ONCOGENES AND THE CONTROL OF GLOBIN GENE EXPRESSION

人类在其血红蛋白表型上经历了两次发育转换， 胚胎向胎儿的转换发生在妊娠早期和胎儿向胎儿的转换 成体转换发生在出生时左右。基因调控有两种 主要成分，即顺式作用的DNA序列和反式作用的分子 (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人白血病细胞株表达 除了成人β-珠蛋白以外的所有珠蛋白基因。之前的工作来自于此 实验室研究表明K562细胞的β-珠蛋白基因具有功能 通常在异源表达系统中。对其机理的阐明 K562细胞中β-珠蛋白基因表达失败的研究可能为 深入了解正常红系细胞中珠蛋白基因的表达和转换。 人腺病毒E1a蛋白产物反式作用于转录 其他病毒基因以及某些常驻细胞基因，如热休克蛋白 70.缺乏增强子的人β-珠蛋白基因不在 HeLa细胞；然而，与E1a基因共转染容易导致 可检测到的β-珠蛋白基因。C-myc蛋白核基质产物具有 与腺病毒E1a蛋白同源性有限，与E1a类似可诱导 HSP70启动子的表达及其与c-ras的互补作用 转化原代细胞。我们建议将一种杂合的myc质粒 将地塞米松反应调控元件(MMTV-XBA-myc)导入K562 细胞，并研究激素对epsilon和β-珠蛋白基因表达的影响 归纳法。混合的epsilon-和beta-cat质粒将被共转染 MMTV-XBA-myc和CAT活性水平的测定 系统。没有合适的人红系细胞株只表达 贝塔-珠蛋白是可用的。这样的线路(S)将使用以下方式建立 Mmtv-xba-myc和含有癌基因的c-ras。类固醇诱导型MMTV 该元件应允许模拟体内c-myc mRNA水平的降低。 可见分化，以及供应成人形成的血红蛋白 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.