Grant: $74,166 - National Institutes of Health - May. 21, 2009
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Award Description: This grant provides RO3 level funding to develop mitochondrial transfection of African trypanosomes. Humans exhibit an innate immunity to the mammalian parasite Trypanosoma brucei brucei. Trypanosoma brucei is the causative agent of African human sleeping sickness and the wasting disease Nagana in cattle. This parasite has the unique ability to developmentally regulate its mitochondrial function during the transmission from the insect vector to the mammalian host. The regulation of mitochondrial biogenesis is necessary for the parasite’s survival and requires the interaction of two genomes (mitochondrial and nuclear) as well as a major RNA modification process in the mitochondrion (RNA editing). While we have some understanding of the regulation of gene expression in the nucleus we know virtually nothing on how gene expression is regulated in the trypanosome mitochondrion. A major hurdle has been the lack of forward and reverse genetics approaches. The overall goal of the research described in this proposal is to develop a transfection system for trypanosome mitochondria using a novel, high throughput method, thereby providing a genetic approach that will allow us to address the questions of how mitochondrial gene expression is regulated during the life cycle of the parasite. In order to develop a mitochondrial transfection system we will: (1) determine the requirements for mitochondrial translation; (2) identify conditions for efficient, transient transfection of mitochondria in vivo and develop a positive selection scheme that allows stable transfection of mitochondria; and (3) Use this transfection system to begin to address the mechanism(s) regulating mitochondrial mRNA stability.
Project Description: The research professional supported by this grant has been working on development of a transfection system for T.b. brucei mitochondria. He has recoded the eGFP gene (meGFP) for mitochondrial expression and purchased the gene construct from GeneArt (Germany). He has added the mitochondrial untranslated regions (5'and 3' UTRs) of cytochrome c oxidase subunit III (COXIII) and ribosomal protein S12 (RPS12) by PCR and cloned the products into the pCR® Blunt II-TOPO® (Invitrogen) vector containing a T7 promotor. He has established flow cytometry analysis for mitochondrial vesicles, which to our knowledge has not been done before. Using percoll gradient purified mitochondria and the mitochondrial specific dye Mitotracker® (Invitrogen) we could distinguish between labeled and unlabeled vesicles.This technique will be critical to the development of an in organellar transfection system to establish optimal sequence environments for transcription and translation of transfection constructs. He developed a transient expression of the recoded eGFP in procyclic mitochondria in vivo.
Jobs Summary: One research professional position was created to carryout these studies. This indivdual is highly trained in trypanosome molecular biology and genetics. He brings uniquely valuable skills to the research laboratory. The availability of funds allowed me to retain this person working on African trypanosomes. Without ARRA funding this person would have either changed research directions in the Hajduk lab or sought other job opportunities. The retention of this individual is potentially valuable to the development of a better understanding of mitochondrial function in trypanosomes. (Total jobs reported: 1)
Project Status: Less Than 50% Completed
This award's data was last updated on May. 21, 2009. Help expand these official descriptions using the wiki below.