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"Gene Expression in Trypanosoma brucei"

by
Stacey Garcia
Stacey Garcia
Microbiology and Molecular Genetics Program
B.Sc. 2006, Ursinus College




Thesis Advisor: Vivian Bellofatto, Ph.D.
Professor
Department of Microbiology and Molecular Genetics

Friday, May 18, 2012
11:00 A.M., ICPH Auditorium


Abstract

Trypanosome RNA synthesis is unique from other eukaryotes in many ways. For example, most protein-encoding genes are transcribed as part of long polycistronic pre-mRNAs that are subsequently processed into mature monocistronic mRNAs. Each open reading frame of the original pre-mRNA can exhibit different steady state levels, indicating that post-transcriptional regulation is important for gene expression. Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes. Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including 5 to 3 exonuclease decay and 3 to 5 exosome-mediated decay. Poly(A)-specific ribonuclease (PARN) is a key deadenylase involved in regulating gene expression in mammals, Xenopus oocytes, and higher plants. Trypanosomes possess three different PARN genes, PARN-1, -2, and -3. We predict that each PARN deadenylates a different subset of mRNAs. Therefore, we characterized PARN-1 and PARN-3 of human infective trypanosome Trypanosoma brucei. Western analysis indicates that PARN-1 and PARN-3 proteins are expressed in procyclic form and bloodstream form T. brucei cells. Fractionation of PARN-1 and PARN-3 indicate that both deadenylases are primarily cytoplasmic. Northern analysis indicates that over-expression of PARN-1 leads to faster deadenylation of epimastigote-stage surface proteins, called BARPs. Generation of a conditional parn-3 knockout cell line indicates that PARN-3 is not essential for procyclic form proliferation. Together these data further elucidate the role of PARN-1 and PARN-3 in T. brucei.


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