Tweeten K A, Molloy G R
Nucleic Acids Res. 1981 Jul 24;9(14):3307-19. doi: 10.1093/nar/9.14.3307.
Hybridization of pulse-labeled RNA from DRB-treated Friend cells to mouse beta-globin cDNA revealed that the appearance of beta-globin mRNA in the cytoplasm was inhibited by greater than 87%. To examine the effect of DRB (125 microM) on HnRNA synthesis, nuclear RNA was electrophoresed in methyl mercuric hydroxide gels, transferred to nitrocellulose, and hybridized with beta-globin specific probes. Full-length nuclear transcripts, while present in untreated cells, were not detected in DRB-treated cells. Using restriction enzymes, the cloned beta-globin gene was divided into fragments proceeding from the 5' gene region to the 3' gene region. RNA labeled in vitro by transcription in nuclei isolated from DRB-treated cells hybridized only to the promoter proximal DNA fragment. Transcripts hybridizing to fragments from both the 5' and 3' regions of the gene were produced in nuclei from untreated cells. Together these results indicate that DRB causes premature termination of transcription within the beta-globin gene.
用脉冲标记的来自经DRB处理的Friend细胞的RNA与小鼠β-珠蛋白cDNA杂交，结果显示β-珠蛋白mRNA在细胞质中的出现受到了超过87%的抑制。为了检测DRB（125微摩尔）对核不均一RNA（HnRNA）合成的影响，将核RNA在氢氧化甲基汞凝胶中进行电泳，转移至硝酸纤维素膜上，并用β-珠蛋白特异性探针进行杂交。全长核转录本在未处理的细胞中存在，但在经DRB处理的细胞中未检测到。使用限制性内切酶，将克隆的β-珠蛋白基因从5'基因区域到3'基因区域分成片段。在从经DRB处理的细胞中分离出的细胞核中通过体外转录标记的RNA仅与启动子近端DNA片段杂交。与基因5'和3'区域片段杂交的转录本在未处理细胞的细胞核中产生。这些结果共同表明，DRB导致β-珠蛋白基因内转录的过早终止。
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