FACTORS CONTROLLING PROMYELOCYTIC EXPRESSION OF DEFENSIN

控制防御素早幼粒细胞表达的因素

• 批准号: 2429916
• 负责人: Yongsheng Ma
• 金额: $ 0.45万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-07 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2429916
• 关键词: DNA footprinting; binding proteins; biochemistry; cell type; complementary DNA; computer assisted sequence analysis; crosslink; defensins; gel mobility shift assay; gene expression; genetic regulation; high performance liquid chromatography; mass spectrometry; molecular site; myelocyte; neutrophil; protein purification; reporter genes; site directed mutagenesis; southern blotting; tissue /cell culture

The study of maturation stage-dependent expression of genes that are specific for the functions of neutrophils is relevant to better understand, mechanistically, myeloid blood cell differentiation, Since acute myelogenous leukemia can be considered to derive from a block in differentiation, such new information may provide more insights into the pathogenesis of this disease as well. Defensins (HNP) are microbicidal peptides and the principal constituents of human neutrophil granules. HNP-1 transcripts are selectively present in promyelocytic leukemia cells (HL-60) and unique temporal regulation occurs during drug-induced differentiation. A 165 bp minimal promoter was identified in the 5'- upstream region of the HNP-1 gene that directs HL-60-specific gene expression in-vivo; no consensus binding sites for known factors are present The main objective of this proposal is to study putative novel factors, or factor combinations, that control promyelocytic expression of defensin. To this end, a three-stage approach will be used. First, control elements will be precisely located, based on their ability to i) drive HL-60 specific expression of reporter genes in transient transfection assays, and ii) give specific band patterns in electrophoretic mobility assays (EMSAs). Second, the consensus recognition sites for the binding proteins will be determined by i) in- vitro footprinting using HL-60 nuclear extracts, and ii) site-directed mutagenesis and functional analysis (as in stage I). Finally, binding factors will be characterized by, in order, i) crosslinking and Southwestern blotting, ii) column chromatographic (EMSA assaying) and gelelectrophoretic purification, iii) microsequencing / mass spectrometric analysis of protein fragments, iv) identification and/or cloning of the cDNA, v) examination of cell specific expression, and vi) examination of transactivating potential in other cell types.

基因的成熟阶段依赖性表达的研究 中性粒细胞功能的特定与更好的功能有关 从机械上理解髓样血细胞分化，因为 可以认为急性髓性白血病是从 差异化，此类新信息可能会提供更多的见解 该疾病也是发病机理。防御素（HNP）是生液 肽和人类嗜中性粒细胞颗粒的主要成分。 HNP-1成绩单有选择地存在于Promyelocytic白血病中 细胞（HL-60）和独特的时间调节在药物诱导的过程中发生 分化。在5'-中鉴定出165 bp的最小启动子 引导HL-60特异性基因的HNP-1基因的上游区域 体内表达；对于已知因素没有共识结合位点是 提出该提案的主要目的是研究推定的小说 控制临时细胞表达的因素或因子组合 防御素。为此，将使用三阶段的方法。第一的， 控制元素将根据其能力精确地位于位置 驱动HL-60在瞬时基因的特异性表达 转染测定法，ii）在 电泳迁移率分析（EMSA）。第二，共识 结合蛋白的识别位点将由i）in-确定 使用HL-60核提取物进行体外足迹，ii）定向 诱变和功能分析（如第一阶段）。最后，绑定 因素的特征是，i）交联和 西南印迹，ii）色谱柱色谱法（EMSA分析）和 凝胶摄取的纯化，iii）微钉 /质量 蛋白质片段的光谱分析，iv）鉴定和/或 克隆cDNA，v）检查细胞特异性表达和VI） 检查其他细胞类型的反式激活潜力。