DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures
DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures
复制标题
大豆原生质体和悬浮培养细胞中的 DNA 复制：指数同步培养和氟脱氧尿苷同步培养的比较
作者：
D. Cress;P. Jackson;A. Kadouri;Y. Chu;K. Lark
大豆（Glycine max (L.)）细胞悬浮培养稳定。（细胞系SB-1）已被用于研究DNA复制。细胞或原生质体将放射性胸腺嘧啶或5-溴脱氧尿嘧啶（BUdR）纳入DNA。DNA已被提取为大分子，可以通过放射自显影术可视化。细胞核被分离并在载玻片上裂解，从而避免了细胞质内切酶对DNA的降解。射线自显像显示，DNA合成发生在多个位点上，这些位点排列在单个DNA分子上，中间距离为10 ~ 30 μm。通过碱性梯度的速度沉积证实了自射线照相中看到的复制区域的长度。通过使用速度沉降，还可以证明复制是通过非常小的（4-6S） DNA中间体的合成进行的，这些中间体连接在一起形成了自射线图中所见的更大的复制子大小的片段。在同步和指数培养中观察到小（4-6S）和大（20-30S）中间体。然而，与氟脱氧尿苷（FUdR）同步后，DNA合成速率降低。由于FUdR处理不会减少中间体的大小，因此可以得出结论，较慢的复制速率是由于串联复制单元数量的减少，而不是它们被延长的速率。经过FUdR处理后，胸腺嘧啶的密度类似物BUdR可以取代DNA中几乎所有的胸腺嘧啶残基，导致CsCl的浮力密度从1.694 g/cm3增加到1.747 g/cm3。利用这种密度模拟物，可以估计模板DNA附着在新的复制位点上的数量。当这样做时，可以显示同步细胞在s开始时在大约5,000个不同的位点开始复制（每个这样的位点将复制到平均20 μm的长度）。BUdR的使用还证实，在复制的早期阶段，存在非常小的复制区域（<8S），这些复制区域被稍后复制的未复制DNA片段分开。这些结论大多与动物细胞的DNA复制模式一致。然而，一个主要的区别似乎是，在用FUdR长期抑制大豆细胞复制后，当复制恢复时，非常小的和复制子大小的中间体积累起来。这表明这些细胞中的复制调控可能与动物细胞不同。
Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 μm. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4–6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4–6S) and large (20–30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm3. Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 μm.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.