TRANSCRIPTION IN TRYPANOSOMATID CRITHIDIA FASCICULATA

昆虫寄生虫丛枝克里迪亚是一种 锥虫科，并与许多独特的特征 相关致病属锥虫和利沙曼原虫。 尽管目前还不知道任何一种 在这些原生动物寄生虫的蛋白质编码基因中，它已经 证明了这些基因中可能所有mRNAs的5‘末端 生物含有和相同的35个核苷酸未翻译的前导 序列(迷你外显子)。该序列是在一个离散的家族中发现的 与任何蛋白质编码无关的串联重复基因 吉恩。这导致了这样一种说法，即在体内合成RNA 锥虫是通过连接两个未连接的 前体RNA分子，一种称为不连续的过程 抄写。 这项研究计划的长期实验目标 是在簇毛锦鸡儿中定义一个转录单位并将其与 不连续转录的过程。鉴于 这种现象的独特性，这项工作可能会导致理性 化疗的靶点。 具体的实验建议是：1)表征 微小外显子基因重复序列及其在簇毛锦鸡儿中的表达 DNA和RNA过滤器杂交和DNA测序；2) 鉴定编码α和β微管蛋白基因的蛋白质 通过构建cDNA和基因组文库来表达它们， DNA和RNA过滤器杂交，以及罕见的特异转录 测量。这些项目将确立 在克里斯蒂亚目中不连续转录，定义区域 转录启动，并提供必要的材料 后续研究。3)开发引入外源基因的方法 将DNA导入卵裂殖道，并进行表达检测。这种转染法 系统将用于定义转录所需的序列 启动和微小外显子加成。4)开发一种体外培养 转录系统，使用克里西迪亚提取液和DNA模板， 作为研究特定转录的另一种方法 事件。 相关分子生物学的研究计划和 提出了生物化学的概念。这项提案的总体目标是 获得必要的教学培训和实验室经验 让研究人员成为一名独立的生物医学工作者 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.