Developmental Myosin Heavy Chain Regulation and Function

描述(申请人提供)：骨骼肌的功能是 产生运动所需的收缩力量。一种关键的蛋白质 参与肌肉收缩的是肌球蛋白，它是一种由两个重的 链条和四条轻链。肌球蛋白重链(MyHC)含有马达 粗丝形成所需的杆状结构域和杆状结构域。有8个 已知的MyHC亚型在横纹肌中表达，2例发育，6例 成年人。而成体亚型的功能和表达一直是 广泛的特征，相对较少的人知道的作用 胚胎和围产期骨骼肌异构体或调节因子的研究 他们的表情。它们的表达在怀孕中期开始，达到 在出生前后达到高峰，并在出生后早期迅速下降 因为它们被成年的MyHC亚型所取代。在产前， 肌肉收缩，但不会发生协调运动。因此，精确的函数 这些异构体的定义并不明确。我们建议对该法规和 两种发育中的MyHC亚型的分子和遗传学功能 接近了。首先，我们建议研究顺式和跨式调节因素。 通过功能分析调控胚胎和围产期MyHC的表达 这两个基因的上游调控区域。将测试启动子的活性 在细胞培养和小鼠体内；在后者中，质粒DNA注射和 瞬时转基因将被用来确定 在肌肉发育过程中调节表达的大小和模式。 其次，我们将使用同源重组来创造小鼠，在其中 胚胎或围产期MyHC基因已被呈现为空，并研究由此产生的 表型。最后，确定胚胎MyHC的运动域的作用 在肌肉发育方面，我们将创造出具有显性优势的转基因小鼠 三磷酸腺苷结合区突变。具体地说，我们将检验这个假设 胚胎MyHC的收缩功能是肌肉发育所必需的。 这些研究将确定发育中的MyHC亚型在 骨骼肌的形态和功能。

DESCRIPTION (provided by applicant): The function of skeletal muscle is to produce the contractile force necessary for movement. One of the key proteins involved in muscle contraction is myosin, a hexamer consisting of two heavy chains and four light chains. Myosin heavy chain (MyHC) contains the motor domain and the rod domain necessary for thick filament formation. There are 8 known isoforms of MyHC expressed in striated muscle, 2 developmental and 6 adult. While the function and expression of the adult isoforms has been extensively characterized, relatively little is known about the role of the embryonic and perinatal skeletal muscle isoforms or of the factors regulating their expression. Their expression is initiated at mid-gestation, reaches a peak around birth, and is rapidly down regulated during early postnatal growth as they are replaced by the adult MyHC isoforms. During the prenatal period, muscle contraction but no coordinated movement occurs. Thus the exact function of these isoforms is poorly defined. We propose to evaluate the regulation and function of the two developmental MyHC isoforms using molecular and genetic approaches. First, we propose to study the cis- and trans-regulatory factors governing embryonic and perinatal MyHC expression by functionally analyzing the upstream regulatory regions of both genes. Promoter activities will be tested in cell culture and in mice; in the latter, both plasmid DNA injection and transient transgenics will be used to identify elements necessary for regulating the magnitude and pattern of expression during muscle development. Second, we will use homologous recombination to create mice in which either the embryonic or perinatal MyHC gene has been rendered null and study the resulting phenotype. Finally, to determine the role of the motor domain of embryonic MyHC in muscle development, we will create transgenic mice harboring a dominant mutation in the ATP binding domain. Specifically, we will test the hypothesis that embryonic MyHC contractile function is necessary for muscle development. These studies will define the role of the developmental MyHC isoforms in skeletal muscle form and function.