Hightower R C, Bliska J B, Cozzarelli N R, Santi D V
Department of Biochemistry, University of California, San Francisco 94143.
J Biol Chem. 1989 Feb 15;264(5):2979-84.
Amplified extrachromosomal DNAs from antifolate-resistant Leishmania are 30-75 kilobase (kb) supercoiled molecules that resolve on orthogonal-field-alternation gel electrophoresis (OFAGE) gels. These DNAs comigrate with smaller supercoiled plasmids (7-8 kb), and their mobility is not a simple function of their size. The properties of the amplified DNAs were investigated to determine if an unusual structure accounts for the observed mobility of the amplified DNAs by OFAGE; however, their topological properties were similar to those of standard Escherichia coli plasmids. The migration of a series of supercoiled plasmids ranging in size from 6 to 91 kb was analyzed by OFAGE, and a triphasic pattern was observed. The mobilities of plasmids between 20 and 60 kb increase with size, whereas the migration of plasmids between 6 and 20 and 60 and 91 kb is inversely proportional to size. Like smaller plasmids, the large supercoiled DNAs show a pulse time-independent mobility by OFAGE. The mobility of amplified DNA from Leishmania is in accord with that of the plasmid markers. Therefore, it is primarily the size of the amplified extrachromosomal DNAs from Leishmania, rather than an unusual superhelical density or topological structure, that results in the previously unexplained migration pattern.
来自抗叶酸利什曼原虫的扩增染色体外DNA是30 - 75千碱基（kb）的超螺旋分子，可在正交交变电场凝胶电泳（OFAGE）凝胶上分离。这些DNA与较小的超螺旋质粒（7 - 8 kb）一起迁移，其迁移率并非其大小的简单函数。对扩增DNA的特性进行了研究，以确定是否存在异常结构导致通过OFAGE观察到的扩增DNA的迁移率；然而，它们的拓扑特性与标准大肠杆菌质粒相似。通过OFAGE分析了一系列大小从6到91 kb的超螺旋质粒的迁移情况，观察到一种三相模式。20至60 kb之间的质粒迁移率随大小增加，而6至20 kb和60至91 kb之间的质粒迁移与大小成反比。与较小的质粒一样，大型超螺旋DNA在OFAGE中显示出与脉冲时间无关的迁移率。来自利什曼原虫的扩增DNA的迁移率与质粒标记物一致。因此，主要是利什曼原虫扩增染色体外DNA的大小，而非异常的超螺旋密度或拓扑结构，导致了先前无法解释的迁移模式。
