Nienhuis A W, Canfield P H, Anderson W F
J Clin Invest. 1973 Jul;52(7):1735-45. doi: 10.1172/JCI107355.
A method for isolating human hemoglobin messenger RNA (mRNA) from bone marrow cells was developed to investigate the molecular basis for the defect in globin synthesis in beta thalassemia. Active mRNA was isolated from the bone marrow cells and peripheral reticulocytes of patients with homozygous beta thalassemia, heterozygous beta thalassemia, sickle cell trait, double heterozygosity for beta thalassemia and sickle cell trait, as well as from a patient with normal hemoglobin synthesis but with an elevated reticulocyte count secondary to hereditary spherocytosis. The mRNA was prepared for assay in an mRNA-dependent rabbit reticulocyte cell-free system and the amount of alpha and beta globin chains synthesized was determined by carboxymethylcellulose column chromatography. The relative synthesis of alpha to beta chains in response to normal hemoglobin mRNA was found to be a function of the amount of mRNA added to the assay system: increasing the amount of mRNA led to a decrease in the alpha-to-beta-chain synthetic ratio. Therefore, assays were carried out at limiting concentrations of mRNA. The molecular defect in homozygous beta thalassemia was shown to be carried in the mRNA of bone marrow cells as well as in the mRNA from peripheral reticulocytes, because much less beta than alpha globin was produced in the cell-free system in response to mRNA from either type of cell. In patients doubly heterozygous for beta thalassemia and sickle cell trait, little or no synthesis of beta(A) globin occurred in the bone marrow cells or the peripheral reticulocytes. The alpha to beta(S) synthetic ratio of the intact bone marrow cells was approximately 1, while the same ratio in the peripheral reticulocytes was between 1.5 and 2. The virtual absence of translatable beta globin mRNA in the mRNA prepared from the cells of these doubly heterozygous patients further demonstrates that the molecular defect produced by the beta thalassemia gene is in the beta globin mRNA.
为了研究β地中海贫血中珠蛋白合成缺陷的分子基础，开发了一种从骨髓细胞中分离人血红蛋白信使核糖核酸（mRNA）的方法。从纯合β地中海贫血、杂合β地中海贫血、镰状细胞性状、β地中海贫血和镰状细胞性状的双重杂合子患者的骨髓细胞和外周网织红细胞中分离出活性mRNA，以及从血红蛋白合成正常但因遗传性球形红细胞增多症导致网织红细胞计数升高的患者中分离出活性mRNA。将mRNA制备用于在依赖mRNA的兔网织红细胞无细胞系统中进行测定，并通过羧甲基纤维素柱色谱法测定合成的α和β珠蛋白链的量。发现正常血红蛋白mRNA刺激下α链与β链的相对合成是添加到测定系统中的mRNA量的函数：增加mRNA量导致α链与β链合成比率降低。因此，在mRNA的极限浓度下进行测定。纯合β地中海贫血的分子缺陷在骨髓细胞的mRNA以及外周网织红细胞的mRNA中均有体现，因为在无细胞系统中，无论哪种细胞的mRNA刺激下产生的β珠蛋白都比α珠蛋白少得多。在β地中海贫血和镰状细胞性状的双重杂合子患者中，骨髓细胞或外周网织红细胞中几乎不合成或不合成β（A）珠蛋白。完整骨髓细胞的α与β（S）合成比率约为1，而外周网织红细胞中的相同比率在1.5至2之间。从这些双重杂合子患者的细胞制备的mRNA中几乎不存在可翻译的β珠蛋白mRNA，这进一步证明了β地中海贫血基因产生的分子缺陷存在于β珠蛋白mRNA中。
