The insect parasite Crithidia fasciculata is a member of the family Trypanosomatidadae, and shares many unique features with the related pathogenic genera Trypanosoma and Leishamania. Though it is not known where transcription initiates for any protein coding gene in these protozoan parasites, it has been demonstrated that the 5' termini of probably all mRNAs in these organisms contain and identical 35 nucleotide untranslated leader sequence (mini-exon). The sequence is found in a discrete family of tandem repeat genes which are unlinked to any protein coding gene. This has led to the suggestion that RNA synthesis in trypanosomatids is accomplished by the ligation of two unlinked precursor RNA molecules, a process termed discontinuous transcription. The long term experimental objectives of this research proposal are to define a transcription unit in C. fasciculata and relate it to the process of discontinuous transcription. In light of the uniqueness of this phenomenon, this work may lead to rational targets for chemotherapy. The specific experimental proposals are to 1) characterize the mini-exon gene repeat and its expression in C. fasciculata using DNA and RNA filter hybridization and DNA sequencing; 2) characterize the protein coding alpha and beta tubulin genes and their expression, through cDNA and genomic library construction, DNA and RNA filter hybridization, and specific transcription rare measurements. These projects will establish the existence of discontinuous transcription in Crithidia, define regions of transcription initiation, and provide necessary material for succeeding studies. 3) Develop a method of introducing exogenous DNA into Crithidia and assay for expression. This transfection system will be used to define sequences required for transcription initiation and mini-exon addition. 4) Develop an in vitro transcription system, using Crithidia extracts and DNA templates, as an alternate approach to studying specific transcriptional events. A program of study in relevant molecular biology and biochemistry is proposed. The overall goal of this proposal is to acquire the didactic training and laboratory experience necessary for the Investigator to become an independent biomedical scientist.
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