TRANSCRIPTION AND RNA PROCESSING IN TRYPANOSOMES

锥虫是毁灭性疾病的病原体， 在人类和牛中显示出许多不寻常的基因表达机制。 目前对它们的转录和 转录后控制基因表达，关于特定的DNA 序列和参与转录的蛋白质因子，以及关于 反式剪接反应的机制。 长期目标是 一项旨在揭示转录和反式剪接的生理机制的提案 并鉴定寄生虫特异性功能， 可以用来对付寄生虫本身。 我们计划：1） 确定哪些调节回路决定稳定RNA的最终输出 来自钙调素基因位点。 转录控制和存在 将通过连续转录（run-on transcription 实验 同时稳定各项成绩单 将通过测量RNA衰变率来确定，我们将研究 反式剪接可能与转录偶联。 （二） 建立一种能准确转录锥虫体外系统 基因. 我们将首先使用Crithidia fasciculata作为模型系统， 因为这种生物非常适合生化研究。 只有 体外转录系统的可用性将允许功能性的 锥虫启动子结构和相关因子的分析 在各种基因的转录中。 通过以下方式获得的信息： 然后应用短柱虫系统在体外建立布氏锥虫 提取物 用于体外转录的布氏锥虫DNA模板 将利用体外运行获得的信息进行设计 测定和体内足迹实验。 3)克隆cDNA编码 布氏锥虫小核核糖核蛋白免疫显性抗原 粒子来获得有关蛋白质一级结构的信息 并且能够产生多克隆单特异性抗体。 这些 抗体将被用来研究snRNP的结构及其 与合成的前mRNA底物结合。 4)识别分子 妨碍利用布氏锥虫前mRNA 3'剪接的缺陷 哺乳动物剪接提取物中的位点。 这将是特别感兴趣的 为了确定锥虫剪接底物的功能障碍 是序列特异性的。 如果我们成功地纠正了缺陷， 使用哺乳动物提取物来重建反式剪接成为可能。 总之，这些实验将为详细的 了解基因表达和反式剪接的机制， 布氏锥虫细胞。

Trypanosomes which are the etiological agents of devastating diseases both in man and in cattle display many unusual mechanisms of gene expression. At present little is known about their transcriptional and post-transcriptional control of gene expression, about the specific DNA sequences and protein factors involved in transcription, and about the mechanism of the trans-splicing reaction. The long term goal of this proposal is to unravel the physiology of transcription and trans-splicing in Trypanosoma brucei and to identify parasite-specific functions which might be exploited against the parasites themselves. We plan to: 1) identify what regulatory circuits determine the final output of stable RNAs from the calmodulin gene locus. Transcriptional control and the presence of unique or multiple promoters will be assayed by run-on transcription experiments. At the same time the stability of the various transcripts will be determined by measuring RNA decay rates and we will investigate the possibility that trans-splicing might be coupled to transcription. 2) establish an in vitro system capable of accurately transcribing trypanosome genes. We will initially use Crithidia fasciculata as a model system, because this organism is ideally suited for biochemical studies. Only the availability of an in vitro transcription system will allow the functional analysis of trypanosome promoter architecture and of the factors involved in the transcription of various genes. The information obtained with the Crithidia system will then be applied to establish a T.brucei in vitro extract. The T.brucei DNA templates to be used for in vitro transcription will be designed utilizing the information obtained by in vitro run-on assays and by in vivo footprinting experiments. 3) clone cDNAs coding for the immunodominant antigens of T.Brucei small nuclear ribonucleoprotein particles to obtain information about the primary structure of the proteins and to be able to generate polyclonal monospecific antibodies. These antibodies will be used to investigate the structure of snRNPs and their binding to synthetic pre-mRNA substrates. 4) identify the molecular defect(s) which preclude the utilization of T.brucei pre-mRNA 3' splice sites in a mammalian splicing extract. It will be of particular interest to determine whether the malfunctioning of trypanosome splicing substrates is sequence specific. If we succeed in correcting the defect(s), it will become possible to reconstitute trans-splicing using a mammalian extract. Together, these experiments will provide the basis for a detailed understanding of the mechanism of gene expression and trans-splicing in T.brucei cells.