IDENTIFICATION OF INTRASPECIFIC AND INTERSPECIFIC LEISHMANIA GENETIC - POLYMORPHISMS BY ARBITRARY PRIMED POLYMERASE CHAIN-REACTIONS AND USEOF POLYMORPHIC DNA TO IDENTIFY DIFFERENTIALLY REGULATED GENES

Arbitrary primed polymerase chain reactions (AP-PCR) were used to ampl ify different polymorphic genomic DNA fragments from various Old World Leishmania species. Using four 10-mer AP primers, geographic isolates of L. donovani and various Old World species of Leishmania could be r eadily distinguished from one another by the pattern of amplified DNA products. Our studies confirmed two important characteristics of AP-PC R: 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 polymerphic DNA fragments that differentiate L. donovani geographic i solates 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 grown L. donovani pro- and ''amastigotes.' ' One of the differentially expressed clones, LdE-6-1, exhibited restr iction length polymorphisms that distinguished L. donovani from L. tro pica and L. major. Comparative Northern blotting revealed that LdE-6-1 was differentially expressed in some members of the L. donovani speci es complex but not in L. major or L. tropica. These results demonstrat e that AP-PCR can be used to generate products reflecting particular g enes in organisms with low-complexity genomes.