IDENTIFICATION OF DIFFERENTIALLY EXPRESSED LEISHMANIA-DONOVANI GENES USING ARBITRARILY PRIMED POLYMERASE CHAIN-REACTIONS

Arbitrarily primed polymerase chain reactions (AP-PCR) were used to am plify polymorphic DNA fragments from the genomes of a variety of geogr aphic isolates of Leishmania donovani (Ld). From the latter, five poly morphic DNA fragments were cloned and sequence analysis identified 15 unique clones. Northern blot analysis showed that 13 of the 15 clones hybridized to transcribed RNAs isolated from Id. Eight of these 13 AP- PCR clones specifically hybridized to Ld RNAs that were differentially expressed in promastigote and 'amastigote' cells. Comparative Norther n analysis of four differentially expressed AP-PCR clones indicated th at two clones, LdS-14-14 and LdI-9-7, were expressed in Ld and several other Leishmania species. However, RNAs corresponding to two other AP -PCR clones, LdE-6-1 and LdI-9-5, were detected only in members of the Ld complex, and not in L. major (Lm) or L. tropica (Lt). Comparative Southern blot analysis of the LdS-14-14 locus revealed numerous restri ction-fragment length polymorphisms (RFLP) distinguishing Lm and Lt fr om the Ld isolates and L. infantum. However, the LdS-14-14 loci were m apped to similar-sized chromosomes observed among all Old World Leishm ania species tested, indicating that localized nucleotide divergence, not chromosomal rearrangement, was responsible for altered Southern bl ot patterns. These results demonstrate that AP-PCR is a very useful me thod for identifying expressed gene sequences in organisms of relative ly low-complexity genomes. Interestingly, the majority of these sequen ces identified in this study correspond to differentially expressed ge nes. The value of this method lies in its ability to rapidly and rando mly sample diverse portions of an organism's genome, in order to ident ify interesting and unique sequences.