CLONING AND EXPRESSION OF THE HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE FROM LEISHMANIA-DONOVANI

The gene encoding the hypoxanthine-guanine phosphoribosyltransferase ( HGPRT) enzyme from Leishmania donovani has been cloned and sequenced. The hgprt open reading frame encoded a polypeptide of 211 amino acids that exhibited 3 regions of significant homology with other eukaryotic HGPRTs and a C-terminal tripeptide compatible with a glycosomal targe ting signal. Northern blot analysis of L. donovani RNA revealed two hg prt transcripts, a 1.9-kb mRNA and a 1.7-kb transcript. The expression of the 1.7-kb hgprt mRNA and the activity of HGPRT enzyme were both a ugmented approx. 5-fold in parasites incubated in the absence of purin es. Southern blots of genomic DNA indicated only a single hgprt locus within the L. donovani genome. Overexpression of L. donovani hgprt in E. coli complemented genetic deficiencies in hypoxanthine and guanine phosphoribosylating activities and yielded abundant quantities of enzy matically active HGPRT. The recombinant HGPRT was purified to homogene ity and recognized hypoxanthine, guanine and allopurinol, but not aden ine or xanthine, as substrates. The hgprt clone and pure HGPRT protein provide essential reagents for validating HGPRT as a therapeutic targ et for the treatment of leishmaniasis and other diseases of parasitic origin.