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.The goal of this proposal is to study the nucleotide sequence variability of human trypanosomes and to correlate these data with the description of their surface antigens, particularly with the presence of the glycosyl-phosphatidyl-inositol (GPI) anchor. Our past efforts have been directed towards the identification of the genes encoding variable GPI-anchored surface components. We have now extended these studies by characterizing, with oligonucleotides selected to represent the DNA variable segments, the corresponding RNA-cDNA and genomic sequences. The nucleotide sequence of the entire GPI-anchored glycoprotein gene from three different strains of human pathogenic Trypanosoma brucei has been determined. All three gene structures were found to be in good correlation with the corresponding proteins. These observations confirm that gene duplication and recombination contribute to the generation of the surface antigen variability in Trypanosoma and that trypanosome genes related to the protein coding region of the invariant merozoite surface glycoprotein (MSP) gene may be found elsewhere on the genome. We are now turning our attention to the characterization of the structure and expression of these additional surface antigens in order to characterize their antigenic variability. The occurrence of the GPI anchor in trypanosomes has not been demonstrated in non-pathogenic trypanosomes. As these parasites are capable of infecting a variety of mammals, it is essential to determine if these species carry the GPI anchor. Using a monoclonal antibody, we have partially characterized a Mr = 77,000 surface antigen that appears to be GPI-anchored in all T. brucei strains and T. congolense stocks examined. This antigen also appears to be the first identified member of a multigene family. The variability of its expression as well as its biogenesis will be studied in order to better understand the nature of this GPI-anchored surface protein. Finally, by developing a cloning vector for T. brucei, we will extend the studies of trypanosome surface components. This vector will be used to study the organization, function and expression of the variant surface glycoprotein genes in T. brucei. These studies will characterize the conservation and diversity of the trypanosome surface proteins and will provide a solid background for analyzing the function of these molecules.Q:
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