The infection of man and his domestic animals by African trypanosomes has highly deleterious social, economic and nutritional effects in the endemic area. Existing drugs for trypanosomiasis are unsatisfactory, vector (tse-tse) control is difficult, and immunization is impractical owing to the ability of the organism to repeatedly change its surface coat. New approaches to treatment are urgently needed.
The aim of this proposal is the cloning and characterization of a small number of nuclear genes encoding metabolic enzymes from Trypanosoma brucei, with strong emphasis on components of the glycosome. This organelle is unique to trypanosomatids: in addition to some of the enzymes essential for pyrimidine synthesis, it contains most of the glycolytic pathway, upon which bloodstream trypanosomes are entirely dependent. This glycosome is therefore a promising target for chemotherapy. A lambda expression library will be screened for glycosomal components using antibody to purified glycosomes, if a suitably specific antiserum can be obtained. In addition, the possibility of cloning trypanosome genes of specific predetermined function by utilizing their ability to complement corresponding yeast mutants will also be thoroughly explored, using trypanosome cDNA in a yeast expression vector. Successful implementation of this latter idea would enable many trypanosome genes to be isolated even if the corresponding products are of very low abundance, and totally uncharacterized. It would also set a powerful precedent for cloning metabolic genes from less experimentally amenable parasites whose enzymes cannot be characterized because insufficient material is available. Alternative approaches will include differential cDNA hybridization to seek genes that are more abundantly transcribed in bloodstream than in procyclic (insect inhabiting) trypanosomes, as is the case for some glycosomal enzymes. Two of the cloned cDNAs, their corresponding gene and mRNAs will be thoroughly characterized to obtain information about protein structure, mRNA processing, and control of transcription. For comparison, a previously cloned sequence which is expressed only in procyclics will also be studied. A start will be made on the study of synthesis, processing and enzymic properties of the encoded products.
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