The infection of man and his domestic animals by African trypanisomes has highly deleterious social, economic and nutritional effects in the endemic area. Existing drugs for trypanosomiasis are unsatisfactory, vector (testse) control is difficult and immuization is impractical owing to the ability of the organisms to change their surface coat. New approaches to treatment are urgently needed. When trypanosomes are transferred from the mammalian bloodstream to the gut of the vector and vice-versa, they must change their energy metabolism drastically in order to adapt to the new environment. In the bloodstream form, energy is generate from glygolysis and substrate-level phosphorylation, the necessary rapid rate of glucose breakdown being failitated by compartmentation of the enzymes involved within a microbody, the glucosome. In the vector (procyclic) form, gltcolysis slower and energy is generated in the mitochondrion. Dramatic changes in the parasite surface proteins are also apparent. The unltimate goal of the project is to understand how trypanosomes regulate their metabolism at the molecular level. We will concentrate on mechanisms controlling the levels of mRNA transcripts, using as models the genes cloned and characterized during the first three years of the project: those encoding fructose bisphophate aldolase, which is induced in bloodstream forms, and procyclic- specific acid repetitive protein (PARP) which is probably on the surface of procyclic forms. Both are present as tendem repeats in the genome; the aldolase genes are interspersed with at least two other transcribed regions whose corresponding mRNA show no developmental regulation. The PARP genes take three forms which are distinguishable by hybridization arranged as two types of tandem repeat. It is possible that the PARP promoter and regulatory functions are specified very close to the 5' end of the upstream genes. The regions surrounding all the genes will be mapped exhaustively. Further genomic clones representing fragments of these regions obtained and used as probes on RNA blots to see if they are homologous to any stable RNA. The precise extent of each RNA will be determined by comparison of genomic and cDNA sequences and by nuclease protection mapping. The genomic clones will be used as probes on genomic DNA blots to see if they are repetitive or single-copy in the genome. Each region will be hybridised with radioactive RNA synthesised in isolated trypanosome nuclei. From the results, an idea of the extenet of the transcription unit and the possible position of promoters may be obtained. If no region is found which is not transcribed additional clones from further upstream will be obtained and analysed as before. The existence of polycistronic transcripts will be investigated by analysing RNA species whose processing is incomplete. Such precursors are synthesized in isolated nuclei; large quantities can be obtained by subjecting trypanosomes to heat shock. If suitable methodology is available, the putative promoter regions will be linked to a gene whose product is readily detectable and the construct transfected into trypanosomes by electroporation to look for promoter function. Detection of RNA may be accomplished directly by, for instance, primer extension analysis; alternatively a protein product may be assayed. One option is to transfect genomic clones containing the PARP gene and upstream DNA in trypanosomatids lacking PARP sequences and assay expression directly using anti-PARP antisera. IF the transfected DNA is expressed, promoters will be defined by deletion and site- directed mutagenesis. If no suitable transfection methodology is available, additional genes encoding glycosomal enzymes which are induced in procyclics will be cloned and characterized. The first one to be attempted will be malate dehydrogenase; alternatives include phosphoenolpyruvate carboxykinase and adenylate kinase.
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