CHARACTERIZATION OF HUMAN BETA-GLOBIN TRANS-ACTING REGULATORY FACTORS

解析遗传调控机制和生化因素 参与人类β-珠蛋白基因开关是研究的目标 在这个应用程序中。转换是一个时间的过程， β-珠蛋白样多肽在发育过程中的表达 从胎儿期到产后期的过渡。特别感兴趣的是 从胎儿血红蛋白到成人血红蛋白的分子转换，据推测， 由于β-珠蛋白链的去阻遏（或诱导）， 出生时γ-珠蛋白转录的产生和抑制。的 这一步骤的临床重要性在于， 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，一个人源性K562，一个来自 小鼠MEL已经展示了从γ-珠蛋白到β-珠蛋白链的转换 生产取决于反式作用因素。两种细胞系不同， 因此，在诱导时它们可以产生的血红蛋白的类型中; 人细胞系为胎儿细胞，小鼠细胞系为成体细胞。的 这项研究将集中在利用新的方法直接克隆 编码参与调节因子的基因 β-珠蛋白基因的转录调控，通过阳性 选择方法冈山-伯格质粒的一个改良版本， 已经建成。新质粒可以在携带 β-珠蛋白基因反式激活因子的cDNA。质粒的选择 依赖于细菌氨基糖苷3 '- 磷酸转移酶基因的转录控制下的人类 β-珠蛋白启动子。我们将构建一个完整的cDNA文库， 细胞系（γ-珠蛋白关闭，β-珠蛋白打开）。此库 随后将其导入另一个细胞系（γ-珠蛋白开启， β-珠蛋白关闭），以鉴定表达反式作用因子的克隆 激活β-珠蛋白基因的转录已知 质粒的cDNA克隆位点将用于 利用PCR技术从人细胞中回收插入物。我们 将扩增的cDNA重新克隆到细菌载体中， 探针以恢复基因的染色体拷贝。我们会对这两个 天然克隆和cDNA克隆。我们将通过超过 在E.纯化的蛋白质将用于 结构功能分析实验。 这些研究将产生 对转换现象的深入了解， 为镰状细胞性贫血的治疗提供了新的方法。

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.