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

反式激活因子和珠蛋白基因表达——直接方法

• 批准号: 3917588
• 负责人: H DAVE
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 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/3917588
• 关键词: Adenoviridae; DNA; cell bank /registry; developmental genetics; gene expression; genetic manipulation; genetic transcription; globin; human tissue; messenger RNA; mouse mammary tumor virus; neoplastic cell culture for noncancer research; nucleic acid sequence; oncogenes; plasmids; simian virus 40; transfection; transforming virus

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.

人类的血红蛋白经历两次发育转变 表型。 胚胎到胎儿的转变在妊娠早期 胎儿到成人的转变是在出生前后进行的。 K562人类 白血病细胞系表达除成人以外的所有珠蛋白基因 β-珠蛋白。 该实验室之前的工作表明 K562 β-珠蛋白基因在异源细胞中正常发挥作用 表达系统。 阐明失效机理 K562 细胞中的 β-珠蛋白基因表达可能提供见解 正常红细胞中珠蛋白基因表达和转换 细胞。 已开发出稳定的转化系统 赋予组织特异性的序列定位 珠蛋白基因的上游区域。 它还将使体内 滴定假定的调节因子，从而严格 证明某些序列的功能意义。 将尝试直接分离反式激活基因 使用导致分离出几种癌基因的策略。 混合β-neo质粒，在低水平表达 K562 细胞将与另一种选择标记共转染 （MDR 基因）。 选择的是后者，其次是 含有非表达 beta-neo 的克隆的鉴定 质粒。 将K562和MEL细胞基因组DNA转染至 这些细胞和β-neo的激活被寻求。 分馏 和/或“拯救”策略将被用来分离基因 的兴趣。 已知的反式作用因子，例如 HTLV-1 的 TAT-1（T 抗原） SV40病毒和腺病毒E1a基因产物将被 在稳定转化体中进行研究。 一种主要具有胎儿表型的新细胞系已被开发出来 特点。 由于没有合适的人类细胞系表达β 球蛋白是可用的，我们将尝试建立这样的线路 使用癌基因和/或起源缺陷的人骨髓细胞 SV40病毒DNA。