This project proposes to determine the functions of genes in Leishmania LD1 locus which are frequently amplified; a hallmark of important genes, such as those responsible for drug resistance. Leishmania from humans will be examined for LD1 amplification and the biological consequences of LD1 amplification will be explored. The subcellular location and abundance of proteins encoded in the LD1 locus will be determined in promastigote and amastigote Leishmania that contain unamplified or various amplified LD1 DNAs. The function of selected LD1 locus genes will be explored by strain comparison, gene knockout, and gene inactivation. These studies will initially focus on orfG, orfD, orfC which appear to function in transport, cell-cycle regulation and cell growth, respectively. Resultant phenotypes will be characterized by a variety of techniques. A regulatable promoter will be used for gene inactivation to study essential genes and monitor phenotype development. Additionally, the putative pol I promoter and transcription terminator in rRNA locus of the 1.2 Mb chromosome will be characterized, including the site that contains duplicated LD1 sequences and which will be used for a regulatable promoter. Gene amplification is a characteristic of genes involved in drug resistance in Leishmania. This suggests that LD1 amplification, perhaps, involves genes important for parasite survival. Thus the investigator hopes that some of the LD1 encoded products may provide potential therapeutic targets for parasite control.
| Martinez-Calvillo, Santiago; Yan, Shaofeng; Nguyen, Dan et al. (2003) Transcription of Leishmania major Friedlin chromosome 1 initiates in both directions within a single region. Mol Cell 11:1291-9 |
