TRANS-ACTIVATING FACTORS AND GLOBIN GENE EXPRESSION--A DIRECT APPROACH

人类的血红蛋白经历了两次发育转换 表型。胚胎到胎儿的转换在怀孕早期和 胎儿和成人在出生的时候会发生转换。K562人类 白血病细胞株表达除成人外的所有珠蛋白基因 β-珠蛋白。这个实验室之前的工作表明， K562β-珠蛋白基因在异源细胞中功能正常 表达系统。阐明了其失效的机理 K562细胞中β-珠蛋白基因的表达可能提供一个新的见解 成珠蛋白基因在正常红系中的表达和转换 细胞。 一种稳定的转化系统已经被开发出来，以允许 赋予组织特异性的序列的定位 珠蛋白基因的上游区域。它还将使体内的 假定调节因子的滴定(S)，从而严格 展示了某些序列的功能意义。 将尝试直接分离反式激活基因(S) 使用的策略导致了几个癌基因的分离。 混合的β-neo质粒，在 K562细胞，将与另一个可选择的标记物共转染 (MDR基因)。将为后者选择，然后是 含非表达β-neo基因克隆的鉴定 质粒组。K562和MEL细胞基因组DNA将被导入 并对这些细胞进行了β-NEO的激活研究。分馏法 将采用和/或“抢救”策略分离基因(S) 感兴趣的人。 已知的反式作用因子，如HTLV-1的TAT-1，T抗原 SV40病毒的E1a基因产物和腺病毒的E1A基因产物 在稳定的转化子中研究。 一种以胎儿为主要表型的新细胞系 特色化的。由于没有合适的人类细胞株表达β 球蛋白是可用的，我们将尝试从 使用癌基因和/或来源缺陷的人骨髓细胞 SV40病毒DNA。

Humans undergo two developmental switches in their hemoglobin phenotype. The embryonic to fetal switch early in gestation and tne fetal to adult switch around the time of birth. The K562 human leukemia cell line expresses all globin genes other than the adult beta-globin. Previous work from this laboratory has shown that the K562 beta-globin gene 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. A stable transformant system has been developed to permit the localization of sequences conferring tissue specificity to the upstream region of globin genes. It will also enable the in vivo titration of putative regulatory factor(s), thereby rigorously demonstrating the functional significance of certain sequences. The direct isolation of trans-activating gene(s) will be attempted using the strategy that led to the isolation of several oncogenes. Hybrid beta-neo plasmids, which are expressed at a low level in K562 cells, will be co-transfected with another selectable marker (the MDR gene). Selection will be for the latter, followed by the identification of clones containing non-expressing beta-neo plasmid. K562 and MEL cell genomic DNA will be transfected into these cells and activation of beta-neo sought. A fractionation and/or "rescue" strategy will be employed to isolate the gene(s) of interest. Known trans-acting factors such as TAT-1 of HTLV-1, the T antigen of SV40 virus and the E1a gene product of adenovirus will be studied in stable transformants. A new cell line with a predominantly fetal phenotype has been characterized. Since no suitable human cell lines expressing beta globin are available, we will attempt to establish such lines from human bone marrow cells using oncogenes and/or origin deficient SV40 virus DNA.