Pyati J, Kucherlapati R S, Skoultchi A I
Proc Natl Acad Sci U S A. 1980 Jun;77(6):3435-9. doi: 10.1073/pnas.77.6.3435.
A human beta-globin gene derived from an established human lymphoblast cell line was introduced into murine erythroleukemia (MEL) cells by cell fusion. The globin genes in MEL cells are inducible by dimethyl sulfoxide (Me2SO); induction leads to the accumulation of mouse globin mRNA and hemoglobin. Globin mRNA was not detected in the cytoplasm of the human lymphoblast cells, even at low levels, whether or not these cells were treated with Me2SO. In cell hybrids that had retained the lymphoblast-derived beta-globin gene, human beta-globin mRNA was induced by Me2SO. Poly(A)-containing 10S human beta-globin mRNA was detected in the cytoplasm of the hybrid cells. Karyologic and isozymic analyses of a series of hybrids and subclones showed that human beta-globin gene expression occurred only in hybrids that had retained human chromosome 11. Analysis of one hybrid bearing a deletion of both the beta-globin and lactate dehydrogenase A genes indicated that the beta-globin gene is located on the short arm of human chromosome 11. No other human chromosomes are required for human beta-globin gene expression in MEL cell hybrids. We conclude that the restricted expression of a globin gene in a human nonerythroid cell can be reversed. Furthermore, all components required for the transcription, processing, and transport to the cytoplasm of a human globin mRNA appear to be present in mouse erythroleukemia cells. Thus cell fusion with MEL cells provides a way to isolate permanent cell lines with functioning human globin genes. The technique should be useful for studying the biochemical basis for abnormal function of mutant globin genes, such as those present in individuals with the thalassemia syndromes.
通过细胞融合,将源自已建立的人淋巴母细胞系的人β-珠蛋白基因导入小鼠红白血病(MEL)细胞。MEL细胞中的珠蛋白基因可被二甲基亚砜(Me2SO)诱导;诱导导致小鼠珠蛋白mRNA和血红蛋白的积累。在人淋巴母细胞的细胞质中未检测到珠蛋白mRNA,即使是低水平,无论这些细胞是否用Me2SO处理。在保留了源自淋巴母细胞的β-珠蛋白基因的细胞杂种中,人β-珠蛋白mRNA被Me2SO诱导。在杂种细胞的细胞质中检测到了含多聚腺苷酸的10S人β-珠蛋白mRNA。对一系列杂种和亚克隆的核型和同工酶分析表明,人β-珠蛋白基因表达仅发生在保留了人类11号染色体的杂种中。对一个同时缺失β-珠蛋白和乳酸脱氢酶A基因的杂种的分析表明,β-珠蛋白基因位于人类11号染色体的短臂上。在MEL细胞杂种中,人β-珠蛋白基因表达不需要其他人类染色体。我们得出结论,人非红细胞中珠蛋白基因的受限表达可以被逆转。此外,人珠蛋白mRNA转录、加工和转运到细胞质所需的所有成分似乎都存在于小鼠红白血病细胞中。因此,与MEL细胞的细胞融合提供了一种分离具有功能性人珠蛋白基因的永久细胞系的方法。该技术对于研究突变珠蛋白基因异常功能的生化基础应该是有用的,例如那些存在于地中海贫血综合征患者中的基因。