Analysis of amplified DNAs from drug-resistant Leishmania by orthogonal-field-alternation gel electrophoresis. The effect of size and topology on mobility

Abstract

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.