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。 胚胎在妊娠早期转变为胎儿， 胎儿到成人的转变是在出生的时候。 的K562 人白血病细胞系表达除 成人β-珠蛋白 这个实验室以前的工作表明， K562 β-珠蛋白基因的功能正常， 异源表达系统 阐明了 K562细胞中β-珠蛋白基因表达的失败可能提供了 了解珠蛋白基因的表达和开关在正常 红系细胞 将尝试直接分离反式激活基因 使用导致分离几种癌基因的策略。 在K562细胞中不表达的杂交β-Neo质粒将 与另一种选择标记（RSV-GPT）共转染。 将通过选择GPT获得稳定的转化体， 通过Southern印迹证实β-Neo的存在。 高 来自K562和MEL细胞的分子量基因组DNA将被 转染到这些克隆中并激活β-Neo G418的选择 基因组DNA将被分离 直到分离出感兴趣的基因。 一种“拯救”战略 将在研究MEL细胞基因组DNA时使用。 C-myc已被研究为β-淀粉样蛋白的假定反式作用因子。 和α珠蛋白基因。 未检测到表达， 异源瞬时测定系统，其使用CAT活性作为 标记。 使用稳定的K562细胞转化体的进一步研究 含有c-myc基因的研究正在进行中， c-myc的潜在阻遏作用。 没有合适的表达β珠蛋白的人细胞系可用。 骨髓红系细胞永生化是不可能的 使用c-myc、c-Ha-ras和E1 A的组合的祖细胞 致癌基因 这被认为反映了次优转染和/或 由于人力资源匮乏而导致的增长条件。 将在给药前使用鼠骨髓尝试优化。 回到人类骨髓研究。

Humans undergo two developmental switches in their hemoglobin phenotype. The embryonic to fetal switch early in gestation and the 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. 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 do not express in K562 cells, will be cotransfected with another selectable marker (RSV-GPT). Stable tranformants will be obtained by selecting for GPT and the presence of beta-Neo confirmed by Southern blotting. High molecular weight genomic DNA from K562 and MEL cells will be transfected into these clones and the activation of beta-Neo sought by G418 selection. The genomic DNA will be fractionated until the gene(s) of interest is/are isolated. A "rescue" strategy will be used when studying MEL cell genomic DNA. C-myc has been studied as putative trans-acting factor for beta and epsilon globin genes. Expression was not detected in heterologous transient assay systems using CAT activity as a marker. Further studies using stable K562 cell transformants containing c-myc are in progress as are studies looking for a potential repressor effect of c-myc. No suitable human cell lines expressing beta globin are available. It has not proven possible to immortalize marrow erythroid progenitors using combinations of c-myc, c-Ha-ras, and E1A oncogenes. This is felt to reflect suboptimal transfection and/or growth conditions resulting from a scarcity of human material. Optimization will be attempted using murine marrow prior to returning to human marrow studies.