Reff M E, Davidson R L
Nucleic Acids Res. 1979 Jan;6(1):275-87. doi: 10.1093/nar/6.1.275.
Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase.
培养中的鼠红细胞白血病细胞在由二甲基亚砜（DMSO）诱导的分化过程中积累细胞质珠蛋白mRNA。从正在转录珠蛋白RNA的DMSO诱导的红细胞白血病细胞中制备染色质，以确定是否可以从染色质体外合成珠蛋白RNA序列。使用5-汞尿苷三磷酸和外源大肠杆菌RNA聚合酶进行体外RNA合成。通过在琼脂糖巯基柱上进行亲和层析，从与内源性染色质相关的RNA中纯化新合成的汞化RNA，并通过与cDNA珠蛋白杂交来测定汞化RNA的珠蛋白RNA序列含量。结果表明，珠蛋白RNA序列的合成确实发生，并且对放线菌素和利福平敏感，对α-鹅膏蕈碱不敏感。相比之下，从未诱导的红细胞白血病细胞制备的染色质中，以及在转录前添加珠蛋白RNA的未诱导细胞染色质中，均未检测到大量的珠蛋白RNA序列合成。通过亲和层析显示，分离的RNA:cDNA珠蛋白杂交体含有汞化RNA。这些结果表明，大肠杆菌RNA聚合酶可以从染色质合成珠蛋白RNA序列。
