This proposal examines the role of Ca2+ dependent regulation in the control of cellular activity in African trypanosomes with emphasis on calmodulin (CaM) and its associated proteins. These components regulate many critical activities in mammalian cells. By contrast, regulation of analogous activities in African trypanosomes, such as motility, secretion, division, energy metabolism and differentiation is not understood. Our preliminary studies indicate that African trypanosomes contain CaM, suggesting that, like host cells, trypanosomes utilize Ca2+ as a broad spectrum regulatory molecule. The long term objectives of this proposal are to identify and characterize components of the Ca2+ dependent regulatory pathways in African trypanosomes and evaluate the contribution of these components to the host-parasite complex. Two approaches will be used. The first is a biochemical characterization of trypanosome CaM (TCaM) and its associated proteins. It has the following specific aims: (a) isolation of TCaM using a hydrophobic affinity matrix; (b) determination of the amino acid sequence of TCaM by automated Edman degradation; (c) assessment of TCaM functions by (i) localizing its site of activity using immunofluorescense and immunoelectron microscopy, (ii) identifying and characterizing specific binding proteins of TCaM using 125Iodinated CaM probes and affinity column chromatography and (iii) surveying TCaM binding proteins for enzymatic activities modulated by CaM in other cell systems; (d) determination of stage specific expression of Ca2+ regulated proteins using a combination of metabolic labelling, cell fractionation and radioimmune assay. The second approach uses recombinant DNA methods to examine organization and expression of the TCaM gene.
Specific aims are: (a) Selection of TCaM containing sequences from trypanosome genomic libraries using a cDNA probe specific for electric eel CaM (pCM 109); (b) Generation of a probe containing TCaM sequences; (c) Investigation of CaM gene organization within the genome using restriction fragments of TCaM coding and flanking sequences and (d) Quantitation and characterization of CaMRNAs during trypanosome differentiation. The significance of this investigation lies in; its contribution towards understanding of cell regulation in African trypanosomes with the ultimate goal of identifying host signals that modify trypanosome function through the Ca2+ dependent pathways; its exploitation of the TCaM system as a model for regulation of gene expression during cell differentiation; and its identification of sensitive targets for the development of novel chemoprophylactics and chemotherapies.
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