大鼠肝细胞核复制位点处DNA的分级分离与特性分析。

Kaufman D G, Cordeiro-Stone M, Rude T H, Nelson K G, Kaufmann W K

Exp Cell Res. 1983 Jul;146(2):339-47. doi: 10.1016/0014-4827(83)90135-0.

Regenerating rat liver nuclei when sonicated and centrifuged in a Cs2SO4 gradient were fractionated into three distinct bands. These bands were designated as light band (LB), middle band (MB), and heavy band (HB) according to their density. LB and MB were shown to consist of large granular particles with varying electron densities, but LB also contained remnants of nuclear membrane. When analysed by gel electrophoresis, LB and MB displayed more than 35 bands of proteins. The third fraction, HB, consisted largely of small chromatin fibers and its proteins were predominantly the four histones of the nucleosomal core particle. Following short pulses with [3H]thymidine in vivo, the specific activity of DNA in LB and MB was significantly higher than that of bulk DNA contained in HB. DNA in all three fractions became equally labelled as the duration of the labelling interval increased beyond 30 min. Newly synthesized DNA was characterized by electrophoresis on analytical 1.7% acrylamide -0.5% agarose composite gels. After a 1-min labelling interval in vivo, 17% of the rapidly labelled DNA from LB and MB was stationary at the gel origin like replication forks from E. coli, but only 3% of HB DNA had zero mobility. Electron microscopy confirmed the presence of DNA replication forks in LB, MB, and HB. With increasing time of synthesis the proportion of labelled DNA exhibiting zero mobility decreased in all three fractions. Denaturation of DNA or digestion of single-stranded DNA with S1 nuclease released newly synthesized DNA from the gel origin. Ribonuclease was without effect. DNA recovered from LB and MB also had a higher molecular weight than the HB DNA. Together these results indicate (1) that LB and MB are enriched in newly replicated DNA; (2) that an increased proportion of newly replicated DNA in LB and MB is associated with DNA replication forks; and (3) that the replicating DNA recovered in LB and MB may be associated with other nuclear constituents in situ because this DNA appears to be protected from the more frequent chain breaks introduced into the bulk of chromatin (HB) by sonication.

对再生大鼠肝脏细胞核进行超声处理并在硫酸铯梯度中离心后，可将其分离为三条不同的条带。根据密度，这些条带分别被命名为轻带（LB）、中带（MB）和重带（HB）。结果显示，LB和MB由具有不同电子密度的大颗粒组成，但LB中还含有核膜残余物。通过凝胶电泳分析发现，LB和MB显示出35条以上的蛋白质条带。第三部分HB主要由小的染色质纤维组成，其蛋白质主要是核小体核心颗粒的四种组蛋白。在体内用[³H]胸腺嘧啶进行短脉冲标记后，LB和MB中DNA的比活性显著高于HB中所含的总DNA。随着标记间隔时间超过30分钟，所有三个部分的DNA标记程度变得相同。通过在分析型1.7%丙烯酰胺-0.5%琼脂糖复合凝胶上进行电泳来表征新合成的DNA。在体内进行1分钟的标记间隔后，来自LB和MB的快速标记DNA中有17%像大肠杆菌的复制叉一样停留在凝胶原点，而HB DNA中只有3%的迁移率为零。电子显微镜证实LB、MB和HB中存在DNA复制叉。随着合成时间的增加，所有三个部分中迁移率为零的标记DNA比例均下降。DNA变性或用S1核酸酶消化单链DNA可从凝胶原点释放新合成的DNA。核糖核酸酶没有作用。从LB和MB中回收的DNA分子量也高于HB DNA。这些结果共同表明：（1）LB和MB富含新复制的DNA；（2）LB和MB中新复制DNA比例的增加与DNA复制叉有关；（3）在LB和MB中回收的复制DNA可能在原位与其他核成分相关，因为这种DNA似乎受到保护，免受超声处理对大部分染色质（HB）造成的更频繁的链断裂影响。