The objective of this proposal is to determine the molecular events at the RNA and DNA level that are responsible for antigenic variation in African trypanosomes. Several projects are planned, most of which are extensions of research currently in progress. The RNA polymerase activities in trypanosome cells are being examined. The separation procedures that have been used to resolve the three RNA polymerase activities in other eukaryotes yield only one peak of RNA polymerase activity in trypanosome extracts. The reason for this difference will be explored by (i) further efforts to purify this activity, (ii) examination of in vitro transcription of purified DNA templates by trypanosome extracts and (iii) characterization of the gene(s) for one or more trypanosome RNA polymerase subunits that have been identified by cross-hybridization with heterologous gene probes. Several additional trypanosome genes that have specific properties of interest will be examined. These genes include ones that code for (i) phosphoglycerol kinase, alcohol dehydrogenase and rRNAs, (ii) three bloodstream VSGs whose order of appearance in a serodeme appears to be either pre-programmed (in one case) or random (in another case) and (iii) VSGs that are synthesized during the metacyclic state of trypanosome development or are unique in some other way. Another series of experiments will investigate the distinctive features of the 5'-termini of many, if not all, trypanosome mRNAs including the VSG mRNAs. The possible occurrence of 5'-CAP structures will be determined, the presence of the 35-mer on structural RNA precursors will be investigated and the potential precursor-product relationship between the transcript of the 35-mer DNA coding region and the 35-mer at the 5'-termini of RNAs will be established. It is anticipated that the results of these projects will contribute to a better understanding of the molecular basis for antigenic variation and facilitate the design of better methods to combat or control the disease caused by this parasite.
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