DNA-BINDING PROTEINS IN TRYPANOSOMA BRUCEI

微生物感染化疗的潜在靶点是 病原体特有的生化途径。 为 非洲锥虫，所有信使的强制性合成 通过双连续转录的RNA代表了这样的靶点。 战略 这项研究的长期目标是分析基因结构 和不连续转录。 的 这里描述的实验将定义蛋白质-DNA相互作用 在a）的转录起始位点附近， 外显子）（5 ′-外显子）和B）代表性蛋白质编码基因（3 ′- 外显子）。 这两类基因代表了 涉及不连续转录的离散遗传位点。 凝胶- 阻滞和核酸外切酶III保护试验将用于 鉴定和表征特定蛋白质-DNA相互作用， 克隆的小外显子基因与来自 T.布鲁塞。 通过色谱法富集的核提取物 特异性DNA结合蛋白将用于DNA足迹法 用于鉴定与蛋白质结合的核苷酸的测定。 活性泛素基因的转录起始位点和泛素的表达 117 VSG基因，两种明显不同的3 '-外显子，将被 通过粘粒克隆鉴定， 核连续试验和核酸酶保护试验。 凝胶阻滞 将使用测定来表征结合至 这些3 '-外显子的转录指示位点。

Potential targets for chemotherapy in microbial infections are the biochemical pathways which are unique to the pathogen. For the African trypanosomes, the obligatory synthesis of all messenger RNAs by dicontinuous transcription represents a target for such a strategy. The long term aim of this research is to analyze gene structure and discontinuous transcription in Trypanosoma brucei. The experiments described here will define protein-DNA interactions in the proximity of the start sites of transcription of a) the mini- exon) (5'-exon) and b) representative protein-coding genes (3'- exons). These two-classes of genes are representative of the two discrete genetic loci involved in discontinuous transcription. Gel- retardation and Exonuclease III-protection assays will be used to identify and characterize specific protein-DNA interactions on a cloned mini-exon gene incubated with crude nuclear extracts from T. brucei. Nuclear extracts enriched by chromatography for the specific DNA-binding proteins will be used in DNA footprinting assays to identify the nucleotides bound by the protein. The transcription initiation sites of active ubiquitin genes and the 117 VSG gene, two apparently different classes of 3'-exon, will be identified by cosmid cloning, the analysis of nascent RNA by nuclear run-on and nuclease-protection assays. Gel retardation assays will be used to characterize proteins binding to the transcription indication sites of these 3'-exons.