Isolation of proteins associated with kinetoplast DNA networks in vivo.
Isolation of proteins associated with kinetoplast DNA networks in vivo.
复制标题
体内与动质体 DNA 网络相关的蛋白质的分离。
DOI：
10.1073/pnas.90.5.1786
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发表时间：
1993
影响因子：
11.1
通讯作者：
Ray,DS
中科院分区：
文献类型：
--
作者：
Xu,C;Ray,DS
动基体DNA（kDNA），锥虫的线粒体DNA，是一个高度浓缩的盘状网络的链状DNA环组成的大环，相当于传统的线粒体DNA，和几千个较小的环状DNA称为小环。在细胞裂解时，kDNA扩增，产生具有接近整个细胞的总直径的链状环的二维网络。为了鉴定与细胞中kDNA的浓缩形式相关的蛋白质，通过甲醛处理使蛋白质可逆地交联至束状短肢虫全细胞中的kDNA。纯化交联的网络，发现其保留了缩合结构，该结构在蛋白酶K处理或通过在65 ℃下加热逆转交联时完全膨胀。五个低分子量蛋白质释放的kDNA热处理，通过聚丙烯酰胺凝胶电泳纯化，并确定其氨基端序列。PCR扩增和序列分析的cDNA序列之间的这些氨基端序列和miniexon（剪接前导）序列存在于5'端的所有C。fasciculata mRNAs预测了9个氨基酸的前序列的存在，这些前序列具有三种蛋白质上线粒体前序列的特征。这些蛋白质中有两种是富含赖氨酸的碱性蛋白质。这些研究结果表明，碱性蛋白质可能发挥作用的kDNA在动基体中的缩合，这些蛋白质进口到动基体的机制，涉及可裂解的前序列。
Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is a highly condensed disc-shaped network of catenated DNA circles consisting of maxicircles, the equivalent of conventional mitochondrial DNA, and several thousand smaller circular DNAs termed minicircles. Upon cell lysis, kDNA expands, giving rise to a two-dimensional network of catenated circles with an overall diameter close to that of the whole cell. To identify proteins associated with the condensed form of kDNA in the cell, proteins were reversibly crosslinked to kDNA in whole cells of Crithidia fasciculata by formaldehyde treatment. Crosslinked networks were purified and found to retain a condensed structure which becomes fully expanded upon proteinase K treatment or reversal of the crosslinks by heating at 65 degrees C. Five low molecular weight proteins released from the kDNA by heat treatment were purified by polyacrylamide gel electrophoresis and their amino-terminal sequences were determined. PCR amplification and sequence analysis of cDNA sequences between these amino-terminal sequences and the miniexon (spliced leader) sequence present at the 5' end of all C. fasciculata mRNAs predicts the presence of 9-amino acid presequences with features characteristic of mitochondrial presequences on three of the proteins. Two of these proteins are lysine-rich basic proteins. These findings suggest that basic proteins may play a role in the condensation of kDNA in the kinetoplast and that these proteins are imported into the kinetoplast by a mechanism involving a cleavable presequence.