Developmental Myosin Heavy Chain Regulation and Function

描述（由申请人提供）：骨骼肌的功能是 产生运动所需的收缩力。一种关键的蛋白质 参与肌肉收缩的是肌球蛋白，一种由两个重链组成的六聚体， 四条轻链。肌球蛋白重链（MyHC）包含马达 域和杆域所必需的粗丝形成。有8 已知的MyHC亚型在横纹肌中表达，2发育和6 成年人了虽然成体同种型的功能和表达已经被研究， 广泛的特点，相对较少的是知道的作用， 胚胎和围产期骨骼肌亚型或调节因子 他们的表情。它们的表达起始于妊娠中期， 在出生前后达到高峰，在出生后早期生长期间迅速下调 因为它们被成人MyHC同种型所取代。在产前期间， 肌肉收缩，但没有协调的运动发生。因此， 这些异构体的定义不明确。我们建议评估该条例， 利用分子和遗传学方法研究两种发育中MyHC亚型的功能 接近。首先，我们建议研究顺式和反式调节因子， 调控胚胎和围产期MyHC的表达， 两个基因的上游调控区。将测试促销活动 在细胞培养物和小鼠中;在后者中，质粒DNA注射和 瞬时转基因将用于确定必要的元素， 调节肌肉发育过程中表达的幅度和模式。 第二，我们将使用同源重组来创建小鼠，在小鼠中， 胚胎或围产期MyHC基因已呈现无效，并研究结果 表型最后，为了确定胚胎MyHC运动域的作用， 在肌肉发育方面，我们将创造出一种转基因小鼠， ATP结合域的突变。具体来说，我们将测试假设 胚胎MyHC收缩功能是肌肉发育所必需的。 这些研究将确定发育中MyHC亚型在 骨骼肌形态和功能。

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.