Functional and evolutionary characterization of the zebrafish ryanodine receptor multigene family

基因和全基因组复制过程是基因组和生物体复杂性进化的基本机制。 从重复事件中继承下来的基因被称为基因家族的成员。基因家族成员的功能多样性是由于后代基因中的随机突变导致的编码区中氨基酸的变化和/或调节结构域的变化的结果。 我们正在研究细胞内钙释放通道基因家族的遗传调控和分子进化，也被称为兰尼碱受体（RyR）在非哺乳类脊椎动物。 我们选择斑马鱼作为我们的模型系统有几个原因。它们具有哈代耐寒、抗病、易饲养等特点，已成为研究脊椎动物遗传和发育的良好模式动物。 斑马鱼基因组计划和最近的序列数据汇编也将使我们能够挖掘基因序列。与鱼类合作的另一个明显优势是，它们具有离散的肌纤维类型。我们以前已经表明，鱼表达纤维类型特异性RyR亚型的骨骼肌的鱼。我们感兴趣的是在确定的空间和时间的表达模式的所有成员的RyR基因家族在斑马鱼。 我们将克隆斑马鱼RyR基因亚型的调控区域，并确定在显微注射斑马鱼胚胎中使用绿色荧光标记的启动子构建体表达的必要区域。我们还将研究RyR基因在发育中的功能作用，通过注射反义吗啉到斑马鱼胚胎中来“敲低”表达。为了研究鱼类骨骼肌中两种异构体的分子进化，我们将调查原始脊椎动物物种包括腔棘鱼和盲鳗的基因组DNA。原始和衍生RyR序列的比较将揭示有关基因的编码和非编码（调控）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.