GENE AMPLIFICATION IN LEISHMANIA DONOVANI

Drug resistance is a major obstacle to the conquest of parasitic diseases,
A thorough understanding of the molecular mechanisms and alterations
associated with drug resistance might suggest rational ways that drug
resistance in parasitic diseases could be circumvented or prevented. In
this application, we propose a thorough biochemical and molecular genetic
dissection of one form of drug resistance in Leishmania donovani, gene
amplification. Three separate gene amplification systems have been
developed in this laboratory: i. a difluoromethylornithine (DFMO)-resistant
cell line that overproduces ornithine decarboxylase (ODC); ii. a
mycophenolic acid-resistant strain that overexpresses inosine monophosphate
dehydrogenase; and iii. a multidrug resistant L. donovani. In this
proposal, we will focus on one of these gene amplification systems, the
DFMO-resistant L. donovani strain that has amplified the ODC gene and
contains two extrachromosomal DNAs. The overall objectives of this
proposal are to examine the biochemical and molecular alterations in the
drug-resistant cells and to analyze the ODC gene and protein in more
detail. We will definitively establish that the NH2 terminus of the ODC
protein predicted from the nucleotide sequence of the cloned ODC gene is
accurate by purifying ODC and sequencing its NH2 terminus. Whether the two
extrachromosomal elements that are present in the DFMO-resistant cells are
circular or linear will be authenticated by physical mapping, CHEF
electrophoresis, and electron microscopy. In addition, other
DFMO-resistant strains,from several Leishmania species will be generated
and their amplicons characterized. Homologies between sequences on the two
amplified DNAs of DFMO-resistant cells and amplified DNAs from other
drug-resistant Leishmania will be ascertained. Restriction maps of both
extrachromosomal DNAs and their chromosomal counterparts will be generated
by chromosome walking in order to analyze the mechanism by which the
amplified DNAs originated as a consequence of selective pressure.
Transcriptional activity and mapping of the extrachromosomes will be
performed, and the relationship between the two leishmanial ODC transcripts
will be determined. Finally, we will insert the leishmanial ODC gene into
a bacterial expression vector and transform it into ODC-deficient E, coli
to prepare large quantities of recombinant ODC protein for kinetic,
structural, and immunological studies. Expression of site directed mutants
will permit a genetic dissection of some of the critical structural
determinants that are required for catalytic activity and DFMO inactivation
of this chemotherapeutically important enzyme.