和生物复杂性进化的基本机制。从复制事件中下来的基因被称为基因家族的成员。基因家族成员在功能上的多样性是编码区氨基酸变化和/或后代基因随机突变导致调节结构域变化的结果。我们正在研究非哺乳动物脊椎动物细胞内钙释放通道基因家族(也称为ryanodine受体(RyR))的遗传调节和分子进化。它们耐寒、抗病、易于饲养，已成为研究脊椎动物遗传和发育的典型模型。此外，斑马鱼基因组计划和最近的序列数据组装也将使我们能够挖掘基因序列。与鱼一起工作的另一个明显优势是，它们有不同类型的肌肉纤维。我们先前已经证明鱼类在鱼的骨骼肌中表达纤维类型特异性的RyR亚型。我们有兴趣确定斑马鱼RyR基因家族所有成员的空间和时间表达模式。我们将克隆斑马鱼RyR基因异构体的调节区，并使用绿色荧光标记的启动子构建物在显微注射斑马鱼胚胎中确定表达的必要区域。我们还将研究RyR基因在发育中的功能作用，通过向斑马鱼胚胎中注射反义吗啉来“下调”表达。为了研究鱼类骨骼肌中两种异构体状态的分子进化，我们将调查包括腔棘鱼和八头鳗在内的原始脊椎动物的基因组DNA。原始和衍生的RyR序列的比较将揭示有关基因编码区和非编码区的重要信息。

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.