Housman D, Forget B G, Skoultchi A, Benz E J
Proc Natl Acad Sci U S A. 1973 Jun;70(6):1809-13. doi: 10.1073/pnas.70.6.1809.
A hybridization assay procedure was devised that makes possible quantitation of the ratio of mRNA of alpha to mRNA of beta globin chains in an RNA sample. The assay uses the radioactive synthetic DNA copies obtained by incubation of RNA-dependent DNA polymerase of avian myeloblastosis virus with rabbit globin mRNA that is 80-90% enriched in mRNA specific for synthesis of alpha or beta globin chains. The rabbit alpha-chain mRNA is obtained from the postribosomal supernatant of rabbit reticulocyte lysates; the rabbit beta-chain mRNA is obtained from the largest polysomes of rabbit reticulocytes treated with L-O-methylthreonine. Sufficient homology exists between rabbit and human globin chains and globin mRNAs that the synthetic DNA copies of chain-specific rabbit globin mRNA hybridize with human globin mRNA. Applied to the study of globin mRNA isolated from reticulocytes of humans with alpha and beta thalassemia, the technique revealed marked quantitative deficiency of alpha-chain mRNA relative to beta-chain mRNA in alpha thalassemia and similar deficiency of beta-chain mRNA relative to alpha-chain mRNA in beta thalassemia. The thalassemia syndromes are therefore characterized by true quantitative deficiency of the mRNA specific for the affected globin chain.
设计了一种杂交测定程序，该程序能够对RNA样品中α珠蛋白链mRNA与β珠蛋白链mRNA的比例进行定量。该测定使用放射性合成DNA拷贝，这些拷贝是通过禽成髓细胞瘤病毒的RNA依赖性DNA聚合酶与兔珠蛋白mRNA孵育获得的，兔珠蛋白mRNA在用于合成α或β珠蛋白链的mRNA中富集了80 - 90%。兔α链mRNA从兔网织红细胞裂解物的核糖体后上清液中获得；兔β链mRNA从用L - O - 甲基苏氨酸处理的兔网织红细胞的最大多核糖体中获得。兔和人珠蛋白链以及珠蛋白mRNA之间存在足够的同源性，使得链特异性兔珠蛋白mRNA的合成DNA拷贝能够与人珠蛋白mRNA杂交。应用于对从α和β地中海贫血患者网织红细胞中分离的珠蛋白mRNA的研究时，该技术显示在α地中海贫血中α链mRNA相对于β链mRNA存在明显的定量缺陷，而在β地中海贫血中β链mRNA相对于α链mRNA也存在类似的缺陷。因此，地中海贫血综合征的特征是受影响的珠蛋白链特异性mRNA存在真正的定量缺陷。
