Kinetoplastid protozoa such as Leishmania spp., Trypanosoma brucei and Trypanosoma cruzi cause a variety of diseases in humans and livestock. The parasites generate mature messenger RNA by the addition of a common 39-nucleotide sequence (termed the mini-exon) to protein-encoding precursor RNAs in a trans-splicing reaction. Trans-splicing has not been identified in the mammalian hosts of these parasites, therefore the process is parasite-specific and a selective target for anti-parasite attacks. The long term aim of this research is to understand common mechanisms of gene expression in these pathogenic protozoa. Because the mini-exon is synthesized as a short, discrete precursor (termed medRNA) in all these species, the research in this proposal is directed to understanding the synthesis of medRNA (in the model organism Leishmania tarentolae). Essential promoter element(s) of the mini-exon gene will be defined. The upstream region of a cloned mini-exon gene, which has been marked with a 40-bp tag, will be subjected 1) to linker-scanning mutagenesis and 2) point mutation analysis of the -67/-58 region. Mutated genes contained on the stable transfection vector pX will be introduced into L. tarentolae by electroporation. Transcription of the episomal genes will be monitored by Northern blotting, nuclease protection, primer extension and nascent RNA analyses. Mutated genes will also be used as templates, in the gel mobility-shift assay, to define the sites of protein binding. Proteins that bind to the essential -67/-58 region will also be characterized by two complementary methods. DNA-binding protein sequence will be inferred from cDNA clones that have been isolated by virtue of their binding a """"""""-67/58"""""""" double-stranded oligonucleotide. Native protein(s) will also be purified from nuclear extracts by column chromatography. Purification of the protein(s) will be monitored by the gel mobility-shift assay. The proposed experiments will define the essential upstream promoter element of the Leishmania mini-exon gene. It will also characterize potential transcription factors that associate with the essential element of this important, parasite-specific gene.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI034536-03
Application #
2003972
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1994-12-01
Project End
1998-11-30
Budget Start
1996-12-01
Budget End
1998-11-30
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Thomas, Sean; Green, Amanda; Sturm, Nancy R et al. (2009) Histone acetylations mark origins of polycistronic transcription in Leishmania major. BMC Genomics 10:152
Hitchcock, Robert A; Thomas, Sean; Campbell, David A et al. (2007) The promoter and transcribed regions of the Leishmania tarentolae spliced leader RNA gene array are devoid of nucleosomes. BMC Microbiol 7:44
Thomas, Sean; Yu, Michael C; Sturm, Nancy R et al. (2006) A non-universal transcription factor? The Leishmania tarentolae TATA box-binding protein LtTBP associates with a subset of promoters. Int J Parasitol 36:1217-26
Thomas, Sean; Westenberger, Scott J; Campbell, David A et al. (2005) Intragenomic spliced leader RNA array analysis of kinetoplastids reveals unexpected transcribed region diversity in Trypanosoma cruzi. Gene 352:100-8
Foldynova-Trantirkova, Silvie; Paris, Zdenek; Sturm, Nancy R et al. (2005) The Trypanosoma brucei La protein is a candidate poly(U) shield that impacts spliced leader RNA maturation and tRNA intron removal. Int J Parasitol 35:359-66
Zeiner, Gusti M; Foldynova, Silvie; Sturm, Nancy R et al. (2004) SmD1 is required for spliced leader RNA biogenesis. Eukaryot Cell 3:241-4
Zeiner, Gusti M; Sturm, Nancy R; Campbell, David A (2003) The Leishmania tarentolae spliced leader contains determinants for association with polysomes. J Biol Chem 278:38269-75
Campbell, David A; Thomas, Sean; Sturm, Nancy R (2003) Transcription in kinetoplastid protozoa: why be normal? Microbes Infect 5:1231-40
Orlando, Tereza C; Rubio, Mary Anne T; Sturm, Nancy R et al. (2002) Intergenic and external transcribed spacers of ribosomal RNA genes in lizard-infecting Leishmania: molecular structure and phylogenetic relationship to mammal-infecting Leishmania in the subgenus Leishmania (Leishmania). Mem Inst Oswaldo Cruz 97:695-701
Yu, Michael C; Orlando, T Cristina; Sturm, Nancy R et al. (2002) Two distinct functional spliced leader RNA gene arrays in Leishmania tarentolae are found in several lizard Leishmania species. Int J Parasitol 32:1411-22

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