STUDIES OF A NOVEL RNA APPROACH THAT PROMOTES HEART DEV

描述：(改编自《调查者摘要》)。的长期目标是 实验室的目的是阐明细胞、分子和遗传学 调控心肌细胞分化的机制及机制 发育中的心脏中的肌原纤维形成。混合型芽孢子虫是一种 研究心脏发育的有趣模型，因为它带有一个 C基因突变通过异常诱导发挥作用 来自前内胚层的突起。双隐性的心(c/c) 突变胚胎不会跳动，因为它们缺乏原肌球蛋白 不含有组织的肌原纤维。但是，可以通过以下方式纠正该缺陷 在正常前内胚层存在的情况下培养突变心脏 以前内胚层为条件的组织，或分离的总RNA 从内胚层或条件培养液中，每一种都促进 突变心脏中的肌原纤维形成。已有单个克隆(#4) 从全条件培养液构建的cDNA文库中鉴定 可作为生物活性RNA模板的RNA，其能够 在器官培养和活体内纠正心脏缺陷。这个 生物活性RNA可以与胚轴中存在的蛋白质结合 心脏和生物活性RNA进入突变心脏的细胞以发挥作用 救援队。据推测，生物活性RNA是一种调节性的 上调突变心脏原肌球蛋白合成的分子 直接或与其复合体促进肌纤维形成 结合蛋白。有四个具体的目的来检验这一假设：1) 生物活性rna的表达将在正常和突变型中进行检测。 应用RT-PCR和原位技术研究不同发育阶段的心脏 杂交。正义和反义RNA的异位表达将 通过创造转基因紫杉醇进行检测；2)体外诱变 将进行克隆4RNA以阐明其解救机制 活性及其与新发现的结合的结合能力 3)突变心脏原肌球蛋白表达上调的研究 被锥体#4的RNA修正；4)全长的cDNA和RNA及其 调节其表达的机制。预计 这些研究将为胰岛素抵抗的机制提供重要的新信息。 正常心肌细胞的活体诱导相互作用 在分子水平上的分化。

DESCRIPTION: (Adapted from Investigator's Abstract). The long term goal of the laboratory is to elucidate the cellular, molecular and genetic mechanisms that regulate myocardial cell differentiation and myofibrillogenesis in the developing heart. Ambystoma mixicanum is an intriguing model for studying heart development because it carries a mutation in gene c thought tot exert its effect via abnormal inductive processes from the anterior endoderm. The hearts of double recessive (c/c) mutant embryos do not beat, as they are deficient in tropomyosin and do not contain organized myofibrils. However, the defect can be corrected by culturing the mutant hearts in the presence of normal anterior endoderm tissue, medium conditioned by the anterior endoderm, or total RNA isolated from endoderm or the conditioned medium, each of which promotes myofibrillogenesis in the mutant hearts. A single clone (#4) has been identified from a cDNA library constructed from total conditioned medium RNA which can act as a template for the bioactive RNA capable of correcting the heart defect in organ culture as well as in vivo. The bioactive RNA can bind with a protein present in the embryonic axolotl heart and the bioactive RNA enters the cells of mutant hearts to effect rescue. It is hypothesized that the bioactive RNA is a regulatory molecules which up regulates tropomyosin synthesis in the mutant hearts for promoting myofibrillogenesis either directly or in complex with its binding protein. There are four Specific Aims to test this hypothesis: 1) The expression of the bioactive RNA will be examined in normal and mutant hearts at various stages of development using RT-PCR and in situ hybridization. Ectopic expression of sense and antisense RNA will be tested by creating transgenic axolotls; 2) In vitro mutagenesis of the clone #4 RNA will be performed to elucidate the mechanism of its rescuing activity as well as its binding ability to the newly-discovered binding protein; 3) Study up regulation of tropomyosin in the mutant hearts corrected by the cone #4 RNA; 4) The full length cDNA and the RNA and its mechanism by which its expression is regulated. It is anticipated that these studies will provide significant new information the mechanism of in vivo inductive interactions responsible for normal cardiac myocyte differentiation at the molecular level.