Trypanosoma brucei and Trypanosoma cruzi, two related parasitic protozoa, cause substantial and economically significant morbidity and mortality throughout the third world. Vaccines are not available and drug therapy is inadequate because of resistance and toxicity. Thus, new targets for chemotherapeutic intervention are being sought. Buthionine sulfoximine, an inhibitor of 7-glutamylcysteinesynthetaseOyGCS), cures mice infected with 7". brucei implicating iGCS as a potential drug target. The gene for the catalytic subunit of -yGCS has been cloned and sequenced from rat and yeast. An alignment of these two sequences was made and degenerate oligonucleotides were designed to two conserved regions. Partial clones of GCS from T. brucei and T. cruzi. were obtained by PCR and were used to screen genomic libraries to obtain full length clones. Both strands of the T. brucei clone were sequenced by subcloning and progressive oligonucleotides revealing the yGCS open reading frame of 2037 bp. Southern DNA analysis showed that T. brucei vGCS is among the minority of trypanosomal single copy genes. RNA analysis revealed a transcript of about 2.3 kb. The deduced amino acid sequence of 669 amino acids shares 45% and 36% identity with mammalian and yeast -yGCS, respectively. The 77.4 kDa trypanosomal enzyme containing an N- terminal His6 tag was expressed in E. coli and purified for kinetic characterization. Further studies of yGCS will be directed toward identifying functional differences between the trypanosomal and mammalian enzymes which might be exploited for design of selective inhibitors.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology