ONCOGENES AND THE CONTROL OF GLOBIN GENE EXPRESSION

人类经历了两次血红蛋白表型的发育转换， 胚胎到胎儿的转换发生在妊娠早期，胎儿到 在出生时发生的成人转换。 基因调节有两个 主要成分，即顺式作用DNA序列和反式作用分子 （可能是以特定方式与DNA序列相互作用的蛋白质 来控制基因表达。 K562人白血病细胞系表达 除了成人β-珠蛋白以外的所有珠蛋白基因。 以前的工作从这个 实验室研究表明，K562细胞的β-珠蛋白基因功能 通常在异源表达系统中。 阐明机制 K562细胞中β-珠蛋白基因表达的失败可能提供了一个新的证据， 对正常红系细胞中珠蛋白基因表达和转换的深入了解。 人腺病毒E1 a蛋白产物反式作用于转录 其他病毒基因以及某些固有的细胞基因如热休克蛋白 70. 增强子缺陷的人β-珠蛋白基因在细胞中不转录， HeLa细胞;然而，与E1 a基因共转染容易导致 可检测的β-珠蛋白mRNA。 c-myc蛋白核基质产物具有 与腺病毒E1 a蛋白有限的同源性，类似E1 a可以诱导 从hsp 70启动子的表达，并可以补充c-ras在 转化原代细胞 我们建议用杂交myc质粒 地塞米松反应性调控元件（MMTV-Xba-myc）导入K562细胞 细胞和研究类固醇后β-珠蛋白和β-珠蛋白基因表达 诱导 将共转染杂合β-CAT和β-CAT质粒 与MMTV-Xba-myc和CAT活性水平测定异源 系统. 无仅表达 β-珠蛋白可用。 这些线路将使用 MMTV-Xba-myc和含c-ras癌基因。 类固醇诱导的MMTV 元件应允许模拟体内c-myc mRNA水平的降低 可见分化，以及供应成人血红蛋白形成 细胞系

Humans undergo two developmental switches in their hemoglobin phenotype, the embryonic to fetal switch occurring in early gestation and the fetal to adult switch occurring around the time of birth. Gene regulation has two main components, namely cis-acting DNA sequences and trans-acting molecules (presumably proteins that interact with DNA sequences in a specific manner to control gene expression. The K562 human leukemia cell line expresses all globin genes other than the adult beta-globin. Previous work from this laboratory showed that the beta-globin gene of K562 cells functions normally in a heterologous expression system. Elucidation of the mechanism of failure of beta-globin gene expression in K562 cells may provide an insight into globin gene expression and switching in normal erythroid cells. The human adenovirus E1a protein product acts in trans on the transcription of other viral genes as well as certain resident cellular genes such as hsp 70. An enhancer deficient human beta-globin gene is not transcribed in HeLa cells; however, cotransfection with the E1a gene results in easily detectable beta-globin mRNA. The c-myc protein nuclear matrix product has limited homology to adenovirus E1a protein and like E1a can induce expression from the hsp 70 promoter and can complement c-ras in transforming primary cells. We propose to transfect a hybrid myc plasmid with a dexamethasone responsive regulatory element (MMTV-Xba-myc) into K562 cells and study epsilon and beta-globin gene expression upon steroid induction. Hybrid epsilon- and beta-CAT plasmids will be cotransfected with MMTV-Xba-myc and the level of CAT activity assayed in heterologous systems. No suitable human erythroid cell lines expressing only beta-globin are available. Such line(s) will be established using MMTV-Xba-myc and a c-ras containing oncogene. The steroid inducible MMTV element should permit mimicry of the in vivo decrease in c-myc mRNA level seen with differentiation, as well as supply an adult hemoglobin forming cell line.