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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI022229-01
Application #
3133100
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1985-04-01
Project End
1988-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Type
Graduate Schools
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Patnaik, P K; Bellofatto, V; Hartree, D et al. (1994) An episome of Trypanosoma brucei can exist as an extrachromosomal element in a broad range of trypanosomatids but shows different requirements for stable replication. Mol Biochem Parasitol 66:153-6
Medina-Acosta, E; Paul, S; Tomlinson, S et al. (1994) Combined occurrence of trypanosomal sialidase/trans-sialidase activities and leishmanial metalloproteinase gene homologues in Endotrypanum sp. Mol Biochem Parasitol 64:273-82
Patnaik, P K; Fang, X; Cross, G A (1994) The region encompassing the procyclic acidic repetitive protein (PARP) gene promoter plays a role in plasmid DNA replication in Trypanosoma brucei. Nucleic Acids Res 22:4111-8
Medina-Acosta, E; Beverley, S M; Russell, D G (1993) Evolution and expression of the Leishmania surface proteinase (gp63) gene locus. Infect Agents Dis 2:25-34
Medina-Acosta, E; Cross, G A (1993) Rapid isolation of DNA from trypanosomatid protozoa using a simple 'mini-prep' procedure. Mol Biochem Parasitol 59:327-9
Hug, M; Carruthers, V B; Hartmann, C et al. (1993) A possible role for the 3'-untranslated region in developmental regulation in Trypanosoma brucei. Mol Biochem Parasitol 61:87-95
Patnaik, P K; Kulkarni, S K; Cross, G A (1993) Autonomously replicating single-copy episomes in Trypanosoma brucei show unusual stability. EMBO J 12:2529-38
Sherman, D R; Janz, L; Hug, M et al. (1991) Anatomy of the parp gene promoter of Trypanosoma brucei. EMBO J 10:3379-86
Clayton, C E; Fueri, J P; Itzhaki, J E et al. (1990) Transcription of the procyclic acidic repetitive protein genes of Trypanosoma brucei. Mol Cell Biol 10:3036-47
Cross, G A; Bellofatto, V; Clayton, C E et al. (1990) Using transfection to study gene expression in trypanosomes. Biochem Soc Trans 18:714-6

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