NOVEL DNA-BINDING FACTOR SPECIFIC TO BLOOD CELLS

血细胞特异性的新型 DNA 结合因子

• 批准号: 2134099
• 负责人: David William O'Neill
• 金额: $ 8.29万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-01 至 1998-12-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2134099
• 关键词: DNA binding protein; animal genetic material tag; blood cells; chemical binding; embryonic stem cell; gene mutation; gene rearrangement; genes; genetically modified animals; hematopoiesis; hemoglobin; human genetic material tag; laboratory mouse; protein purification; site directed mutagenesis

Since the expression of globin genes is regulated in both a temporal and tissue-specific manner, the globin gene family represents a model system for the study of gene regulation in mammalian development. Insights into the molecular basis of hemoglobin switching may also lead to new therapies for inherited anemias such as sickle cell disease and beta- thalassemia. We have recently identified a DNA binding factor (pyr factor, or PYR-F) which is restricted to blood cells late in development and which binds to a putative regulatory site for fetal-to-adult globin gene switching. The PYR-F binding site is capable of adopting a complex secondary structure composed of triple-and single-stranded segments (H- DNA), which is believed to be important in gene regulation. The highly restricted tissue- and developmental stage-specific pattern of PYR-F DNA binding activity, the location of its binding site, and the ability of the binding site to adopt a non-B DNA structure suggests that PYR-F may function in hematopoietic cell development and hemoglobin switching. This proposal outlines a plan to determine the structure and function of PYR-F by: (1) studying the effect of deleting the PYR-F binding site on the control of human hemoglobin switching in transgenic mice; (2) purification of PYR-F to homogeneity, with the ultimate goal of using amino acid sequence data to obtain a cDNA clone; (3) using the mouse PYR- F cDNA clone is to isolate the human cDNA and mouse and human genomic DNA clones; and (4) studying the function of PYR-F in blood cell development by creating a targeted mutation of the PYR-F gene in mouse embryonic stem cells.

由于球蛋白基因的表达在时间和时期都受到调节 组织特异性的方式，球蛋白基因家族代表模型系统 用于研究哺乳动物发育中的基因调节。 深入了解 血红蛋白转换的分子基础也可能导致新 遗传性贫血的疗法，例如镰状细胞疾病和β- 地中海贫血。 我们最近确定了DNA结合因子（Pyr 因素，或pyr-f），该因素仅限于发育后期的血细胞 它与胎儿到成年环球蛋白的推定监管部位结合 基因切换。 pyr-f结合位点能够采用复合物 二级结构由三重和单链段组成（h- DNA），被认为在基因调节中很重要。 高度 PYR-F DNA的组织和发育阶段特异性模式受限 结合活动，其结合位点的位置以及 采用非B DNA结构的结合位点表明Pyr-f可以 在造血细胞发育和血红蛋白转换中的功能。 该提案概述了确定的计划和功能 Pyr-f作者：（1）研究删除pyr-f结合位点对 转基因小鼠中人血红蛋白转换的控制； （2） 将PYR-F纯化为同质性，其最终目的是使用 氨基酸序列数据以获得cDNA克隆； （3）使用小鼠pyr- F cDNA克隆是要分离人cDNA，小鼠和人基因组DNA 克隆； （4）研究PYR-F在血细胞发育中的功能 通过在小鼠胚胎茎中创建PyR-F基因的靶向突变 细胞。