Gene Expression During Trypanosome Differentiation

Hajduk, Stephen

Abstract

I plan to initiate studies on the expression of mitochondrial and nuclear genes involved in mitochondrial biogenesis during the developmental cycle of Trypanosoma brucei. The developmental stages of T. brucei found in the insect vector, or in culture at 26C have a fully funcional mitochondrion with Krebs cycle enzymes and functional cytochromes. In the bloodstream of the mammalian host the trypanosome's mitochondrion is devoid of cristae and Krebs cycle enzymes and cytochromes are not detectable. The bloodstream trypanosomes share with yeast the unusual ability to completely repress mitochondrial biogenesis and to survive entirely by glycolysis. There are several possible molecular mechanisms by which mitochondrial activities are repressed during the differentiation process. By analysis of the genomic DNA, transcripts and protein products with recombinant DNA probes and antibodies it will be possible to distinguish between the following possible mechanisms. (A) Transcription of genes in the nucleus or the mitochondrion of the trypanosome, coding for products essential for mitochondrial biogenesis, may be completely repressed in the mammalian host. The termination of transcription may be associated with rearrangements in the gene or flanking nucleotide sequences or catabolic repression. (B) Transcription may occur but processing events, such as splicing, may be altered in the bloodstream resulting in either a non-translated message or nonfunctional product. (C) Translation may be unaltered but the protein is not trans-located into the mitochondrion. (D) Finally, it is possible that a processing or assembly step in the mitochondria is altered resulting in a non-functional protein. I have chosen to study the structure of the cytochrome c and cytochrome b genes and the regulation of their expression. Both genes are developmentally regulated in trypanosomes and their expression appears to be closely linked. Cytochrome c is coded for entirely by the nuclear genome and cytochrome b is coded for by the mitochondrial genome. Additional studies on the development of an in vitro mitochondrial transcription system will be initiated. The long-term objective of the research described is to determine basic molecular mechanisms involved in the regulation of gene expression in a complex, differentiating system.