Opposite replication polarities of transcribed and nontranscribed histone H5 genes.
Opposite replication polarities of transcribed and nontranscribed histone H5 genes.
复制标题
转录和非转录组蛋白 H5 基因的复制极性相反。
DOI：
10.1128/mcb.8.4.1657-1663.1988
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发表时间：
1988
影响因子：
5.3
通讯作者：
Leffak,M
中科院分区：
文献类型：
--
作者：
Trempe,JP;Lindstrom,YI;Leffak,M
我们使用体外核径流复制测定来分析禽类细胞中活性和非活性组蛋白 H5 基因的复制方向。在胚胎红细胞中，转录的组蛋白 H5 基因对内源性核酸酶切割表现出敏感性。相反，在相同条件下，该基因在类淋巴母细胞系 MSB-1 的细胞核中对内源性核酸酶消化不敏感，并且通过 MSB-1 细胞 RNA 的斑点印迹分析无法检测到组蛋白 H5 基因转录本。当从胚胎红细胞中分离出细胞核并与溴脱氧尿苷三磷酸一起孵育时，内源性核酸酶切割位点的径流复制导致密度标记 DNA 中组蛋白 H5 基因 3' 端附近的片段相对富集。然而，在 MSB-1 细胞或鸡胚成纤维细胞的细胞核中，来自限制性内切酶切割位点（或 MSB-1 细胞核中诱导的内源性核酸酶切割位点）的径流复制导致致密 DNA 中靠近 H5 基因 5' 端的片段相对富集。基于体外径流复制测定中溴脱氧尿苷增强地掺入 DNA 起始远端区域，我们得出结论，胚胎红细胞中的活性组蛋白 H5 基因优先从 5'-侧翼 DNA 起点沿转录方向复制，而 MSB-1 细胞和鸡胚成纤维细胞中的非活性对应基因优先沿相反方向复制。
We used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocytes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3′ end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5′ end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, we conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5′-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction.