Department of Bacteriology, Iowa State University, Ames, Iowa 50010.
Genetics. 1972 Feb;70(2):215-32. doi: 10.1093/genetics/70.2.215.
The aim of this study was to determine whether there are specific weak points in the Bacillus subtilis chromosome and if so whether the replication point is the site of breakage. To answer these questions, B. subtilis chromosomes were partially labeled with 5-bromodeoxyuridine (5-BUdR). Sheared or unsheared preparations of partially labeled chromosomes which may or may not contain replication forks were analyzed for the distribution of genetic markers in a CsCl density gradient. Two sets of experiments based upon the density transfer experiments of Yoshikawa and Sueoka (1963) were performed: (1) experiments in which the origin of the chromosome was labeled and (2) experiments in which the terminus of the chromosome was labeled. In the first experiment, strain 23 (thy(-), his(-)) spores were germinated in the presence of 5-BUdR for various lengths of time and then transferred to fresh medium containing phenethyl alcohol (PEA) and thymidine (TdR). The DNA was isolated before and after transfer to PEA and TdR. In the second experiment strain 23 (thy(-), his(-)) spores were germinated in the presence of TdR and then PEA was added. After various lengths of time transfer was made to fresh medium containing PEA and 5-BUdR. The DNA was extracted by an extremely gentle technique to avoid breakage and centrifuged in a CsCl density gradient. PEA was added to the germinated spores to prevent dichotomous replication, but PEA did not prevent dichotomous replication in any of these experiments. This contradicts the conclusion of others that PEA prevents the chromosome from entering a new round of replication, but allows the chromosome to complete the round of replication already begun. The following observations offer support for the hypothesis that the replication point is a weak point in the chromosome: (1) when conditions were created to obtain partially labeled chromosomes with replication points: (a) labeled markers appeared at the hybrid density, (b) unlabeled markers appeared at the light density, (c) shearing of the DNA had little effect on the CsCl density gradient, except on a small proportion of labeled markers which had not appeared at the hybrid density prior to shearing; (2) when conditions were created to obtain partially labeled chromosomes with no replication points: (a) the majority of DNA molecules appeared at an intermediate density between the hybrid and the light densities, (b) the labeled and unlabeled markers appeared in the intermediate peak with approximately the same ratio as in the DNA preparations, (c) the labeled markers were found in the intermediate peak except where dichotomous replication had occurred, (d) after shearing, the labeled markers appeared at the hybrid density and the unlabeled markers appeared at the light density. Thus it is concluded that the replication point is a weak point in the B. subtilis chromosome where breakage easily occurs.
本研究的目的是确定枯草芽孢杆菌染色体是否存在特定的弱点，如果存在，复制点是否是断裂的部位。为了回答这些问题，我们用 5-溴脱氧尿苷（5-BUdR）对枯草芽孢杆菌染色体进行了部分标记。对可能含有复制叉或不含有复制叉的部分标记染色体的剪切或未剪切的制备物进行分析，以在 CsCl 密度梯度中分析遗传标记的分布。进行了两组基于 Yoshikawa 和 Sueoka（1963）密度转移实验的实验：（1）标记染色体起点的实验；（2）标记染色体末端的实验。在第一个实验中，用 5-BUdR 在不同时间点培养 23 株（thy(-)，his(-))孢子，然后转移到含有苯乙醇（PEA）和胸苷（TdR）的新鲜培养基中。在转移到 PEA 和 TdR 之前和之后，分离 DNA。在第二个实验中，用 TdR 培养 23 株（thy(-)，his(-))孢子，然后加入 PEA。在不同的时间后，转移到含有 PEA 和 5-BUdR 的新鲜培养基中。通过极其温和的技术提取 DNA，以避免断裂，并在 CsCl 密度梯度中离心。向发芽的孢子中加入 PEA 以防止二分复制，但在这些实验中，PEA 并没有阻止染色体进入新的一轮复制。这与其他人的结论相矛盾，即 PEA 阻止染色体进入新一轮复制，但允许染色体完成已经开始的一轮复制。以下观察结果为复制点是染色体弱点的假设提供了支持：（1）当创造条件获得带有复制点的部分标记染色体时：（a）标记标记出现在杂交密度处，（b）未标记标记出现在轻密度处，（c）DNA 的剪切对 CsCl 密度梯度几乎没有影响，除了一小部分标记标记物在剪切之前尚未出现在杂交密度处；（2）当创造条件获得没有复制点的部分标记染色体时：（a）大多数 DNA 分子出现在杂交密度和轻密度之间的中间密度处，（b）标记和未标记标记以与 DNA 制剂大致相同的比例出现在中间峰中，（c）除二分复制发生的地方外，标记标记物出现在中间峰中，（d）剪切后，标记标记物出现在杂交密度处，未标记标记物出现在轻密度处。因此，可以得出结论，复制点是枯草芽孢杆菌染色体的一个弱点，容易发生断裂。
