Gene Expression During Trypanosome Differentiation

Hajduk, Stephen

Abstract

African trypanosomes undergo a series of complex development changes in the mammalian host and the insect vector. Perhaps the most dramatic of these changes involve the pathways for ATP production. In the bloodstream developmental stages mitochondrial electron transport and oxidative phosphorylation are suppressed and ATP production is entirely from glycolysis. The insect midgut developmental stage has a fully operative Krebs cycle and electron transport chain. The overall goal of the experiments described in this grant will be to define the molecular mechanisms involved in the control of mitochondrial activities during Trypanosoma brucei development. To approach this problem four fundamental aspects of the molecular biology of trypanosomes will be investigated: 1) Transcription of the maxicircle will be studied to determine the primary transcription units and the DNA sequences involved in the control of maxicircle transcription during trypanosome development. Factors which interact with these regulatory sequences will be isolated and the role of these factors in the developmental control of maxicircle transcription determined. To achieve this goal an in vitro transcription system will be established using purified trypanosome mitochondrial RNA polymerase. 2) The in vivo organization of the kinetoplast DNA (kDNA) will be studied, the role of polyamines and DNA binding proteins in the packaging will be determined and the potential role of the bent helical portion of the minicircle in directing the packaging in vitro determined. 3) The developmental control of cytochrome c translation and subsequent transport into mitochondria will be examined. In vitro and in vivo studies on the translation of cytochrome c will be undertaken to determine the regulatory step in the assembly of this protein in the mitochondrion of T. brucei. 4) Transcription of the minicircle component of the kDNA network will be studied to determine the nature of the primary transcript and the DNA sequences controlling minicircle transcription. The sequence of the minicircle transcript will be determined from cDNAs, the putative protein product isolated and the possible role of the protein in kDNA packing determined. The results of these studies will help to elucidate the basic molecular mechanisms controlling mitochondrial biogenesis in African trypanosomes and will eventually lead us to the environmental factors modulating trypanosome development. This knowledge will be useful in the development of new strategies to control these important parasites.