ASSEMBLY OF ISOFORMS OF THE MYOSIN ROD

胚胎和新生儿肌球蛋白重链的同源二聚体亚型是 在肌肉发育过程中共表达，但不形成杂交。这个 拟议的研究试图确定是热力学因素还是生物因素 确定亚单位关联。孤立的杆段将被变性， 复性并检测杂二聚体的形成。高度的 杆状亚基亚基异构体之间的序列同源性需要应用 检测混血的新方法。其中包括使用硫醇- 封闭/二硫化物形成方法与非还原十二烷基硫酸钠-PAGE联用，a 共振能量转移程序和固定化亚单位 杂交技术。如果没有检测到杂交体，棒状物就会异构体 将在大肠杆菌中表达，并在 已知序列不同的区域。该化验结果将用于 确定同源二聚体中的每个突变对异源二聚体的影响 队形。如果检测到杂交种，类似的突变研究也将被检测到 以确定序列中与其关联的那些特征 视情况而定。了解肌球蛋白二聚的这些结构特征是 在描述肌原纤维如何在体内组装和发挥作用方面很重要 收缩过程。

The embryonic and neonatal homodimeric isoforms of myosin heavy chain are coexpressed during muscle development but do not form a hybrid. The proposed study seeks to ascertain if thermodynamic or biological factors determine subunit association. Isolated rod segments will be denatured, renatured and assayed for formation of the heterodimer. The high degree of sequence homology between the rod subunit isoforms necessitates application of new methods to detect the hybrid. These include use of a thiol- blocking/disulfide-forming method coupled with non-reducing SDS-PAGE, a resonance energy transfer procedure, and an immobilized subunit hybridization technique. If hybrids are not detected, the rod isoforms will be expressed in E. coli and subjected to site specific mutagenesis in regions where the sequences are known to differ. The assay will be used to determine the effects of each mutation in the homodimer on heterodimer formation. If hybrids are detected, similar mutagenesis studies will be done to determine those features of the sequence upon which association depends. Understanding these structural features of myosin dimerization is important in describing how myofibrils assemble and function in the contractile process.