Single stranded DNA Binding Proteins in Trypanosoma cru*

克鲁锥虫中的单链 DNA 结合蛋白*

• 批准号: 6401784
• 负责人: Noreen Williams
• 金额: $ 3.75万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6401784
• 关键词: DNA binding protein; SDS polyacrylamide gel electrophoresis; South America; Trypanosoma cruzi; affinity chromatography; confocal scanning microscopy; cooperative study; fluorescence microscopy; gel mobility shift assay; gene expression; genetic recombination; genetic regulation; genetic transcription; immunofluorescence technique; messenger RNA; northern blottings; nucleic acid repetitive sequence; protein degradation; protein localization; protein purification; protein sequence; protein structure function; protozoal genetics; reporter genes; southern blotting; western blottings

DESCRIPTION (provided by applicant): American trypanosomiasis, or Chagas' disease is a major public health problem among poor rural populations in Latin America, where 16-18 million persons suffer from this disease, and 90 million are at risk. Sharp differences in regulation of gene expression between trypanosomatids and their mammalian hosts have been described, suggesting this process as an interesting target for drug design. In these parasites, the presence of polycistronic transcription means that post-transcriptional regulation of gene expression is the main step in determining the abundance of mRNAs. Using nuclear protein fractions from Trypanosoma cruzi epimastigotes, the group in Uruguay demonstrated the formation of at least three complexes that specifically recognize two dinucleotide repeated sequences (poly[dT-dG] and poly[dC-dA]) which are abundant in the vicinity of coding regions of T. cruzi genes. The specificity and preliminary estimation of equilibrium constants of these complexes are in good agreement with reported data for gene regulatory proteins. The goal of this project is to determine whether these dinucleotide repeats and the proteins that bind to them have a role in regulation of gene expression, transcription or in selective transcript processing, degradation or stabilization or recombination. The specific aims of this FIRCA project are: 1. to characterize the proteins interacting with the cis target (purification and microsequencing), to determine the fine subcellular localization of these proteins by immunofluorescence microscopy and to determine if they show stage specific expression by western analysis. 2. to characterize the genes encoding these proteins (cloning, sequencing and analysis by southern blot and chromosomal location as well as northern blot analysis of their expression). 3. to examine the dinucleotide sequence for effect on expression of reporter constructs. The location and the minimum length of the repeat sequence and, if it corresponds, the stage at which expression is altered [posttranscriptional, translational, etc.] will be examined. These experimental approaches will lead to an understanding of the role of these dinucleotide repeated sequences and the function and significance of the proteins that specifically recognize them providing insights into the regulation of gene expression in T. cruzi.

描述（由申请人提供）：美国锥虫病，或Chagas' 疾病是拉丁裔农村人口贫困人口的主要公共卫生问题 美国，16-1800万人患有这种疾病，9000万人 有风险。在调节基因表达之间的急剧差异 已经描述了锥虫及其哺乳动物宿主，这表明了这一点 过程是药物设计的有趣目标。在这些寄生虫中 多余的转录的存在意味着转录后 基因表达的调节是确定丰度的主要步骤 mrnas。使用Cruzi Epimastigotes的核蛋白馏分， 乌拉圭的小组展示了至少三个配合物的形成 特别识别两个二核苷酸重复序列（poly [dt-dg] 和poly [dc-da]），它们在T的编码区域的附近很丰富。 克鲁兹基因。平衡的特异性和初步估计 这些复合物的常数与报告的基因数据非常吻合 调节蛋白。该项目的目的是确定是否 二核苷酸重复和与它们结合的蛋白质在 调节基因表达，转录或选择性转录本 处理，降解或稳定或重组。具体目的 这个FIRCA项目是：1。表征与蛋白质相互作用的蛋白质 顺式目标（纯化和微链测序），以确定罚款 通过免疫荧光显微镜和 确定它们是否通过西方分析表明特定于阶段的表达。 2 表征编码这些蛋白质的基因（克隆，测序和 通过Southern印迹和染色体位置以及Northern印迹分析 分析其表达）。 3。检查二核苷酸序列 对记者构建体表达的影响。位置和最低 重复序列的长度，如果对应，则是该阶段 表达会改变[转录后，翻译等] 检查。这些实验方法将导致对 这些二核苷酸重复序列以及功能和意义的作用 专门认识它们的蛋白质提供了见解 调节Cruzi T. Cruzi中的基因表达。