The research in the initial three years of this grant focused on the specificity of mammalian RNA maturation pathway(s). We demonstrated that chimeric RNAs which were transcribed by RNA polymerases III or I and contained complete or partial copies of protein-encoding genes were not substrates for the RNA maturation pathways normally utilized by RNA polymerase II transcripts. We next turned our efforts to investigation of RNA maturation in trypanosomatids, lower eukaryotes in which RNA production appears to involve unprecedented mechanisms, including the apparent trans- splicing of a common 35 nucleotide leader segment (that is no encoded in the same DNA region as the gene body) onto the 5' terminus of each mRNA. In trypanosomatids, definition of transcriptional control elements and even of transcription initiation sits has proven elusive for some RNAs, these elements evidently lie >50 kb upstream of the gene body. To effectively study transcription and RNA processing events in trypanosomatids, we have now succeeded in transfecting a trypanosomatid gene into T. brucei cells and demonstrating specific expression of this re- introduced gene. With this system, we shall study in detail the promoter of one T. brucei gene (the rRNA gene) and we shall also attempt to obtain transcription of other transfected trypanosomatid genes to enable study of the trans-splicing process. Most promising is the analysis of a variant surface antigen (VSG) gene, no only because it is through successive expression of different members of this gene family that the host immune system is evaded, but also because these genes are thought to be transcribed by RNA polymerase I. In addition, a variety of other trypanosomatid genes will be examined. In a companion approach, we will utilize Crithidia, an insect parasite that can be grown inexpensively in essentially unlimited amounts in culture. We have already demonstrated that this organism utilizes 5' trans-splicing mRNA leaders and we have cloned and characterized representative Crithidia mini-exon genes. We shall now attempt to develop a DNA transfection system analogous to that already demonstrated for T. brucei and in vitro transcription and translation systems using the mini-exon and other Crithidia genes in order to study the mechanism of mini-exon trans-splicing and the potential function of the mini- exon sequence in translation.
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