MYOSIN GENE DIVERSITY AND FUNCTION

肌球蛋白基因多样性和功能

• 批准号: 6483266
• 负责人: Leslie Anne Leinwand
• 金额: $ 0.55万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6483266
• 关键词: developmental genetics; gene expression; gene targeting; genetically modified animals; immunocytochemistry; in situ hybridization; laboratory mouse; linkage mapping; molecular cloning; muscle contraction; muscle metabolism; myoblasts; myogenesis; myosins; protein structure function; striated muscles; tissue /cell culture

DESCRIPTION: Muscle contraction requires the orderly assembly and functional interaction of numerous proteins in the sarcomere. The most abundant sarcomeric protein is the motor protein, myosin, which in mammals is represented by at least eight closely related isoforms. Expression of these genes is precisely regulated in space and in time, but their functional relationships remain largely unknown. In Aim I of the proposed experiments, transgenic approaches will be used to define the role of these genes in muscle development and function. Mice null for the expression of the two major skeletal myosin heavy chains (MyHC), fast IIb and fast IId have been obtained. Common phenotypes include decreased body mass and limb weakness. Distinct phenotypes include kyphosis, histopathology and physiological defects. Interestingly, both null strains exhibit compensation by other MyHC genes, but the compensating gene is different between the two strains. The basis for these phenotypes will be explored and the molecular basis for, and the timing of compensation will be determined. Because compensation has occurred, and yet the mice have strong phenotypes, the PI will test whether the MyHCIIa gene can functionally substitute for the MyHCIId gene by "knocking" the IIa coding region into the IId locus. The sequence of the coding regions for all six human skeletal MyHC genes has been completed and these will be used in Aim II to determine the biochemical properties of the skeletal isoforms, including the roles of the two highly variable loops in the motor domain. To accomplish this goal, the motor domains will be expressed in baculovirus and their enzymatic and motile properties will be characterized. In addition to myosin's motor activity, it is a structural protein, self-assembling into the thick filament. The PI will continue to define the determinants of thick filament assembly using biochemical, phage display and cell culture approaches. Finally, in Aim III, the molecular and cellular biology of an unusual contractile cell type, the myofibroblast, which has features of both muscle and nonmuscle cells, will be explored. In vivo, these cells participate in tissue injury and in wound healing. The PI has shown that these cells express an extensive array of sarcomeric proteins (including six skeletal MyHC genes) and two myogenic regulatory factors, and yet they are not terminally or morphologically differentiated. The number, distribution and gene expression profiles of these cells will be explored in vivo in normal and pathological settings. Using cultured myofibroblast model systems, the role and organization of sarcomeric proteins in myofibroblast contractility will be determined.

描述：肌肉收缩需要有序的组装和 肌动中许多蛋白质的功能相互作用。 最多 丰富的肉瘤蛋白是运动蛋白，肌球蛋白，它在 哺乳动物至少由八个密切相关的同工型表示。 这些基因的表达在空间和及时受到调节， 但是他们的功能关系在很大程度上仍然未知。 在目标 提出的实验将使用转基因方法来定义 这些基因在肌肉发育和功能中的作用。 老鼠无效 为了表达两个主要的骨骼肌球蛋白重链（MyHC）， 快速IIB和快速IID已获得。 常见表型包括 减少体重和肢体弱点。不同的表型包括 Kybhosis，组织病理学和生理缺陷。 有趣的是，两者俩 无菌株通过其他MyHC基因表示补偿，但是 两种菌株之间的补偿基因不同。基础 这些表型将被探讨，并为分子基础和 赔偿时间将被确定。 因为薪酬有 发生了，但小鼠具有强烈的表型，PI将测试 Myhciia基因是否可以在功能上代替myhciid 通过将IIA编码区“敲打”到IID基因座中来进行基因。 这 所有六个人类骨骼MYHC基因的编码区域序列均具有 已完成，这些将用于AIM II来确定 骨骼同工型的生化特性，包括 电机域中的两个高度可变循环。 实现这一目标 目标，运动域将在杆状病毒及其中表达 将表征酶促和动力的特性。 此外 肌球蛋白的运动活动是一种结构蛋白，自组装 进入厚丝。 PI将继续定义 使用生化，噬菌体显示的厚丝组件的决定因素 和细胞培养方法。 最后，在AIM III中，分子和 不寻常的收缩细胞类型的细胞生物学，肌纤维细胞， 将探索具有肌肉和非肌肉细胞的特征。 在体内，这些细胞参与组织损伤和伤口愈合。 PI表明这些细胞表达了广泛的阵列 肉瘤蛋白（包括六个骨骼MYHC基因）和两个肌原性 监管因素，但在终端或形态上却没有 分化。 数量，分布和基因表达曲线 这些细胞将在正常和病理中在体内探索 设置。 使用培养的肌纤维细胞模型系统，角色和 在肌纤维细胞收缩力中的肉瘤蛋白组织将 可以确定。