Identification of intra- and interspecificLeishmania genetic polymorphisms by arbitrary primed polymerase chain reactions and use of polymorphic DNA to identify differentially regulated genes
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Arbitrary primed polymerase chain reactions (AP-PCR) were used to amplify different polymorphic genomic DNA fragments from various Old WorldLeishmania species. Using four 10-mer AP primers, geographic isolates ofL. donovani and various Old World species ofLeishmania could be readily distinguished from one another by the pattern of amplified DNA products. Our studies confirmed two important characteristics of AP-PCR: its abilities to amplify a consistent pattern of DNA fragments from the genomes of different isolates of a single species and to identify genetic polymorphisms between the species isolates. We selected three polymorphic DNA fragments that differentiateL. donovani geographic isolates for further analysis. Sequence analysis of the clones derived from these three polymorphic fragments revealed eight unique sequences. Six of eight unique clones hybridized to distinct RNAs upon Northern-blot analysis. Three of these six clones hybridized to RNAs expressed differentially in in vitro grownL. donovani pro- and “amastigotes”. One of the differentially expressed clones, LdE-6-1, exhibited restriction length polymorphisms that distinguishedL. donovani fromL. tropica andL. major. Comparative Northern blotting revealed that LdE-6-1 was differentially expressed in some members of theL. donovani species complex but not inL. major orL. tropica. These results demonstrate that AP-PCR can be used to generate products reflecting particular genes in organisms with low-complexity genomes.
KeywordsNorthern Blotting Genetic Polymorphism Geographic Isolate Restriction Length Polymorphic Fragment
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