PACKAGING AND REPLICATION OF MITOCHONDRIAL DNA

拟议研究的目标是继续研究两种不同的 包装形式的大鼠肝脏线粒体DNA。 这两种形式包括： (1)线粒体DNA的一种复制静止的紧密形式，可以分离 基于其高S值，以及（2）复制分子， 结合单一多肽种类（P16）（MR = 16，000）的比率为 每个mtDNA分子至少60个多肽。 的本质特征 复制中间体是组成新生链是高度 在亲本断裂期间稳定防止分支迁移损失 股。 这两种形式的mtDNA将通过温和的裂解而释放， 根据沉积速度进行分离。 每个物种都将 通过疏水分子筛从任何未结合的污染物中纯化 柱层析并通过琼脂糖凝胶电泳监测。 复合物的超微结构方面将通过电子显微镜检查。 显微镜 线粒体DNA的紧密形式将被化学探测， 酶法测定其RNA、脂质、蛋白质和 多胺 非DNA成分在稳定细胞中的作用 将建立紧凑的结构。 mtDNA-P16复制 复合物将通过化学交联研究进一步检查， 泄露关于边界P16的相对并列的信息 分子。 有效地利用将玻璃纤维过滤器制成绑定 选择性地检索P16结合的环以及特异性的 限制，代表显性结合的mtDNA片段 序列的 P16将从线粒体裂解物中大量分离， 已知含有过量的P16，并通过分子筛纯化， 制备等电聚焦然后纯蛋白质将 用脱蛋白DNA重组，深入研究 结合相互作用和建立功能关系 P16和复制环对分支的稳定性之间的关系 迁移

The goal of the proposed research is to continue the study of two different packaged forms of rat liver mitochondrial DNA. These two forms consist of (1) a replicatively quiescent compact from of mtDNA that can be separated on the basis of its high s value, and (2) replicating molecules to which is bound a single polypeptide species (P16) (MR = 16,000) in a ratio of at least 60 polypeptides per mtDNA molecule. The essential feature of the replictive intermediates is that the constituent nascent strands are highly stabilized against branch migratonal loss during scission of the parental strands. These two forms of the mtDNA will be released by gentle lysis and separated on the basis of sedimentation velocity. Each species will be purified from any unbound contaminants by hydrophobic, molecular sieving column chromatography and monitored by agarose gel electrophoresis. Ultrastructural aspects of the complexes will be examined by electron microscopy. The compact form of mtDNA will be probed chemically and enzymatically to determine its content of RNA, lipid, protein, and polyamines. The role of the non-DNA costituents in the stabilization of the compact structure will be established. The mtDNA-P16 replication complexes will be further examined by chemical crosslinking studies to divulge information regarding the relative juxtaposition of the bound P16 molecules. Effective use will be made of a glass fiber filter binding method to selectively retrieve P16-bound circles as well as specific restriction, fragments of mtDNA that represent the dominant binding sequences. The P16 will be isolated in bulk from mitochondrial lysates, known to contain excess P16, and purified by molecular sieving and preparative isoelectric focusing. The pure protein will then be reconstituted with deproteinized DNA to examine in depth the selectivity of the binding interaction and to establish the functional relationship between P16 and the stability of the replication loops against branch migration.