CONSERVATION OF LOW-COPY GENE LOCI IN OLD-WORLD LEISHMANIAS IDENTIFIES MECHANISMS OF PARASITE EVOLUTION AND DIAGNOSTIC MARKERS

Genome plasticity. has been hypothesized to be a driving force behind parasite speciation. We have evaluated divergence in single and low-co py genes in terms of locus organization, chromosomal localization and gene expression in Leishmania infantum, L. major, L. tropica and three widely divergent geographic isolates of L. donovani. Seventeen genes of low to moderate copy number (1-4 copies/haploid genome) were analyz ed to identify restriction fragment length polymorphisms (RFLPs) provi ding heritable markers distinguishing Old World (OW) leishmanias. Thes e RFLP markers were conserved in parasite isolates from primary infect ions demonstrating their utility as diagnostic tools. The species desi gnations established by RFLP analysis of field isolates was confirmed by use of monoclonal antibodies. All 17 genes were present in each OW leishmania analyzed except LSIP (A45), which was absent from L. infant um. The 17 genes were found to be distributed among 9 distinct chromos omes. However, in spite of variations in chromosome karyotypes among t he various OW leishmanias, individual gene probes localized to a simil ar sized chromosome from each isolate. These observations coupled with a molecular tree derived from RFLP data suggest that the OW leishmani as comprise a monophyletic lineage, with species associated with cutan eous disease exhibiting the greatest level of divergence. Data from th is study supports previous observations that species causing cutaneous and visceral disease have diverged primarily by nucleotide substituti ons. Such nucleotide divergence may not only lead to changes in protei n function and antigenicity, but may also alter gene regulation progra ms as exemplified by the finding that the LdI-9-5 and LdE-6-1 genes we re expressed only in visceralizing leishmanias.