Synthesis and distribution of primer RNA in nuclei of CCRF-CEM leukemia cells.
Synthesis and distribution of primer RNA in nuclei of CCRF-CEM leukemia cells.
复制标题
CCRF-CEM 白血病细胞核内引物 RNA 的合成和分布。
DOI：
10.1021/bi00466a004
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Fernandes,DJ
中科院分区：
文献类型：
--
作者：
Paff,MT;Fernandes,DJ
摘要：检测了引物RNA和RNA引发的新生DNA在CCRF-CEM白血病细胞核中的分布，并对从这些细胞核基质中纯化的引物RNA进行了表征。在存在接近生理浓度的剩余核糖和脱氧核苷三磷酸的情况下，通过将全细胞裂解物与[a-32 P] ATP和[3 H] dTTP孵育来放射性标记RNA引发的新生DNA。用氯化铯密度梯度离心法纯化引物RNA，聚丙烯酰胺凝胶电泳分析。核亚分级研究表明，至少94%的引物RNA和RNA引发的新生DNA位于细胞核的不溶性基质部分。从核基质中分离的主要引物RNA长度为8-10个核苷酸，并且几条证据表明该寡核糖核苷酸是功能性引物RNA。基本上所有的基质引物RNA都共价连接到新复制的DNA上，如其在氯化铯梯度中的浮力密度、磷酸转移分析和对DNase I的敏感性所证明的。从[a-32 P] dTTP转移的32 P分析表明，在引物RNA的3 '端的核糖核苷酸的随机分布。从混合实验中获得的数据表明，RNA引发的新生DNA与核基质的缔合不是这些片段与核基质聚集的结果。没有显着量的引物RNA，RNA引发的新生DNA，或磷酸盐转移中检测到的高盐溶性（非基质）部分的核，虽然nonmatrix fraction containedmost的新复制的DNA。这些观察结果提供的证据表明，在CCRF-CEM细胞中，引物RNA和RNA引发的冈崎片段的合成发生在核基质上，并且引物RNA在新生DNA迁移离开基质结合的DNA复制位点之前被降解。e不连续合成的DNA（冈崎）片段的滞后链的复制叉是一个重要的
Revised Manuscript Received December 13, 1989 abstract: The distribution of primer RNA and RNA-primed nascent DNA in nuclei of CCRF-CEM leukemia cells was examined, and the primer RNA purifiedfrom the nuclear matrices of these cells was characterized. RNA-primed nascent DNA was radiolabeled by incubating whole-cell lysates with [a-32P] ATP and [3H] dTTP in the presence of approximatelyphysiological concentrations of the remaining ribo-and deoxyribonucleoside triphosphates. The primer RNA was purified by cesium chloride density gradient centrifugation and analyzed bypolyacrylamide gel electrophoresis. Nuclear subfractionation studies revealed that at least 94% of the primer RNA and RNA-primed nascent DNA were located within the insoluble matrix fraction of the nucleus. The predominant primer RNA isolated from the nuclear matrix was 8-10 nucleotides in length, and several lines of evidence indicated that this oligoribonucleotide was the functional primer RNA. Essentially all of the matrix primer RNA was covalently linked to the newlyreplicated DNA as demonstrated by its buoyant density in cesium chloride gradients, phosphate-transfer analysis, and sensitivity to DNase I. Analysis of 32P transfer from [a-32P] dTTP revealed a random distribution of ribonucleotides at the3'-end of the primer RNA. Data obtained from mixing experiments indicated that theassociation of RNA-primed nascent DNA with the nuclear matrix was not the result of aggregation of these fragments with the nuclear matrix. No significant amount of either primer RNA, RNA-primed nascent DNA, or phosphate transfer was detected in the high-salt-soluble (nonmatrix) fraction of the nucleus, although the nonmatrix fraction containedmost of the newly replicated DNA. These observations provide evidence that in CCRF-CEM cells the synthesis of both primer RNA and RNA-primed Okazaki fragments takes place on the nuclear matrix, and that the primer RNA is degraded prior to the migration of the nascent DNA away from the matrix-bound DNA replication sites. e discontinuous synthesis of DNA (Okazaki) fragments on the lagging strand of the replication fork is an important