TRANSCRIPTION IN TRYPANOSOMATID CRITHIDIA FASCICULATA

昆虫寄生虫Crithidia fasciculata是 家族锥虫科，并分享许多独特的功能， 相关的致病性属锥虫属和利沙原虫属。 虽然不知道转录从哪里开始， 蛋白质编码基因在这些原生动物寄生虫，它已被 证明了这些细胞中可能所有mRNA的5'末端 生物体含有相同的35个核苷酸的非翻译前导序列 小外显子（mini-exon）。 该序列是在一个离散的家庭中发现的 串联重复的基因， 基因 这导致了这样的建议，即RNA合成在 锥虫是通过连接两个未连接的 前体RNA分子，这一过程被称为不连续的 转录。 本研究提案的长期实验目标 在C中定义一个转录单位。fasciculata并将其与 不连续转录的过程。 考虑到 这种现象的独特性，这项工作可能会导致理性 化疗的目标。 具体的实验建议是：1）表征 mini-exon基因重复序列及其在C. fasciculata使用 DNA和RNA滤膜杂交和DNA测序; 2） 表征编码α和β微管蛋白基因的蛋白质， 它们的表达，通过cDNA和基因组文库构建， DNA和RNA过滤杂交，特异性转录罕见 测量. 这些项目将确定 不连续的转录在Crithidia，定义区域 转录起始，并提供必要的材料， 后续研究。 3)开发一种方法， 将DNA导入Crithidia并测定表达。 该转染 系统将用于定义转录所需的序列 起始和小外显子添加。 4)开发一种体外 转录系统，使用Crithidia提取物和DNA模板， 作为研究特定转录的替代方法， 事件 相关分子生物学的研究计划， 生物化学提出。 本提案的总体目标是 获得必要的教学培训和实验室经验 研究者成为独立的生物医学 科学家

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.