CHARACTERIZATION OF HUMAN BETA-GLOBIN TRANS-ACTING REGULATORY FACTORS

人 β-珠蛋白交易调节因子的表征

• 批准号: 5213659
• 负责人: ZARRINTAJ ALIABADI
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5213659
• 关键词: DNA footprinting; Escherichia coli; aminoglycoside antibiotics; complementary DNA; gene complementation; genetic library; genetic promoter element; genetic regulation; genetic transcription; globin; human genetic material tag; messenger RNA; molecular cloning; nuclear runoff assay; nucleic acid probes; phosphotransferases; plasmids; polymerase chain reaction; protein structure function; tissue /cell culture; transcription factor; transfection; transfection /expression vector

Resolving the genetic regulatory mechanism(s) and biochemical factors involved in human beta-globin gene switching is the goal of research outlined in this application. Switching is the process of temporal expression of beta-globin-like polypeptides during the developmental transition from fetal to postnatal life. Of particular interest is the molecular switch from fetal hemoglobin to adult hemoglobin, presumably, as a result of derepression (or induction) of beta-globin chain production and suppression of gamma-globin transcription upon birth. The clinical importance of this step lies in the fact that elevated amounts of fetal hemoglobin can provide considerable protection to patients suffering from sickle cell anemia. Cell fusion studies involving two erythroleukemia cell lines, one human-derived, K562, and one derived from mouse, MEL, has shown the switch from gamma-globin to Beta-globin chain production depends on trans-acting factors. The two cell lines differ, therefore, in the type of hemoglobin they can produce upon induction; fetal for the human cell line and adult for the mouse cell line. The research proposed will focus on utilizing novel methods to directly clone the gene(s) encoding the regulatory factors involved in the transcriptional regulation of the beta-globin gene through positive selection methods. A modified version of the Okayama-Berg plasmid has been constructed. The new plasmid can be selected for when carrying the cDNA of a beta-globin gene trans-activator. The selection of the plasmid is dependent on the expression of a bacterial aminoglycoside 3'- phosphotransferase gene under the transcriptional control of the human beta-globin promoter. We will construct total cDNA libraries from one cell line (gamma-globin off, beta-globin on) in our plasmid. This library will subsequently be introduced into another cell line (gamma-globin on, Beta-globin off) to identify clones which express trans-acting factors that actIvate the transcription of the Beta-globin gene. The known sequences flanking The cDNA cloning site of the plasmid will be used to retrieve the insert from the human cells utilizing PCR techniques. We will reclone the amplified cDNA into bacterial vectors to be used as a probe to recover the chromosomal copy of the gene. We will sequence both the native and the cDNA clones. We will purify the transfactor(s) by over expressing the gene in E. coiL The purified protein will be used in structure-function analysis experiments. These studies will produce considerable insight into the switching phenomenon which ultimately could provide new approaches to management of sickle cell anemia.

解析遗传调控机制(S)与生化因素 参与人类β-珠蛋白基因切换是研究的目标 在本申请中概述。转换是一个暂时的过程 β-珠蛋白样多肽在发育过程中的表达 从胎儿过渡到出生后的生活。特别值得关注的是 从胎儿血红蛋白到成人血红蛋白的分子开关，据推测， 作为β-珠蛋白链去抑制(或诱导)的结果 出生时γ-珠蛋白转录的产生和抑制。这个 这一步骤的临床重要性在于，升高的 胎儿血红蛋白可以为患者提供相当大的保护 患有镰状细胞性贫血。细胞融合研究涉及两个 红白血病细胞系，一个来自人，K562，一个来自 小鼠梅尔已经显示出从伽马球蛋白链到贝塔球蛋白链的转换 生产依赖于相互作用的因素。这两种细胞系不同， 因此，在诱导时它们能产生的血红蛋白类型； 人类细胞系为胎儿，小鼠细胞系为成体。这个 建议的研究将集中在利用新方法直接克隆 编码调节因子的基因(S) 阳性对β-珠蛋白基因转录调控的研究 选择方法。冈山-伯格质粒的改良版本具有 已经建造好了。当携带新的质粒时，可以选择新的 一种β-珠蛋白基因反式激活剂的cdna。质粒的选择 依赖于细菌氨基糖苷3‘-的表达 人类转录调控下的磷酸转移酶基因 β-珠蛋白启动子。我们将从一个 我们的质粒中的细胞系(关闭了伽马珠蛋白，打开了β珠蛋白)。这个图书馆 随后将被导入另一细胞系(伽玛珠蛋白， β-珠蛋白关闭)以识别表达反式作用因子的克隆 来激活贝塔-珠蛋白基因的转录。已知的 该质粒的cdna克隆位点两侧的序列将用于 利用聚合酶链式反应技术从人类细胞中检索插入片段。我们 会将扩增的cdna重新克隆到细菌载体中，作为 恢复基因的染色体拷贝的探针。我们将对这两种情况进行排序 自然克隆和cDNA克隆。我们将提纯转因子(S)。 在大肠杆菌中表达该基因，纯化的蛋白将用于 结构功能分析实验。这些研究将产生 对切换现象有相当深的洞察力，最终可能 为镰状细胞性贫血的治疗提供新的方法。