TRANSCRIPTION AND RNA PROCESSING IN TRYPANOSOMES

锥虫是毁灭性疾病的病原体 在人和牛身上显示了许多不寻常的基因表达机制。 目前对它们的转录和表达情况知之甚少 关于特定DNA的基因表达的转录后控制 参与转录的序列和蛋白质因子，以及关于 反式剪接反应的机理。这样做的长期目标是 建议是解开转录和反式剪接的生理学 在布鲁氏锥虫中，并鉴定寄生虫的特异性功能 可能被用来对付寄生虫本身。我们计划：1) 确定哪些调节电路决定稳定RNA的最终输出 来自钙调蛋白基因座。转录控制和存在 的唯一或多个启动子将通过连续转录进行检测 实验。同时，各种成绩单的稳定性 将通过测量RNA衰减率来确定，我们将研究 反式剪接可能与转录有关。2) 建立准确转录锥虫的体外系统 基因。我们最初将使用丛枝克里迪亚作为一个模型系统， 因为这种生物非常适合进行生化研究。只有 体外转录系统的可用性将使功能 锥虫启动子结构及其影响因素分析 在各种基因的转录过程中。使用获取的信息 然后应用克里迪亚系统在体外建立布鲁氏毛滴虫 提取。用于体外转录的布鲁氏锥虫DNA模板 将利用体外试管获得的信息进行设计 测定和体内足迹实验。3)克隆编码的cDNA 布鲁氏毛滴虫小核糖核蛋白的免疫优势抗原 颗粒以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 抗体将被用于研究SNRNPs的结构和它们的 与合成的前信使核糖核酸底物结合。4)分子鉴定 阻碍布氏毛滴虫Pre-mRNA3‘剪接利用的缺陷(S) 哺乳动物剪接提取物中的位点。它将会特别引起人们的兴趣 以确定锥虫体剪接底物的故障 是特定于序列的。如果我们成功地纠正了缺陷(S)，它就会 使用哺乳动物提取物重建反式剪接成为可能。 总而言之，这些实验将为详细的 对基因表达和反式剪接机制的理解 布氏毛滴虫细胞。

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.