Chapter 12 The replicon.

Chapter 12 The replicon

12. 1 Introduction. 12. 2 Replicons can be linear or circular. 12
Introduction Replicons can be linear or circular Origins can be mapped by autoradiography and electrophoresis The bacterial genome is a single circular replicon Each eukaryotic chromosome contains many replicons Isolating the origins of yeast replicons D loops maintain mitochondrial origins The problem of linear replicons Rolling circles produce multimers of a replicon Rolling circles are used to replicate phage genomes The F plasmid is transferred by conjugation between bacteria Conjugation transfers single-stranded DNA Connecting bacterial replication to the cell cycle Cell division and chromosome segregation The division apparatus consists of cytoskeletal and regulatory components Partitioning involves membrane attachment and (possibly) a motor Multiple systems ensure plasmid survival in bacterial populations Plasmid incompatibility is determined by the replicon The ColE1 compatibility system is controlled by an RNA regulator 第4部分 DNA 12 复制子 349 射线自显影法和电泳可以对起始原点绘图 ,431 细菌的基因组是单个的环状复制子 ,433 每个真核的染色体都包含多个复制子 ,436 分离酵母复制子中的起始点 ,438 D环包含线粒体起始点 ,440 线性复制子的难题 ,442 旋转循环可形成一个复制子的多聚体 ,445 单链染色体组的形成可供细菌的结合 ,449 细菌复制与细胞循环的联合 ,453 细胞分化和染色体分离 ,455 多种体制确保质粒在菌落中的存活 ,460 质粒的不相容性与拷贝数有关 ,463

Replicon：复制子。基因组中一个能被独立复制的DNA片段单位，其中含有复制起驶点。在细菌和质粒DNA中只含有一个复制子。
12.1 Introduction Replicon is a unit of the genome in which DNA is replicated; contains an origin for initiation of replication. 复制子无论只有一个染色体的原核细胞，还是有多个染色体的真核细胞，对每个细胞分裂期而言，整个基因组都必须精确地复制，那么复制是怎样与细胞生长同期联系起来的呢? 复制状态与细胞生长周期作比较需要用两个主要原理 1DNA复制的起始使细胞(无论原核或真核)进一步分裂，根据这点，细胞分裂的数目由是否起始DNA决定。 2如果复制开始，接下来的分裂直到复制完成才开始，事实上，复制的完成也许给细胞分裂提供了一个触发器，然后复制的基因被分给每个子细胞，分配之后的基因组就成了染色体。细胞周期调节基因控制着起始DNA复制的开关和触发分裂的开关，在原核细胞中，复制的起始只与细胞染色体的一个特殊位置有关，分裂的过程是通过隔膜形成完成的，在真核细胞中，复制的起始以S区开始为标志，经过DNA合成的延长期，延长期涉及到许多单个的启动子，分裂是通过细胞有丝分裂的重组完成。在这一章中，我们着重介绍DNA复制的调节，复制周期是怎样被起始的，什么控制它的进程，它的终止信号怎样；在第30章中，我们讨论真核细胞的调节过程，控制S区和有丝分裂的进入，以及能够推迟这些行动直到获得合适的条件。单独复制的DNA单元称为复制子，每个复制子只复制一次，在每个细胞周期中只出现一次，复制子通过它的控制元素确定，它在复制开始的地方起始，也许在复制终制的地方终止。任何涉及到起始的序列，或者更准确地说，不是从末端分离出的被复制成作为复制子的一部分，起始端是顺式作用的位置，只能影响它所在的DNA分子，原核生物中复制子把它看成一个既包括起始端又包括蛋白质的调节基因的单位，然而，现在复制子通常用作真核染色体来描述包括起始位点的复制单位；反式作用的调节蛋白也许在其他地方被复制。原核细胞中的基因只进行一次复制，复制和分离的单位是一致的，最大的复制子就是细菌染色体本身，在单个的起始位点启动触发了整个基因的复制，对每个细胞分裂一次。每个单倍体生物只有一个染色体，因此这种类型的复制就称为单倍体复制。细菌也许以质粒的形式包含其他基因信息，质粒是一个自发环绕的DNA，构建成一个复制子，一个质粒复制子也可能复制细菌基因组，也可能有不同的控制，当质粒的复制数目超过细菌染色体组时，复制处于综合控制下，每个噬菌体或病毒DNA也能充作一个复制子，在传染过程中也多次启动复制，也许有一种更好的方式看待原核生物复制子使范围扩大化：任何在细胞里包含复制起始位点的DNA都可看成复制子。

Replicon：复制子。基因组中一个能被独立复制的DNA片段单位，其中含有复制起驶点。在细菌和质粒DNA中只含有一个复制子。
12.1 Introduction Replicon is a unit of the genome in which DNA is replicated; contains an origin for initiation of replication. 复制子是一个可移动的单位，在细菌染色体里，它被双链DNA用作复制产物，但它也可以单对等位或多对等位形式用作复制单链噬菌体或质粒基因组，复制子的复制方式取决于发生在起始阶段的内部作用的实质，普遍原则是复制在起始阶段被控制，一旦复制开始，它就会持续下去直到整个基因组被复制，起始率是由调节蛋白与结合位点的内部作用来控制的。在细菌和真核基因的复制中可以看见它们组织上的差异，每个真核细胞染色体都包含有大量的复制子。因此分离单位包括许多复制单位，这从另一角度增加了对复制的控制，在细胞周期里，一条染色体上所有复制子都必须被激发，虽然它们不是同时激活，但在一个相当长的时间里才被激活，然而在细胞周期里每个复制子激活不超过一次。一些信号必须把已经复制的复制子从没有复制的复制子区分出来以便复制子不被激活两次，因为许多复制子都能单独被激活，因此必须存在另一个信号以便确定什么时候整个复制过程所有的复制子都复制完成。我们已经开始收集关于单个复制子的信息，但我们对复制子之间的联系仍然知之甚少，我们不知道在每个细胞周期里，复制子的组成是否一样，是所有的起始位点都使用，还是一些位点不用?起始位点总是以相同的顺序激活吗?如果有不同的起始位点，那怎么区分他们呢? 和核内染色体相比，核内染色体是单独控制，线粒体和叶绿体的DNA也许象质粒那样是综合控制。每个细胞DNA都有许多复制的染色体，DNA复制的控制必须与细胞周期相关。在这些体系中，关键问题是确定能充当起始位点的序列和决定怎样被用作复制的蛋白质识别，我们从考虑复制子的基本结构和他们的不同形式开始，然后考虑起始位点，接着转向基因组的复制是怎样和细胞的分裂一致，以及谁负责把细胞的基因分给子细胞。

12.1 Introduction Bacteria may contain additional genetic information in the form of plasmids. A plasmid is an autonomous circular DNA genome that constitutes a separate replicon (see Figure 11.2). A plasmid replicon may show single-copy control, and replicate with the bacterial genome pari passu. Or it may be under multicopy control, when it is present in a greater number of copies than the bacterial chromosome. Each phage or virus DNA also constitutes a replicon, able to initiate many times during an infectious cycle. Perhaps a better way to view the prokaryotic replicon, therefore, is to reverse the definition: any DNA molecule that contains an origin can be replicated autonomously in the cell. Figure 11.2 Several types of independent genetic units exist in bacteria.

12.2 Origins can be mapped by autoradiography and electrophoresis
Replication fork： Replication fork is the point at which strands of parental duplex DNA are separated so that replication can proceed.

A molecule of DNA engaged in replication has two types of regions. Figure 12.1 shows that when replicating DNA is viewed by electron microscopy, the replicated region appears as an eye within the nonreplicated DNA. The nonreplicated region consists of the parental duplex; this opens into the replicated region where the two daughter duplexes have formed. 在电子显微镜下观察DNA复制，复制区域在非复制的DNA中象一只眼睛，然而，它的外观不能区分是单向还是双向复制，正如图14.2描绘的那样，“眼睛”能代表两种结构中的任何一种，如果是单向复制，那么“眼睛”就代表一个固定的起始位点和一个运动的复制叉，如果是双向复制，“眼睛”就代表一对复制叉，无论哪种情况，复制的过程都是“眼睛”进行扩展直到它最后走完整个复制子。 Figure 12.1 Replicated DNA is seen as a replication eye flanked by nonreplicated DNA.

The appearance of a replication eye does not distinguish between unidirectional and bidirectional replication. As depicted in Figure 12.2, the eye can represent either of two structures. If generated by unidirectional replication, the eye represents one fixed origin and one moving replication fork. If generated by bidirectional replication, the eye represents a pair of replication forks. In either case, the progress of replication expands the eye until ultimately it encompasses the whole replicon. 设想一个DNA分子参与复制，它有两种类型的部位，图14.1证明没有参与复制的区域构成平行的双链，它在两条子链形成的区域开放成复制区，在复制形成的位点称为复制叉(有时也被称为生长点)，一个复制叉沿着DNA依次移动，从起始位点开始。复制也许是单向的或是双向的，这要取决于起始位点形成了一个还是两个复制子，在单向复制中，一个复制叉离开起始位点沿着DNA前进，在双向复制中形成两个复制叉，他们从起始位点开始从相反的方向出发。 Figure 12.2 Replicons may be unidirectional or bidirectional, depending on whether one or two replication forks are formed at the origin.

Figure 12.3 A replication eye forms a theta structure in circular DNA.
12.2 Origins can be mapped by autoradiography and electrophoresis When a replicon is circular, the presence of an eye forms the -structure drawn in Figure The successive stages of replication of the circular DNA of polyoma virus are visualized by electron microscopy in Figure 12.4. 当一个复制子是圆形时，“眼睛”的存在使之形成了θ结构，如图14.3， Figure 12.3 A replication eye forms a theta structure in circular DNA.

Figure 12.4 The replication eye becomes larger as the replication forks proceed along the replicon. Note that the "eye" becomes larger than the nonreplicated segment. The two sides of the eye can be defined because they are both the same length. Photograph kindly provided by Bernard Hirt. When a replicon is circular, the presence of an eye forms the -structure drawn in Figure The successive stages of replication of the circular DNA of polyoma virus are visualized by electron microscopy in Figure 12.4. 接下来多形瘤病毒的圆形DNA复制阶段可以通过电子显微镜识别，如图14.4。一个复制“眼睛”有一个还是两个复制叉能用两种方法确定，其方法的选择取决于DNA是一个细胞基因的有限的可确定分子还是不确定区域。如果是有限的线性分子，我们使用电子显微镜测量出“眼睛”的两端与DNA末端的距离，然后在有不同尺寸“眼睛”的分子中比较“眼睛”两端的位置，如果复制是单向的，那么 “眼睛”只有一端能够移动，另一端是固定的，如果复制是双向的，两边都移动，起始位点是两端的中间位置。

Figure 12.5 Different densities of radioactive labeling can be used to distinguish unidirectional and bidirectional replication. With undefined regions of large genomes, two successive pulses of radioactivity can be used to label the movement of the replication forks. If one pulse has a more intense label than the other, they can be distinguished by the relative intensities of labeling. These can be visualized by autoradiography. Figure 12.5 shows that unidirectional replication causes one type of label to be followed by the other at one end of the eye. Bidirectional replication produces a (symmetrical) pattern at both ends of the eye. This is the pattern usually observed in replicons of eukaryotic chromosomes (Huberman and Riggs, 1968). 在大型基因的不确定区域中，进行两次连续脉冲能标记复制叉的运动，如果一次脉冲比另一种有更强的标志，他们就能通过标记强度区分出来，这能通过自动放射自显影照像区分出来，图14.5就显示出单向复制两端不同类型的标记，双向复制两端只产生一种类型的标记。这种方法常用来观察真核染色体的复制。

Figure 12.6 The position of the origin and the number of replicating forks determine the shape of a replicating restriction fragment, which can be followed by its electrophoretic path (solid line). The dashed line shows the path for a linear DNA. A more recent method for mapping origins with greater resolution takes advantage of the effects that changes in shape have upon electrophoretic migration of DNA. Figure 12.6 illustrates the two dimensional mapping technique, in which restriction fragments of replicating DNA are electrophoresed in a first dimension that separates by mass, and a second dimension where movement is determined more by shape. Different types of replicating molecules follow characteristic paths, measured by their deviation from the line that would be followed by a linear molecule of DNA that doubled in size. 一个用来绘制起始点的有很好的分辩率的方法是充分利用DNA的电泳迁移产生变化的结果。图14.6说明了两个双向绘制技术，在此技术中，复制DNA的限制性碎片在第一维和第二维上都被电泳，第一维通过质量而分离，在第二维上的运动更多是由形状而决定。不同类的复制分子通过不同的路径，此特殊路径需通过偏移谱系的偏差来测量，而该谱系又被在尺寸上增长一倍的线性DNA分子跟随着。一个简单的Y结构跟随一条连续的路径，在此Y结构中，复制叉是沿着线性碎片移动的。当三个分支具有同一长度时，便会出现回折点，因此，结构最大范围地从线性DNA上偏离。类似的想法决定了双倍Y结构及泡的路径，不对称的泡经历一条不连续的路径，当叉跑向末端时，泡在间断处被改变成Y结构。总的来说，对于独特复制的DNA，不同的技术表明起始点最常用于起始双向复制。从分辨水平着手，我们随后必须进行分子水平的研究，以便鉴定组成起始点的顺式序列和识别顺式序列的转录作用因子。

12.3 The bacterial genome is a single circular replicon
Termination in E. coli has the interesting features reported in Figure We know that the replication forks usually meet and halt replication at a point midway round the chromosome from the origin. But two termination regions (terE,D,A and terC,B) have been identified, located ~100 kb on either side of this meeting point. Each contains multiple terminators. Each terminus is specific for one direction of fork movement, and they are arranged in such a way that each fork would have to pass the other in order to reach the terminus to which it is susceptible. This arrangement creates a "replication fork trap;" if for some reason one fork is delayed, so that the forks fail to meet at the usual central position, the more rapid fork will be trapped at the ter region to wait for the arrival of the slow fork. 细菌基因组是单一的循环复制子要使遗传被正确地进行，细菌复制子应发挥如下作用： ◆ 起始复制循环 ◆ 控制起始发生的频率 ◆ 将复制后的染色体分配到子细胞中前两个功能都是起始点所具的特性。分离可能是个独立的功能，但在原核系统中，分离通常伴随起始点附近的序列而休止。在真核细胞中，起始点没有分离的功能，但它也只与复制相关。作为普通的原则，那就是组成起始点的DNA能被其能力所分离出来，这种能力是维持连接它本身的任何DNA序列的复制。当DNA从起始点被克隆到缺乏起始点的分子中时，只要DNA从起始点起就包含用来识别它自身是对复制的一个真实起始点的所有序列，那么重组将产生一个有能力自行复制的质粒。虽然在较高等真核生物中的起始点被鉴定出来，但在细菌、酵母、叶绿体和线粒体中的起始点现已被鉴定了。其一般的特征是组成富含A·T的一段综合序列，我们估计这与需要解开DNA双螺旋而起始复制有关。大肠杆菌的基因组从单一的起始点起被双向复制，该起始点被鉴定为oric基因座。增加oric到任何DNA片段上将产生一个在大肠杆菌中能复制的人工质粒。通过减少oric克隆碎片的尺寸，被要求用来起始复制的区域已与245bp的碎片同等化了。(在下一章我们将更详细地讨论oric的特性及其与复制装置的相互作用。) Figure 12.7 Replication termini in E. coli are located beyond the point at which the replication forks actually meet.

12.3 The bacterial genome is a single circular replicon
正确地起始的质粒有可能无规则地进行分裂，但它能通过产生附加序列而被固定。这样，当细菌群体分离时，被要求用作起始的区域就不再运载足够的信息了，这信息是用来使复制DNA分子能够分配到子细胞中去的。有关分配的功能可被一些独特的序列鉴定，这些序列在质粒中起作分裂稳定性的作用。原核生物的复制子通常是环状的——DNA形成一个没有自由末端的封口环。环状结构包括细菌的染色体本身，所有质粒及许多噬菌体。它们在叶绿体及线粒体DNA中也是很普通的。环状分子的复制避免了线性分子遇到的末端复制问题，却又面临另一个问题：即如何终止复制。细菌染色体的复制是作为一个单位从唯一的复制起点开始，双向进行的。两个复制叉从起点(oric)开始，以大致相同的速度，沿基因组移动，直至在某点相遇，最后终止复制。复制过程中有一个有趣的问题：是什么因素确保DNA的复制恰好在复制叉汇合的区域进行。DNA本身复制完成后，需要一种控制DNA高度有序结构的酶参与，以完成两条子代染色体的分离。控制复制的序列叫终止位点，该位点包括一个约23bp的短核酸序列，引发体外复制的终止。终止序列只单向发生作用，DNA的复制需要tus基因产物的参与，该基因编码一种蛋白,识别共同序列，并阻止复制叉继续前进。 E.coli中的复制还有个有超的特点(见图14.7)。众所周知，复制叉一般从染色体复制起点开始双向复制，中途在某点相遇而终止复制。然而，复制叉交汇点的两侧各约100kb的区域，定义为终止区域(terD.A和terC.B)，每一个末端对于复制叉运动的一个方向是特异的，并且他们的排列方式使每一个复制叉为了能够到达敏感的末端，必须通过另一个复制叉。这种排列产生一个“复制叉带”；如果由于某种原因，一个复制叉延迟了，这样复制叉便不能在通常的中心位置相遇了。更多的比较快的复制叉将在终止端堵塞，以等待较慢的复制叉的到来，复制对DNA结合蛋白的影响是十分有趣的。如果一个复制叉遇到与DNA结合的阻遏，因子会发生什么呢?我们假定复制叉能够置换阻遏因子(在此之后，阻遏因子必须与复制位置的两条链结合，以维持基因表达的控制)。一个特殊的有趣的问题是当复制叉遇到转录时出现的RNA聚合酶时会怎么样，复制叉的移动速度会比RNA聚合酶快10倍以上，如果他们朝同一方向移动的话，每个复制叉必须置换聚合酶，或者它必须减速下来以等待RNA聚合酶到达其终止子，这表明DNA聚合酶与RNA聚合酶同方向移动时，其能够通过它并且不阻碍转录，但我们不明白这种反应的基础。当复制叉遇到一个与其反向移动的即朝向他的RNA聚合酶，便会产生一个问题，它能够置换RNA聚合酶吗?或者是复制与转录都将终止?来自E.coli染色体的组织研究表明这些相遇并不能很容易解决。几乎所有有活性转录系统都是定向的，这样是为了当复制叉通过它们时，它们能在相同的方向表达。这种假说也包括所有的那些不经常表达的小的转录单位，包含高表达基因的传代倒位的困难，表明复制叉与一系列的转录RNA聚合酶头与头相遇可能是致死性的。 Figure 12.7 Replication termini in E. coli are located beyond the point at which the replication forks actually meet.

12.4 Each eukaryotic chromosome contains many replicons
S phase: 每个真核染色体包含许多复制子。在真核细胞中，DNA的复制局限于细胞周期中的一部分，S期出现于中期，且在高级真核细胞中持续一段时间，包含于真核细胞染色体中的大量DNA的复制通过将其分为许多单独的复制子完成的。在S期任何一点复制时仅仅有这些复制子的一部分参与，我们可认为在整个S期中一个特殊时间每一个复制子具有活性，尽管这种提法的证据并不充分。重要的一点是S期的起始是通过第一个复制子的激活为标记的，在此后的几个小时内，起始事件发生于其它复制子。因此S期的控制包括两个过程：细胞脱离G1期过程和单独复制子按照有序的方式进行复制的起始。 S phase is the restricted part of the eukaryotic cell cycle during which synthesis of DNA occurs.

The start of S phase is signaled by the activation of the first replicons. Over the next few hours, initiation events occur at other replicons in an ordered manner. Much of our knowledge about the properties of the individual replicons is derived from autoradiographic studies, generally using the types of protocols illustrated in Figure 12.5 and Figure Chromosomal replicons usually display bidirectional replication. 我们关于单独复制子性质的许多知识是由放射自显影技术获得的，通常是用图14.5和14.6所示的操作方式，染色体复制子通常为双方复制。复制子的平均大小是多少?在基因组中的个数是多少? Figure 12.5 Different densities of radioactive labeling can be used to distinguish unidirectional and bidirectional replication.

Figure 12.6 The position of the origin and the number of replicating forks determine the shape of a replicating restriction fragment, which can be followed by its electrophoretic path (solid line). The dashed line shows the path for a linear DNA. The start of S phase is signaled by the activation of the first replicons. Over the next few hours, initiation events occur at other replicons in an ordered manner. Much of our knowledge about the properties of the individual replicons is derived from autoradiographic studies, generally using the types of protocols illustrated in Figure 12.5 and Figure Chromosomal replicons usually display bidirectional replication.

How large is the average replicon, and how many are there in the genome? A difficulty in characterizing the individual unit is that adjacent replicons may fuse to give large replicated eyes, as illustrated in Figure The approach usually used to distinguish individual replicons from fused eyes is to rely on stretches of DNA in which several replicons can be seen to be active, presumably captured at a stage when all have initiated around the same time, but before the forks of adjacent units have met. 给独立单位定性的困难是连接在一起的复制子可能熔成巨大的复制眼，如图14.8所示。区分独立复制子与熔化眼的常用方法是依靠DNA的伸直，因为在其中几个复制子能够发现具有活力，假设当所有的复制子在相同时间内已经起始时在同一状态时被观察，但必须在连接单位的复制叉相遇之前。 Figure 12.8 Measuring the size of the replicon requires a stretch of DNA in which adjacent replicons are active.

"Regional" controls might produce this sort of activation pattern, in which groups of replicons are initiated more or less coordinately, as opposed to a mechanism in which individual replicons are activated one by one in dispersed areas of the genome. Two structural features suggest the possibility of large-scale organization. Quite large regions of the chromosome can be characterized as "early replicating" or "late replicating," implying that there is little interspersion of replicons that fire at early or late times. And visualization of replicating forks by labeling with DNA precursors identifies 100300 "foci" instead of uniform staining; each focus shown in Figure 12.9 probably contains >300 replication forks. The foci could represent fixed structures through which replicating DNA must move. 有一些证据表明“区域”控制可能产生这种活性形式，在这种形式下，成群的复制子或多或少的协调一致起始，与其相反的一种机理是在基因组不同分散区域，独立的复制子是一个接一个的被激活，两个结构特征表明大规模有组织性的可能性。染色体相当大的区域能够区分为“早期复制”或“晚期复制”，这就暗示早期晚期至少都有一些分散的复制子在复制。正在复制的复制叉可用DNA前体标志，这样可视度为100—300“foc1”，这样便可不用常规染色法。图14.9所示的点可能包含大于300个复制叉，复制叉可认为是固定的结构，由此可知复制DNA必须移动。在成群的活性复制子中，这种单位的平均大小是按起始端间的距离测量的(也就是说，按连接复制子的中点之间的距离)，复制叉移动的速率可以用在一给定时间内的放射自显影最大移动轨迹来估测。 Figure 12.9 Replication forks are organized into foci in the nucleus. Cells were labeled with BrdU. The leftmost panel was stained with propidium iodide to identify bulk DNA. The right panel was stained using an antibody to BrdU to identify replicating DNA.

真核复制子与原核位置的对比见于表14.1，独立的真核复制子相对较小。它们复制的速率比微生物复制叉运动速率要慢的多。我们很想知道是什么促成每次复制的开始，是否是一个特殊的DNA序列，其长度如何，不同的起点之间的顺序如何?是否一个起点与一个在染色体内的高度有顺序的结构的特定形态有关。在图14.1中所指出的复制速率中，显示出，在所有复制功能都激的情况下，一个乳腺细胞的基因在1小时之内复制完。但在典型的体细胞中，S期确要持续6个小时以上。这说明：不超过15%的复制过程在任何给定的情况下活动，(有一些特例，如早期Drosophila胚胎的胚胎细胞的分裂当中，S期的长度被大量复制的刺激功效压缩了)。在S期的不同时间内，要开始复制的起点如何选定?是不是在起点序列中包含着使用时间的信息?有一个有趣的问题：即复制机制中如何将已复制过的起点和未被复制的起点分别开来，是否每个起点在S期都能一次且仅一次的应用，与它有关的是蛋白质或DNA的成分(例如：看其甲基化的成分)。已用的根据表明，染色体复制不是因为复制叉停止运动或从DNA上脱落下来而停止。更有可能的是，一个复制叉直到它碰到一个相邻复制的复制叉时才停下来，我们曾经指出：在复制叉的连接处加入一段合成DNA的逻辑可能性的问题。

12.5 Isolating the origins of yeast replicons
分离酵母复制的起点任何具有起点的DNA片段都可以被复制。因此，尽管在eukaryotes中质粒很少，但在vitro中可能用合适的操纵子来组建它们，这在酵母中已经完成了，尽管在高一些的eukaryotes中还来。 S.cerwisiac变异可以通过添加带有野生型基因版本的DNA来转导入野生型，一些酵母DNA片段(当为环状时)可以很有效的对感受态细胞进行转导这些片段在细胞内以不完整(自立)的状态可以保存下来，也就是自我复制质粒。一类高转导频率的片段有特定序列使其有能力在酵母内高效复制，这个序列称为ARS(独立复制序列)ARS因子在确切的起点中位置靠前；在染色体当中，ARS所在位置是首位。做为复制起点的一样的平均频率，是ARS功能的体现结果。当ARS被系统的在扩展的染色体区域内描绘出来后，似乎只有一部分是用来起动复制。其它的都无用或很少用到。如果某些起点的用途可能性有变化的话。那就可以推出，在复制环中无固定终点。在这种情况下，不同的细胞环境，对于一个给定的染色体区域会有不同的复制起点。一个ARS因子是由富含A～T结构的区域组成的，其中包括某些诱变剂影响起点功能的间断位点。图14.10显示一个诱变剂沿起点长度的系统分析，当诱变剂作用在一个14bp的核心位置时，起点功能完全丧失，这个核心区域包括11bp完全由A·T碱基对构成的序列，这种统一序列在已知的ARS因子中是唯一的homology。除了在核心中的一致序列外，还有不完美的附加序列，特别是在9/n位保持一致，在这些序列中变异减少，但不能完全消除起点功能。它们的功能假定为部分重叠，使其中任何一个都不是必不可少，(它们的减少服从序列一致并且它们的重叠一起说明：为什么它们一开始并未被定义为起点的成份，只有到后来才指出是对起点功效有贡献的附加位点。起始位点也有一个转录因子结合位点。这个因子可能不能激活转录，它在起始复制中的作用可能与在转录中的作用相化人，那就是和结合在此位点的其它蛋白相互作用，有助于稳定聚合在那里的复合体一个分子量为π400KD的蛋白复合体结合到起始位点，它包括6个不同蛋白而被称为起始识别复合体(ORC)，这个结合需ATP，整个细胞循环中ORC一直与ARS成分协作，因此起始可能依赖于环境的改变而不是与起始位点协作的改变，在高等真核细胞中也找到了相当于ORC成份。关于核心保持序列和起始中的分子行为的关系，有许多重要问题有待解决，DNA最初在哪融解，新生链〓〓在哪起始合成? ARS元素与保质的起始位点区域一致，是一段起始DNA复制的正激活序列，高等真核生物中能找到相似的成分吗?在找与ARS成份相似的序列时遇到的困难，表明起始位点更复杂，有提议说，一些动物细胞复制子可能有复制杂的起始模式，一些情况下在一个区域能找到许多小的复制泡，产生了是否存在两个或多个复制起始的问题，可公正地说高等真核的复制子起始位点的本质有待确定。 Figure An ARS extends for ~50 bp and includes a consensus sequence (A) and additional elements (B1-B3).

12.6 D loops maintain mitochondrial origins
D loop is a region within mitochondrial DNA in which a short stretch of RNA is paired with one strand of DNA, displacing the original partner DNA strand in this region. The same term is used also to describe the displacement of a region of one strand of duplex DNA by a single-stranded invader in the reaction catalyzed by RecA protein. D loop: 线粒体复制起始点保持D-loop结构真核和原核的染色体上的复制子的起始位点都是保守结构：他们包含复制识别和在适宜时起始复制的DNA序列。起始需要解开DNA链和双向DNA的合成。但在其它基因组中起始位点普遍存在不同种的重排，最显著的是在线粒体中。

12.6 D loops maintain mitochondrial origins
Replication starts at a specific origin in the circular duplex DNA. But initially only one of the two parental strands (the H strand in mammalian mitochondrial DNA) is used as a template for synthesis of a new strand. Synthesis proceeds for only a short distance, displacing the original partner (L) strand, which remains single-stranded, as illustrated in Figure The condition of this region gives rise to its name as the displacement or D loop (for review see Clayton, 1982; Clayton, 1991). 环状双链DNA的复制起始于一个特殊的位点。但最初仅一条母链用作新链合成的模板。合成仅进行一小段距离，置换出保持单链的初始链。如Figure 14.11。根据此区域的形状命名为置换或D-loop结构。单个D-loop结构在动物线粒体上是一段 bp的开放序列。维持D-loop结构的短链是不稳定的且回转；它经常解链和重合成以保持此位点处双链的开放。有些线粒体DNA拥有几个D-loop结构，反映出其有多个起始位点。叶绿体DNA也有同样的机制，高等植物的叶绿体DNA有两个D-loop结构。动物线粒体DNA，D-loop结构上的短链被延伸。原L链被置换区域变得更长，D-loop结构被扩大。扩大持续至它达到环三分之二处。这段区域的复制暴露出被置换链上的起始位点。一个H链从此处开始合成，合成围绕被取代链朝L链合成方向相反的方行进行。由于开始时缓慢，当L链合成结束时，H链仅绕环三分之一圈。这样就释放出一个完整的双链环的一个带裂沟的环，此环保持部分单链直至H链完全合成。最后新链被修补成为完整链。滚环和D-loop结构的存在表明了一个普遍准则。起始位点可能是一段用单链作为模板起始合成的DNA序列。双链的打开不一定导致另一条链复制的起始。至于线粒体DNA的复制，互补链的复制起始点处于不同位置。 Figure The D loop maintains an opening in mammalian mitochondrial DNA, which has separate origins for the replication of each strand.

12.7 The problem of linear replicons
Strand displacement is a mode of replication of some viruses in which a new DNA strand grows by displacing the previous (homologous) strand of the duplex. Strand displacement: 线形复制子的末端复制问题到目前为止我们讨论的复制子没有一个有线形末端：它们不是环形的(如E.coli或线粒体基因组)就是长单位(如真核染色体)的一部分。但某些情况下线形复制子作为染色体外的部分出现，当然是在真核染色体的末端。所有已知核苷酸聚合酶的合成方向都是从5'-3'，这就产生了线形复制子末端DNA合成问题。如图Figure 14.12所示的两条母链。下面的链没问题：以它作模板合成子链，子链直接到达末端，末端处聚合酶离开。但要合成上面链末端的互补序列，合成必须从最后一个碱基开始(否则这条链在连续的复制过程中变得更短)。我们还不知线形DNA末端起始合成是否可行。我们通常认为聚合酶结合在碱基进入位置的周围。因此线形复制子末端的复制必有一个特殊的机制。四种解决方案可满足复制一个末端的需要： ◆将复制子变成一个环形或分子可避开这个问题。噬菌体如T4或λ噬菌体是采用这种方法。 ◆DNA形成异常结构，例如末端形成发夹结构从而没有自由末端，Paramecium的线形线粒体DNA复制时生成交联键。 ◆末端可以变化，而不是精确的确定。真核染色体可能采用这种方案，染色体DNA末端的短重复序列的拷贝数不固定。增加或减少重复单位使得不必恰从最末端开始复制。 ◆A蛋白质干扰使在真正的末端开始。几种病毒线形核酸有与5′末端碱基共价相连的蛋白。最典型的例子是腺病毒DNA，噬菌体29和脊髓灰质炎病毒。线形末端起始的一个主要的例子是腺病毒和29DNAs，它们实际上使用如Figure 14.13所示的链置换的方法，从两个末端起始复制。同样的步骤独立地在两个末端进行。在一个末端合成一条新链，取代前一次在双链中配对的同源链。当复制叉分子的另一端时，释放出被置换的链为自由链。自由链独立地复制：这需要通过分子末端的短互补序列间碱基配对形成双链。

The ability of all known nucleic acid polymerases, DNA or RNA, to proceed only in the 5 3 direction poses a problem for synthesizing DNA at the end of a linear replicon. Consider the two parental strands depicted in Figure The lower strand presents no problem: it can act as template to synthesize a daughter strand that runs right up to the end, where presumably the polymerase falls off. But to synthesize a complement at the end of the upper strand, synthesis must start right at the very last base (or else this strand would become shorter in successive cycles of replication). Figure Replication could run off the 3 end of a newly synthesized linear strand, but could it initiate at a 5 end?

A prime example of initiation at a linear end is provided by adenovirus and 29 DNAs, which actually replicate from both ends, using the mechanism of strand displacement illustrated in Figure The same events can occur independently at either end. Synthesis of a new strand starts at one end, displacing the homologous strand that was previously paired in the duplex. When the replication fork reaches the other end of the molecule, the displaced strand is released as a free single strand. It is then replicated independently; this requires the formation of a duplex origin by base pairing between some short complementary sequences at the ends of the molecule. 线形复制子的末端复制问题到目前为止我们讨论的复制子没有一个有线形末端：它们不是环形的(如E.coli或线粒体基因组)就是长单位(如真核染色体)的一部分。但某些情况下线形复制子作为染色体外的部分出现，当然是在真核染色体的末端。所有已知核苷酸聚合酶的合成方向都是从5'-3'，这就产生了线形复制子末端DNA合成问题。如图Figure 14.12所示的两条母链。下面的链没问题：以它作模板合成子链，子链直接到达末端，末端处聚合酶离开。但要合成上面链末端的互补序列，合成必须从最后一个碱基开始(否则这条链在连续的复制过程中变得更短)。我们还不知线形DNA末端起始合成是否可行。我们通常认为聚合酶结合在碱基进入位置的周围。因此线形复制子末端的复制必有一个特殊的机制。四种解决方案可满足复制一个末端的需要： ◆将复制子变成一个环形或分子可避开这个问题。噬菌体如T4或λ噬菌体是采用这种方法。 ◆DNA形成异常结构，例如末端形成发夹结构从而没有自由末端，Paramecium的线形线粒体DNA复制时生成交联键。 ◆末端可以变化，而不是精确的确定。真核染色体可能采用这种方案，染色体DNA末端的短重复序列的拷贝数不固定。增加或减少重复单位使得不必恰从最末端开始复制。 ◆A蛋白质干扰使在真正的末端开始。几种病毒线形核酸有与5′末端碱基共价相连的蛋白。最典型的例子是腺病毒DNA，噬菌体29和脊髓灰质炎病毒。线形末端起始的一个主要的例子是腺病毒和29DNAs，它们实际上使用如Figure 14.13所示的链置换的方法，从两个末端起始复制。同样的步骤独立地在两个末端进行。在一个末端合成一条新链，取代前一次在双链中配对的同源链。当复制叉分子的另一端时，释放出被置换的链为自由链。自由链独立地复制：这需要通过分子末端的短互补序列间碱基配对形成双链。 Figure Adenovirus DNA replication is initiated separately at the two ends of the molecule and proceeds by strand displacement.

Figure The 5 terminal phosphate at each end of adenovirus DNA is covalently linked to serine in the 55 kD Ad-binding protein. In several viruses that use such mechanisms, a protein is found covalently attached to each 5 end. In the case of adenovirus, a terminal protein is linked to the mature viral DNA via a phosphodiester bond to serine, as indicated in Figure 应用这些机制的几个病毒中发现一个蛋白附着每个5′末端。腺病毒复制时一个末端蛋白通过键联到成熟的DNA，如图Figure 14.14所示。

How does the attachment of the protein overcome the initiation problem? The terminal protein has a dual role: it carries a cytidine nucleotide that provides the primer; and it is associated with DNA polymerase. In fact, linkage of terminal protein to a nucleotide is undertaken by DNA polymerase in the presence of adenovirus DNA. This suggests the model illustrated in Figure The complex of polymerase and terminal protein, bearing the priming C nucleotide, binds to the end of the adenovirus DNA. The free 3OH end of the C nucleotide is used to prime the elongation reaction by the DNA polymerase. This generates a new strand whose 5 end is covalently linked to the initiating C nucleotide. (The reaction actually involves displacement of protein from DNA rather than binding de novo. The 5 end of adenovirus DNA is bound to the terminal protein that was used in the previous replication cycle. The old terminal protein is displaced by the new terminal protein for each new replication cycle.) 蛋白的连接是怎样克服起始问题的呢?末端蛋白有双重作用：运载提供引物的核苷酸C；和DNA聚合酶协作。事实上末端蛋白与核苷酸连接是由DNA酶催化的。这符合Figure 14.15描述的模型。聚合酶与末端蛋白的复合物，载有引物C核苷酸，结合到腺病毒DNA末端。C核苷酸的自由末端3-OH通过DNA聚合酶引导延伸反应。这就产生了一条新链，其5′末端与起始C共价相连。(这个反应实际上包括蛋白质从DNA上移走而不是结合。腺病毒5′末端结合前一次复制循环中使用的末端蛋白上。对于每一个新循环，旧蛋白被新蛋白所取代。) 末端蛋白结合到离DNA末端9-18bp的区域。这个核心序列在宿主DNA中也能找到。邻近区域，17-48之间是宿主蛋白、核因子1(起始反应所需)结合所必须的。因此起始物可能在9-48之间形成，一个离DNA末端固定的距离。 Figure Adenovirus terminal protein binds to the 5 end of DNA and provides a C-OH end to prime synthesis of a new DNA strand.

12.8 Rolling circles produce multimers of a replicon
Rolling circle is a mode of replication in which a replication fork proceeds around a circular template for an indefinite number of revolutions; the DNA strand newly synthesized in each revolution displaces the strand synthesized in the previous revolution, giving a tail containing a linear series of sequences complementary to the circular template strand. Rolling circle: 滚环产生复制子多聚物复制产生的结构依赖于模板与复制叉的关系。最主要的特点是模板是圆形的还是线形的，复制叉是处是DNA双向还是单向合成。

Replication of only one strand is used to generate copies of some circular molecules. A nick opens one strand, and then the free 3OH end generated by the nick is extended by the DNA polymerase. The newly synthesized strand displaces the original parental strand. The ensuing events are depicted in Figure 复制叉是处是DNA双向还是单向合成。仅一条链的复制用于生成一些环形分子的拷贝，一个切刻打开一条链，随后切刻产生的自由3-OH末端被3-OH聚合酶延伸。新合成的链取代原母链。Figure 14.16描述了这些过程。这种结构被称为滚环，因为延伸点围绕环形模板链滚动。这能很精确地持续下去。当它移动时，复制叉延伸到外链取代前面的链。因为新合成的物质和新合成的物质共价相连，被置换的链的5′末端有原单位基因组。每次滚环取代前次循环中合成的物质。 Figure The rolling circle generates a multimeric single-stranded tail.

An example is shown in the electron micrograph of Figure The rolling circle is put to several uses in vivo. Some pathways that are used to replicate DNA are depicted in Figure Figure 14.17的电子显微摄影图显示了一个例子。 Figure A rolling circle appears as a circular molecule with a linear tail by electron microscopy.

Figure Rolling circles can be used for varying purposes, depending on the fate of the displaced tail. Cleavage at unit length generates monomers, which can be converted to duplex and circular forms. Cleavage of multimers generates a series of tandemly repeated copies of the original unit. Note that the conversion to double-stranded form could occur earlier, before the tail is cleaved from the rolling circle An example is shown in the electron micrograph of Figure The rolling circle is put to several uses in vivo. Some pathways that are used to replicate DNA are depicted in Figure 在体内滚环有几种用途。Figure 描述了DNA复制的几条途径。一个长末端的粘接生成原环形复制子的线形形式的拷贝。线形可能保持单链或因互补链 (其序列与原环的模板链一致)的合成转为双链。滚环复制为扩大原复制子提供一条途径。在Xenopus的卵母细胞中这种方式可产生放大的rDNA.rDNA.的基因形成大量邻接的重复片段。从基因组得到的单个重复单位转变成滚环。被取代的尾巴包含许多重复单位转变为双链DNA；接着它从环上分割下来，这样两个末端能被连接起来产生一个大的扩增的rDNA.环。因此扩大的物质包含大量的一样的重复单位。

Figure fX174 RF DNA is a template for synthesizing single-stranded viral circles. The A protein remains attached to the same genome through indefinite revolutions, each time nicking the origin on the viral (+) strand and transferring to the new 5 end. At the same time, the released viral strand is circularized. Replication by rolling circles is common among bacteriophages. Unit genomes can be cleaved from the displaced tail, generating monomers that can be packaged into phage particles or used for further replication cycles. A more detailed view of a phage replication cycle that is centered on the rolling circle is given in Figure Phage durham consists of a single-stranded circular DNA, known as the plus (+) strand. A complementary strand, called the minus ( ) strand, is synthesized. This action generates the duplex circle shown at the top of the figure, which is then replicated by a rolling circle mechanism. 滚环复制在噬菌体中普遍存在。单位基因组能丛置换的尾部分割出来，产生的单体能包装成噬菌体质粒或用于进一步复制循环。在Figure 14.19能更详细地看到一个以滚环复制为中心的噬菌体复制循环。噬菌体X174包括单链环形DNA，称为正链。其互补链即负链被合成。这个过程产生了双链环形，如图的顶端所示，然后以滚环方式复制。双链环形结构转变成。噬菌体的基因组编码的A蛋白切割双链DNA的正链，切割后A蛋白与5′末端相连而3′末端由DNA结合酶延伸。 DNA的结构在这个反应中有重要的作用，因为DNA只有在变性时可能被切割A蛋白能结合到包围在切刻周围的DNA片段。切割产生3′末端和5′末端这两个末端在噬菌体复制中都起作用。由于滚环复制切刻的3-OH末端进入一条新链，这条链围绕负链延伸直至它到达起始点取代原来的链此时A蛋白又其作用。它和滚环连接，因此当延伸点回到最初位置时，它就在附近，这样同一个A蛋白又可以识别起始位点并切割它，然后附着在新切刻的末端形成单链基因组便于细菌集结。

12.9 Single-stranded genomes are generated for bacterial conjugation
A large (~33 kb) region of the F plasmid, called the transfer region, is required for conjugation. It contains ~40 genes that are required for the transmission of DNA; their organization is summarized in Figure The genes are named as tra and trb loci. Most of them are expressed coordinately as part of a single 32 kb transcription unit (the traY-I unit). traM and traJ are expressed separately. traJ is a regulator that turns on both traM and traY-I. On the opposite strand, finP is a regulator that codes for a small antisense RNA that turns off traJ. Its activity requires expression of another gene, finO. Only four of the tra genes in the major transcription unit are concerned directly with the transfer of DNA; most are concerned with the properties of the bacterial cell surface and with maintaining contacts between mating bacteria. 细菌接合时形成单链基因组复制与遗传单位增殖间的联系还有一个例子，即细菌接合。细菌接合时，质粒基因组或宿主染色体从一个基因组转移到另外一个基因组。拼命以F粒为介导。F质粒是经典的游离体，它可以以自由的环形质粒存在，也可以作为线形序列整合到细菌染色体上。F质粒是一个大的环形DNA，约100kb长，它的遗传图按坐标从1到100F的系统相应完成的。 F因子从E.coli染色体多个位点整合进去，通常是借助宿主染色体与F质粒上特定的共同序列的重组(称为IS序列)。当以游离形式存在时，F质粒使用自身的起始位点(oriV)和调控系统，且维持每条染色体只有一个拷贝的水平。当它整合进细菌染色体时，自身复制系统被抑制，F因子DNA只能作为染色体的一部分被复制。 F质粒的存在，无论是游离的还是整合的对宿主细菌都有重要的影响。F+细菌能与F-细菌拼命(或交配)。接合包括供体与受体的接触；接触在F因子转移之后。如果F因子在供体细菌中作为游离质粒而存在，它就被作为一个质粒被转化，转化过程使F-受体变为F+。如果F因子在供体细菌中为整合态，转染过程可能使得一些或全部细菌染色体被转染。许多质粒有接合系统，以相似的方式运作，但F因子是第一个被发现的且一直作为这种遗传转染的例证。 F质粒上一个大的区域称为转染区域，它是接合所必需的。它包括约四十个DNA转移所需的基因，Figure 14.20总结了基因的组织形式。基因tra和trb。TraM和traJ是独立表达的traJ是启动traM和traY4的调控子。在相反的链上，finP是编码一小段关闭traJ基因的反义RNA的调控子。它的活性需要另一个基因finO的表达。只有四个tra基因与DNA的转染直接相相；大多数与细菌细胞表面的性质和交配细菌的接触有关。 F+细菌吸附着的蛋白菌毛是由F因子编码的。基因traA编码亚单位蛋白菌毛蛋白，菌毛蛋白聚合成菌毛。至少12个tra基因是菌毛蛋白修饰、组装成菌毛所必需的。F菌毛结构与头发相似，2-3m长，从细菌表面突起。典型的F因子有2-3个个菌毛。菌毛蛋白亚单位聚合成一个中空的圆锥体，直径约8nm。 Figure The tra region of the F plasmid contains the genes needed for bacterial conjugation.

Mating is initiated when the tip of the F-pilus contacts the surface of the recipient cell. Figure shows an example of E. coli cells beginning to mate. A donor cell does not contact other cells carrying the F factor, because the genes traS and traT code for "surface exclusion" proteins that make the cell a poor recipient in such contacts. This effectively restricts donor cells to mating with F-negative cells. (And the presence of F-pili has secondary consequences; they provide the sites to which RNA phages and some singlestranded DNA phages attach, so F-positive bacteria are susceptible to infection by these phages, whereas F-negative bacteria are resistant.) 当F菌毛顶端接触到受体表面，交配就开始Figure 14.21是一个E-coli开始交配的例子。供体细胞不和其它有F质粒的细胞接触，因为基因TraS和TraT编码“表面排斥”蛋白。这有效的限定了细胞只能与F细胞交配。供体与受体最初的接触很容易被中断，但其它的tra基因拉近交配细胞的距离，对结合起稳定作用。F因子的菌毛是起始结合所必须的。DNA肯定是通过一个通道转染的，但菌毛似乎不提供这样的通道；时至今日，这个通道仍然没有验证身份。TraD是F细菌中的一个内膜蛋白，它是DNA转移必需的，因此它有可能提供或是通道的一部分。 Figure Mating bacteria are initially connected when donor F pili contact the recipient bacterium.

Transfer of the F factor is initiated at a site oriT, the origin of transfer, which is located at one end of the transfer region. The transfer process may be initiated when TraM recognizes that a mating pair has formed. Then TraY binds near oriT and causes TraI to bind. TraI is a relaxase, like X174 A protein. TraI nicks oriT at a unique site (called nic), and then forms a covalent link to the 5 end that has been generated. TraI also catalyzes the unwinding of ~200 bp of DNA (this is a helicase activity; see later). Figure shows that the freed 5 end leads the way into the recipient bacterium. A complement for the transferred single strand is synthesized in the recipient bacterium, which as a result is converted to the F-positive state (for review see Frost et al., 1994). F因子的转化是从位点oriT开始的，oriT位于转化区域的一端。当TraM识别到交配双链形成时，转染过程就起始。然后TraY结合在OriT附近激发了TraI的结合。TraI是一个释放酶，例似于X174的A蛋白。TraI于唯一的位点切割OriT然后与产生的5′末端共价相连。TraI还有螺旋酶活性，解开长约200bp的DNA序列。Figure 14.22显示5′自由末端先进入受体细菌。在受体细菌内合成被转染的单链的互补链，结果是它转变为F。在供体细菌中必须合成互补链以取代已被转移的链。如果这与转录同时发生，F质粒形成如图所示的形状的DNA一般呈滚环形，但复制，单链转录独立于DNA合成。有一点我们还不明白的是，即使在滚环复制进行时，仅有单位长度的F因子被转化到受体细菌中。这意味着某个因素在第一次复制后终止了复制，然后F质粒恢复价键的完整。 Figure Transfer of DNA occurs when the F factor is nicked at oriT and a single strand is led by the 5 end into the recipient. Only one unit length is transferred. Complementary strands are synthesized to the single strand remaining in the donor and to the strand transferred into the recipient.

A complementary strand must be synthesized in the donor bacterium to replace the strand that has been transferred. If this happens concomitantly with the transfer process, the state of the F plasmid will resemble the rolling circle of Figure (and will not generate the extensive singlestranded regions shown in Figure 12.22). Conjugating DNA usually appears like a rolling circle, but replication as such is not necessary to provide the driving energy, and single-strand transfer is independent of DNA synthesis. Only a single unit length of the F factor is transferred to the recipient bacterium. This implies that some (unidentified) feature terminates the process after one revolution, after which the covalent integrity of the F plasmid is restored (for review see Ippen-Ihler and Minkley, 1986; Willetts and Skurray, 1987). Figure The rolling circle generates a multimeric single-stranded tail.

Figure Transfer of chromosomal DNA occurs when an integrated F factor is nicked at oriT. Transfer of DNA starts with a short sequence of F DNA and continues until prevented by loss of contact between the bacteria. When an integrated F plasmid initiates conjugation, the orientation of transfer is directed away from the transfer region, into the bacterial chromosome. Figure shows that, following a short leading sequence of F DNA, bacterial DNA is transferred. The process continues until it is interrupted by the breaking of contacts between the mating bacteria. It takes ~100 minutes to transfer the entire bacterial chromosome, and under standard conditions, contact is often broken before the completion of transfer (for review see Lanka and Wilkins, 1995). 但当完整的F质粒起始接合时，转化方向偏离转化区域而进入细菌染色体。Figure 14.23显示在F DNA的一小段先导序列后面有转入的细菌DNA。这个过程一直延续，直至被细菌间连接的中断所终止。转化整个细菌体染色体需约100分钟，在一般情况下转化完成之前连接被中断。当oriT位点整合的F因子缺刻时，染色体DNA发生转移，由于染色体DNA向远离gra区的方向转移，所以只有FDNA的一条短链进入受体，DNA的转移一直进行，直到细菌间的接触丧失才被阻止，随着一条互补链的合成，转移的DNA将和细菌的染色体在受体中重组。F因子在orit位置缺刻，5′末端引导单链DNA进入受体菌体的接触被打破在两菌中单链转化成双链供体DNA和受体基因组的重组。进入受体菌的供体DNA被转变成双链形式，可能与受体菌的染色体发生重组。(重组的发生要求有供体DNA的扦入)。这种染色体DNA的结合提供了一种细菌间遗传物质的交换方式大肠杆菌Ecoli的一个菌株与一个整合的F因子以相对高的频率支持这种重组(相对于缺乏整合F因子的菌株而言)；这种菌株被称为Hfr(高频重组)。对于一个受体菌染色体，F因子的每一个整合位置，对于一个不同Hfr菌株的重组频率提高，他们都具有转移细菌标记的典型模式。结合菌体间的接触通常在ONA的转移完成前分开。这种结果导致细菌染色体被转移的可能性主要依赖其与oriT区的距离。离F整合因子位点近的细菌基因先进入受体菌，并且比那些距离远的后进入受体菌的基因表现出较大的转移频率，这样就形成从F整合因子超递减的围绕染色体的转移频率的梯度。供体菌染色体的标记位置能够按转移发生的时间顺序被检测，同时，这也提供了一种大肠杆菌Ecoli染色体图谱的标准描绘，这种图谱按时间分成100分钟。对于每个Hfr菌株该图谱都提供了一套特定的转移时间。显然，转移梯度频率对每个Hfr菌株都是〓〓的。

12.10 Connecting bacterial replication to the cell cycle
Multiforked chromosome: Multiforked chromosome (in bacterium) has more than one replication fork, because a second initiation has occurred before the first cycle of replication has been completed.

Figure The fixed interval of 60 minutes between initiation of replication and cell division produces multiforked chromosomes in rapidly growing cells. Note that only the replication forks moving in one direction are shown; actually the chromosome is replicated symmetrically by two sets of forks moving in opposite directions on circular chromosomes. Consider the example of cells dividing every 35 minutes. The cycle of replication connected with a division must have been initiated 25 minutes before the preceding division. This situation is illustrated in Figure 12.24, which shows the chromosomal complement of a bacterial cell at 5minute intervals throughout the cycle. 细菌复制和细胞循环的联接关系细菌的复制和细胞的生长有两个联系： ◆复制循环的启始频率通过调整与细胞的生长相适应。 ◆复制循环的完成与细胞的分裂相结合。细菌生长率的倍增时间也就是细胞数量倍增的时间，倍增时间越短，细菌生长的越快，大肠杆菌Ecoli的生长倍增时间范围能从1分钟那么快到180分钟那么慢。由于细菌染色体是一个单复制子，复制循环的频率被单启始点启始的次数多少所控制。复制循环按两个常量定义。 ◆复制完全的细菌染色体时间约40min，这个固定时间被定义为C。这个持续时间和复制叉以50000bp/min移动速率相一致(在一个恒定的温度，DNA的合成速率变化小；在DNA的前体供应受限之前，DNA一直以相同的速率合成)。 ◆D是固定的20min，代表在复制完成一轮和细胞分裂间流失的时间。这段时间或许代表细胞分裂，各种必要成分组装的时间(常数C和D能被看作细菌有能力完成这些过程的最大速度。它们适用于18到60分钟的倍增时间之间的所有生长速率。但当细胞循环时间大于60min时，这两个常数时间都要相应的变长。)。细胞分裂前，一个染色体的复制循环必须启始固定的时间：C+D=60min，对那些比每60min分裂一次更频繁的细菌，一个复制循环必须在细胞分裂结束之前启始。让我们考虑一个每隔35min细胞分裂一次的例子。复制循环和一个细胞分裂的联接必须在细胞分裂进行前25min启始。这种状态在图14.24有图解说明，该图表明在整个循环中一个细菌细胞染色体的互补有5min的间期。在分裂启始，(35.0 mins)，细胞收到一个特殊复制的染色体，复制叉继续向前，10min时，当这个老复制叉还没有到达终点站，在特殊复制的染色体的两个源头再次发生复制的启始，这些新复制叉的开始，创造了一个多叉染色体。在15min，也就是另一次分裂之前20min，旧的复制叉到了终点。它的到达引起两个子染色体的分裂，每一个子染色体都已被新的复制又复制(现在是唯一的复制叉)，就些复制叉继续前进。

Figure The fixed interval of 60 minutes between initiation of replication and cell division produces multiforked chromosomes in rapidly growing cells. Note that only the replication forks moving in one direction are shown; actually the chromosome is replicated symmetrically by two sets of forks moving in opposite directions on circular chromosomes. 图：14.24在快速生长的细胞中，复制启始和细胞分裂间固定的60min间隔产生了多复制叉染色体。图中复制叉仅朝一个方向移动，实际上环状染色体以相反方向的两套复制叉对称复制。细胞分裂时，两个经特殊复制的染色体分离。然后又开始新一轮的循环单复制叉变老，并在15min时终止，在那以后20min有一次分裂。我们认为复制的启始发生在它所伴随的细胞分裂的1个(25/35min)细胞循环之前。复制启始和细胸循环间联系的一般规则是：当细胞生长快时(循环短)，启始事件发生在一个细胞循环次数增加的相关细胞分裂之前，在每个单独的细菌中相应的有较多的染色体，这种关系可被看作一种细胞反应的无能性，细胸想要保持与较短细胞循环时间一致的步调，却无能减少固定的C和D时间。细胞是如何知道复制循环启始时间的呢?复制启始以一个固定比率发生，即细胞团和染色体复制起点数量的比率。细胞生长的越快，这个比率越大，复制的起点也就越多，细菌的生长能够按单元细胞来描述，每个单元细胞都是17um长的实体。一个菌体的每个单元细胞中包含一个起点，有两个复制起点的生长较快的细胞将有17～34um长。参考图1424就是在细胞分裂后10min细胞团已经长大到是以在两个可行的复制起点去支持复制的启始。为滴定细胞集团，提出了两种模式。在细胞循环的整个阶段一种启动子蛋白能连续合成，启动子蛋白的大量积累将引发启动。或者在一个固定点可能合成一种抑制子蛋白，它因细胞容量的增加而被冲淡，从而导致其浓度低于有效水平，有理由表明一种滴定模式能调控启动，但是数据还不足以区分启动子的积累和抑制子的冲淡(稀释)。当前主要考虑启动子，其蛋白的合成对复制启始是必要的。

12.11 Cell division and chromosome segregation
Septum constitutes the material that forms in the center of a bacterium to divide it into two daughter cells at the end of a division cycle.

The formation of the septum is preceded by the organization of the periseptal annulus. This is observed as a zone in E. coli or S. typhimurium in which the structure of the envelope is altered so that the inner membrane is connected more closely to the cell wall and outer membrane layer. As its name suggests, the annulus extends around the cell. Figure summarizes its development (for review see de Boer et al., 1990). 细胞分裂和染色体的分离由于DNA可能在染色体中高度卷曲，而且可能包含相当少量的其它成份，所以人们对菌体中染色体的分离有着特殊的性趣(相比较而言，真核细胞染色体通过复杂的有丝分裂器完成分裂)，细胞器是相当准确的，但是，一个细菌群中有小于003%的无核细胞。细菌分裂成两个子细胞的同时伴随着一个横隔的形成，该横隔结构是由于外周膜的内陷而在细胞的中心构成。被横隔分开的两部分细胞互相隔离，并提供了子细胞最后完全分裂的位点，两个相关问题指出分裂中横隔的作用：①什么决定横隔形成的位置；②什么因素能确保子染色体能平均位于横隔相对的两边。横隔形成后，周边隔膜环也开始形成。这在coli或Styphimurium中被观察到：为了使内膜同细胞壁和外膜层的联连更紧密，(外)被膜的结构被改变。正如其名子所表明的那样，周边隔膜环沿着细胞周围延伸。图14.25概括了这种发展。周隔环能在一个新细胞的中央被观察到。随着细胞的生长发生了两件事。①细胞中心的横隔膜被周隔环限制。②在最初周隔环的两边都形成新的周隔环，这些新的周隔从细胞中心移到细胞长度1/4和3/4的位置，并将成为下一次分裂后细胞的中心位置。我们还不清楚它们的位移是怎么发生的，或许是由于中心位置外膜的生长去推动周隔离开，也有可能是被拉向两极，为确保细胞分裂成相同大小的子细胞，周隔环移动到正确的位置或许是十分重要的，当细胞的长度固定(ZL)且新的周隔环间的长度保持为L时，细胞开始分隔图14.25细胞周隔环的复制和移位。细胞中心周隔的开始新周隔环的产生新隔环向两极移动 ——停止移动中心周隔环发展成横隔细胞分裂侧视表明周隔环沿细胞周围沿伸横切图表明周隔环与膜连接我们不知道细胞是如何测量长度的，但相关的参数表现了这样的线性距离关系(非二维或三维)。 Figure Duplication and displacement of the periseptal annulus give rise to the formation of a septum that divides the cell.

The behavior of the periseptal annulus suggests that the mechanism for measuring position is associated with the cell envelope. It is plausible to suppose that the envelope could also be used to ensure segregation of the chromosomes. A direct link between DNA and the membrane could account for segregation. If daughter chromosomes are attached to the membrane, they could be physically separated when the septum forms. Figure shows that the formation of a septum could segregate the chromosomes into the different daughter cells if the origins are connected to sites that lie on either side of the periseptal annulus. 横隔和细胞外膜的组成成份相同：外周胞质层包含一严格的肽葡聚糖层，该葡聚糖层是通过三肽或五肽——二糖单元聚合构成，包括两种类型亚基的联接(转肽和转糖基)细菌的类杆状通过PBP-2和RodA的活动维持，PBP-2和RodA的活动对外膜的延伸是重要的，它们俩中任一个蛋白的基因突变都将导致细菌失去其延伸形状，成为圆形。这就证明了形状和严格度能被聚合结构的简单延伸所决定，这是一个重要的规则，横隔中用于产生肽葡聚糖的酶是PBP3(盘尼西林结合蛋白3)，一种催化位点在外周胞后中的膜界蛋白。横隔最初以一种双层肽葡聚糖的形式形成。为使子细胞能分裂，蛋白EnvA被用于破坏双层横隔间的共价连接。外周隔膜环的行为表明测量位置机械结构是与细胞外膜有联系的。我们设想外膜能保证染色体的分离，这似乎是合理的。一种DNA和膜直接连接的旧模式可能解释染色体的分裂如果子染色体们能与膜接触，他们能随着他们间膜的生长而被分离。图14.26表明横隔的形成或许能自动的把染色体分离到不同的子细胞中。例如，如果复制起点被结合到外周隔膜环的每一边的话，这种情况就能被保证。影响细胞分裂的突变体的分离是个别的(零星的，少的)。一个困难是临界功能的突变或许是致命的或多效性的。例如，如果外周隔膜环的形成点发生在对整个外膜的生长都非常重要的位置，去区分因外周隔膜环的形成所引起特异干涉导致的突变和一般性阻止外周膜生长的突变是困难的。图细胞DNA与膜接触能提供一种分裂结构模式正复制的染色体开始同膜结合子染色体和膜结合了染色体间形成横隔横隔分裂细胞染色体分配到子细胞中 Figure Attachment of bacterial DNA to the membrane could provide a mechanism for segregation.

12.12 The division apparatus consists of cytoskeletal and regulatory components
Minicell is an anucleate bacterial (E. coli) cell produced by a division that generates a cytoplasm without a nucleus.

A difficulty in isolating mutants that affect cell division is that mutations in the critical functions may be lethal and/or pleiotropic. For example, if formation of the annulus occurs at a site that is essential for overall growth of the envelope, it would be difficult to distinguish mutations that specifically interfere with annulus formation from those that inhibit envelope growth generally. Most mutations in the division apparatus have been identified as conditional mutants (whose division is affected under nonpermissive conditions; typically they are temperature sensitive). Mutations that affect cell division or chromosome segregation cause striking phenotypic changes. Figure and Figure illustrate the opposite consequences of failure in the division process and failure in segregation: 图14.27 细胞分裂失败产生多核线状物质。图片由sita Hiraga友情提供 ◆当横隔形成被抑制时长线结构形成，但染色体的复制不受影响。细菌继续生长，甚至继续到分离它们的子染色体，但横隔不形成，因而细胞构成了一咱很长的多核线状结构。 ◆当横隔形成的大频繁或位置错误时构成小细胞，有些新的子细胞还因此而缺失染色体。小细胞相当小，缺失DNA，但形态表现正常。当染色体分裂异常时形成无核细胞，像小细胞那样，它们缺失一条染色体，但由于横隔形成正常，它们的尺寸未改变。突变体引起细胞分裂成两种一般集合(通常以一种表型通过异常细胞的外观被揭示) ◇fts 突变表明一种温度敏感的线状细菌。通常细胞形成无横隔的线状，多核规则的沿细胞长向分配。这表明缺陷在于细胞分裂过程本身。 ◇par (分裂)突变展示突变缺失位于染色体分裂，这是由于染色体DNA在细胞中的异常分配，无核细胞因此形成。这种缺陷可能位于DNA的操作。细胞分裂的激活子之一是基因ftsz,ftsz基因是一大束包含在生产和分裂基因中的一部分。ftsz基因的突变阻遏横隔的形成并产生了线状细胞；Ftsz基因的过份表达通过引起每单元细胞基因分裂数的增加，导致小细胞的形成。 Ftsz活性基因的数量似乎与细胞分裂频率有直接关系，这种关系使Ftsz基因在细胞分裂启动中起重要作用。 Figure Failure of cell division generates multinucleated filaments. Photograph kindly provided by Sota Hiraga.

Figure E. coli generate anucleate cells when chromosome segregation fails. Cells with chromosomes stain blue; daughter cells lacking chromosomes have no blue stain. This field shows cells of the mukB mutant; both normal and abnormal divisions can be seen. A difficulty in isolating mutants that affect cell division is that mutations in the critical functions may be lethal and/or pleiotropic. For example, if formation of the annulus occurs at a site that is essential for overall growth of the envelope, it would be difficult to distinguish mutations that specifically interfere with annulus formation from those that inhibit envelope growth generally. Most mutations in the division apparatus have been identified as conditional mutants (whose division is affected under nonpermissive conditions; typically they are temperature sensitive). Mutations that affect cell division or chromosome segregation cause striking phenotypic changes. Figure and Figure illustrate the opposite consequences of failure in the division process and failure in segregation: 图14.28 Ecoli产生无核细胞染色体分离失败，细胞的染色体染兰色，子细胞缺失染色体无兰色，表明mukB基因发生突变，正常和异常的分裂均可见。一些ftsz突变体导致横隔形成的缺失，ftsz作用于不同的阶段，从外周隔膜环的移位到横隔的形态发生。然而，还没有突变被确定去影响细胞分裂的第一阶段，即外周隔膜环的产生。由于它们最接近外周环形成的调控问题，而且是否它直接同外膜的生长和DNA的分裂有直接联系，因而成为大家最感兴趣的问题。 Ftsz的一些有趣性后表明ftsz基因参与横隔形成的早期阶段。在细胞分裂循环早期，ftsz基因在细胞质中无所不在。当细胞伸长并开始在中部收缩时，ftsz基因分布于沿细胞周围的圆环上，特别是如图1425那样分布于中心外同隔膜环上。如果细胞的分裂依赖ftsz基因的浓度，ftsz基因环的浓度将成为横隔形成的台阶限制。Ftsz基因有GTP酶活性，所以一种可能性是GTP的分裂被用于支持寡聚ftsz单体结合成环状结构。

FtsZ functions at an early stage of septum formation. Early in the division cycle, FtsZ is localized throughout the cytoplasm. As the cell elongates and begins to constrict in the middle, FtsZ becomes localized in a ring around the circumference, essentially in the position of the midcenter annulus in Figure The formation of this ring could be the ratelimiting step in septum formation. FtsZ has GTPase activity, so one possibility is that GTP cleavage is used to support the oligomerization of FtsZ monomers into the ring structure. The structure of FtsZ resembles tubulin, suggesting that assembly of the ring could be akin to the formation of microtubules. FtsZ binds to ZipA, which is an integral protein that forms a ring in the inner membrane. 小细胞突变已经提供了一些关于横隔位置的信息。小细胞突变启始位于minB基因位置。由于minB基因的缺失，从而引起在细胞中央和两极的分裂，产生了小细胞。这表明细胞具有在中央或两极产生横隔的能力，同时宽型minB基因的位置作用是在两极抑制分裂。按图14.25所描述的那样，这暗示着新生细胞在中央和两极有外周隔膜环的地方都有潜在的分裂位点。细胞的一极是由上一次的分裂形成的，另一极则是代表更上一次分裂时的横隔。或许是因为两极还保持一些分裂前外周膜环的残余，这些残余能有助于核分裂。 Figure Duplication and displacement of the periseptal annulus give rise to the formation of a septum that divides the cell.

The minB locus consists of three genes, minC,D,E. Their roles are summarized in Figure The products of minC and minD form (or are necessary for formation of) a division inhibitor. Expression of MinCD in the absence of MinE, or overexpression even in the presence of MinE, causes a generalized inhibition of division. The resulting cells grow as long filaments without septa. Expression of MinE at levels comparable to MinCD confines the inhibition to the polar regions, so restoring normal growth. MinE protects the midcell sites from inhibition. Over-expression of MinE induces minicells, because the presence of excess MinE counteracts the inhibition at the poles as well as at midcell, allowing septa to form at both locations (de Boer et al., 1989). 图14.29 MinC/D是分裂抑制子，它们被MinE限制，而只能在两极发挥作用。 MinB基因位点由三个基因min C.D.E构成。他们的作用在图14.29中有概括说明。minC. D基因的产物构成分裂抑制子。在缺乏基因minE时minC.D的表达或当minE基因存在时minC.D的过份表达。将引起分裂抑制的强化。当没有横隔时细胞生长成长线性。当minE基因的表达同mine.D的表达成适当比例时，将限制对极区的抑制，细胞恢复正常的生长。同时，minE基因保护细胞中央位点免受抑制。minE基因的过份表达将抵抗细胞中央和两极的分裂抑制作用，在中央和两极都形成横隔，引发小细胞的形成。因此MinC.D和minE基因的比例将成为在细胞通常位置(细胞中央)分裂的决定性因素。宽型水平阻止两极分裂，却允许细胞中央分裂。MinC/D和MinE的影响成反比例关系，MinCD的缺乏或MinE过分表达引起非损伤性分裂，形成小细胞。MinCD过多或minE不是对细胞中央和两极横隔形成都有抑制作用，结果导致线状细胞。细胞分裂是两个子染色体在横隔形成的每一边找到各自位置的过程，在一般分裂中需注意两件事。 ◇两个子染色体必须相互释放以便它们能随分裂的终止而分裂，这要求终点附近互相卷绕的DNA区域解开缠绕。大多数突变影响用于编码局部异构酶基因的分裂图谱，这种异构酶能从DNA链间通过。突变阻止子染色体的分裂，导致DNA在细胞中央成为一大团。横隔的形成释放了一个无核细胞和一个包含两倍染色体的细胞。这就告诉我们细菌为将染色体分裂到不同的子细胞而必须部分解开它们缠绕的染色体。 ◆影响分裂过程本身的突变是少见的，我们预期有两类： DNA序列的Cis功能突变是分裂过程的目标。基因中用于编码蛋白质的突变是trans突变(翻译突变)，这种蛋白能引发细胞分裂，其中可能包含着可能与DNA结合的蛋白或着控制DNA可能外膜结合位点的活性。(已经发现两种类型的突变在■粒分裂系统中对其负责。仅有翻译突变功能在细菌染色体中被发现，下面将详述)。 Figure MinC/D is a division inhibitor, whose action is confined to the polar sites by MinE.

12.13 Partioning involves membrane attachment and (possibly) a motor
The original form of the model for chromosome segregation shown in Figure suggested that the envelope grows by insertion of material between the attachment sites of the two chromosomes, thus pushing them apart. But in fact the cell wall and membrane grow heterogeneously over the whole cell surface. Furthermore, the replicated chromosomes are capable of abrupt movements to their final positions at ? and ? cell length. If protein synthesis is inhibited before the termination of replication, the chromosomes fail to segregate and remain close to the midcell position. But when protein synthesis is allowed to resume, the chromosomes move to the quarter positions in the absence of any further envelope elongation. This suggests that an active process, requiring protein synthesis, may move the chromosomes to specific locations. 图14.26所展示的最简单的染色体分裂模式表明外膜通过在两个染色体接合位点扦入而生长。这或许太简单，因为外膜的生长似乎不受这些接合位点的限制，同时被复制的染色体能够猝然移到它们的最终位点，也就是细胞长度1/4和3/4的地方。如果蛋白质的合成在复制终止前被抑制，染色体将保持接近细胞中央的位置而不能分裂。但当蛋白质的合成重新开始时，染色体将移到不再延伸外膜的1/4位置，这表明为了使染色体移到特定位置，需要蛋白质的合成激活这个过程。另一类是muk基因的突变打断分裂，使无核后代出现的频率增加：两条子染色体保留在横隔的同一边而没分裂。muk基因的分裂是非致命的，或许能确定分裂染色体的细胞器的成份。mukA基因同已知的外膜蛋白基因tolc是一致的，它的产物能和染色体一起被卷入外膜，从而与外膜结合。基因mukB编码巨球蛋白(180KD)。基因mukB有些序列与编码机械化学酶发动蛋白有关，这种蛋白能为微管结合物提供动力。这表明mukB基因可能是染色体相对外膜自然移动的发动机。几年来我们一直怀疑细菌DNA和膜间存在体连接，但还没有直接理由。细菌DNA能在膜的碎片中找到，在遗传标记物中，细菌DNA以起点，复制叉到终点依次增多。在膜的这些部分中的蛋白质或许被干涉复制启始的突变影响。生长点可能是一种膜上结构，它的起点和复制起始点相连。膜和复制起点结合对复制是必要的，我们应当推出一种什么样的标准才满意呢?DNA顺式作同位点和与他们结合的反式作用因子的突变一定是一致的，这种一致性也一定同这些因子联系DNA同膜(直接/间接)结合的能力相符。尽管近来对膜结合的第一功能重要性有报道，但这些标准还是不令人满意。(参看15章)。 Figure Attachment of bacterial DNA to the membrane could provide a mechanism for segregation.

Functions involved in partition were first identified in plasmids. The components of a common system are summarized in Figure Typically there are two trans-acting loci and a cis-acting element located just downstream of the two genes. ParA is an ATPase. It binds to ParB, which binds to the parS site on DNA. Deletions of any of the three loci prevent proper partition of the plasmid. It seems likely that the ParAParB oligomer binds to some cellular structure, so that parS effectively behaves as a centromere. 多系统确保后粒在细菌中存活当一个分裂单元含许多复制子时，在每一个复制循环过程中每一个复制子的责任将被限制去仅对一个启始信号做出一次反应。但当一个基因组包含一个复制子时，复制次数也决定子代数。复制子也一次包含分裂子代成子细胞的任何系统责任。现在我们思考这些系统和复制本身的联系。我们已提过每种质粒在寄主菌中维持一定的拷贝数。单拷贝控制系统类似于细菌染色体在每次细胞分裂中只复制一次。单拷贝质粒能有效维持其与细菌染色体间的等量性。拷贝控制系统充许每个细胞循环中多次复制启始，导致每个细菌中有多个质粒拷贝。每个细菌染色体中多拷贝质粒都有典型数量(10—20个) 多拷贝数主要是复制控制结构类型的结果，复制启始的责任系统决定细菌中有多少个复制起点。因为每一个质粒构成一个单复制子，复制起点数与质粒分子数相同。单拷贝质粒有一复制控制系统，其作用近似于对菌染色体的控制。一个单起点被复制一次，然后子复制起点被分离到不同的子细胞中。多拷贝质粒有一允许多复制起点存在的系统。如果这个数目足够大(每个细菌中大于10个起点)，一个活跃的分裂系统变的多余，因为甚至从统计分配上讲，质粒在分配给子细胞时仅以低于10 -6的频率缺失。质粒在菌体中维持非常低的缺失率，(甚至对一个单拷贝质粒来说，一般在每个细胞分裂中都少于10 -7)。控制质粒分裂的系统们能通过增加缺失频率的突变取得一致，但对复制本身极用。一些机械结构被用于保证质粒在细菌中的存活，对一个质粒来说，在几个不同类型的系统中运输是普通的，这几个系统都独立作用来确保质粒的存活。这些系统中的一些间接作用，另一些在分裂中直接作用。然而按进化论，它们都是为确保子代细菌中质粒最大数量的永续性。 Figure A common segregation system consists of genes parA and parB and the target site parS.

Figure 9.23 Sporulation involves successive changes in the sigma factors that control the initiation specificity of RNA polymerase. The cascades in the forespore (left) and the mother cell (right) are related by signals passed across the septum (indicated by horizontal arrows). Proteins related to ParA and ParB are found in several bacteria. In B. subtilis, they are called Soj and SpoOJ, respectively. Mutations in these loci prevent sporulation, because of a failure to segregate one daughter chromosome into the forespore (see Figure 9.23). In sporulating cells, SpoOJ localizes at the pole and may be responsible for localizing the origin there. SpoOJ binds to a sequence that is present in multiple copies, dispersed over ~20% of the chromosome in the vicinity of the origin. It is possible that SpoOJ binds both old and newly synthesized origins, maintaining a status equivalent to chromosome pairing, until the chromosomes are segregated to the opposite poles. In C. crescentus, ParA and ParB localize to the poles of the bacterium, and ParB binds sequences close to the origin, thus localizing the origin to the pole. These results suggest that a specific apparatus is responsible for localizing the origin to the pole. The next stage of the analysis will be to identify the cellular components with which this apparatus interacts (Mohl and Gober, 1997).

12.14 Multiple systems ensure plasmid survival in bacterial populations
Because the multiple copies of a plasmid in a bacterium consist of the same DNA sequences, they are able to recombine. Figure demonstrates the consequences. A single intermolecular recombination event between two circles generates a dimeric circle; further recombination can generate higher multimeric forms. Such an event reduces the number of physically segregating units. In the extreme case of a single-copy plasmid that has just replicated, formation of a dimer by recombination means that the cell only has one unit to segregate, and the plasmid therefore must inevitably be lost from one daughter cell. To counteract this effect, plasmids often have site-specific recombination systems that act upon particular sequences to sponsor an intramolecular recombination that restores the monomeric condition. Mutations in these systems increase plasmid loss, and therefore have a phenotype that is similar to partition mutants. (The bacterial chromosome itself may have a similar system to deal with the consequences of recombination occurring between homologous sequences in the daughter chromosomes produced by a replication cycle.) 图细胞间重组合并单体成二体，细胞内重组从寡体中释放一个单元。由于细菌中一个质粒的多拷贝构成同样的DNA序列，他们能重组。图14.30证明了这种结果。在两个循环间的一个单细胞间重组产生了一个二体循环，进一步的重组能产生更高级的多体形式，这将导致分裂单元数的减少。对一个被复制的单拷贝质粒的极端情况是：通过重组形成的二体也就意味着：细胞只有一个单元分裂，质粒也就必然从一个子细胞中丢失。为了抵制这种影响，质粒通常有一种位点特异性重组系统，该系统作用于特殊DNA序列去支持能恢复单体状态的分子内重组。同时，该系统的突变会增加质粒的丢失，因而也就具有一种近似于分裂突变体的表现型。 (细菌染色体本身或许有一近似系统，这一系统用来处理通过复制循环产生的子代染色体里同源序列间的重组序列问题。) Figure Intermolecular recombination merges monomers into dimers, and intramolecular recombination releases individual units from oligomers.

Addiction systems, operating on the basis that "we hang together or we hang separately," ensure that a bacterium carrying a plasmid can survive only so long as it retains the plasmid. There are several ways to ensure that a cell dies if it is "cured" of a plasmid, all sharing the principle illustrated in Figure that the plasmid produces both a poison and an antidote. The poison is a killer substance that is relatively stable, whereas the antidote consists of a substance that blocks killer action, but is relatively short lived. When the plasmid is lost, the antidote decays, and then the killer substance causes death of the cell. So bacteria that lose the plasmid inevitably die, and the population is condemned to retain the plasmid indefinitely. These systems take various forms. One specified by the F plasmid consists of killer and blocking proteins. The plasmid R1 has a killer that is the mRNA for a toxic protein, while the antidote is a small antisense RNA that prevents expression of the mRNA. 图没有质粒的存在并合成长效毒素和短效解毒剂，细菌就不能存活。 “我们悬挂在一起或我们分开悬挂”，以此为基础操纵的“成瘾”系统，是为了确保一个细菌有而且只要保留有一个质粒就能存活。图14.32阐示了一种规则，质粒既产生毒素也产生抗毒素，因而有几种方式确定一个细胞的死亡，如果这个细胞曾被一个质粒“治疗”过。质料产生的毒素是一种具有相当稳定性的致命物质，反之抗毒素虽能抑制毒素的活动，但其活性稍差。当细胞的质粒丢失，而抗毒素又过期时，致命的毒素将引起细胞的死亡，该细胞群因保留不明确的质粒而被淘汰。这些系统有不同的形式：F质粒特异系统由毒素和抗毒蛋白构成；质粒R1有一编码有毒蛋白的MRNA，其表达受到一种小反感RNA解毒剂的抑制。作用于DNA分子复制的真正分裂系统能确保DNA分子在细胞分裂时位于横隔的两边。 Figure Plasmids may ensure that bacteria cannot live without them by synthesizing a long-lived killer and a short-lived antidote.

True partition systems act upon duplicate DNA molecules to ensure that they reside on either side of the septum at cell division. Probably all low copy number plasmids have such a system. Systems that have been characterized for the plasmids F, P1, and R1 have the generally similar organization depicted in Figure There are two trans-acting proteins and a single cis-acting site. In the cases of both P1 and F, the smaller of the two proteins binds to the cis-acting site. In spite of their overall similarities, there are no significant sequence homologies between the corresponding genes or cis-acting sites (for review see Hiraga, 1992). 图一些包含分裂系统的质粒由相邻基因和顺式作用位点构成。除了已知质粒编码并与顺式作用位点结合的蛋白质对分裂是必须的，我们几乎对分裂的结构一无所知。质粒保证所有子细胞都获得复制质粒的重要性，通过能保证分裂的单独质粒中存在多重独立系统而得到强调。可能所有的低拷贝数质粒都有这种系统。图14.32描绘了F，P1和R1质粒具有典型的该系统，他们一般都有相似的组织。图中有两个反式作用蛋白和一个顺式作用位点。在F和P1质粒中，两个反式作用蛋白中较小的那个结合在顺式作用位点。尽管他们全部相似，但在相关基因成顺式作用位点并没有显著的序列同源性。一个真正的复制系统是如何把复制的质粒分裂到不同的子细胞的呢?我们或许可设想两种类型：质粒一定结合的外在结构是有限的。单拷贝质粒模式同图14.26所示相同。细胞仅保留两个位点，质粒的每一个拷贝同一位点结合，这两个位点可能是膜界限或仅构成细菌染色体本身的区域。这种模式的不足是：在细菌群中发现了许多不同类型的质粒，这使我们有必要假设对每种类型的质粒都有一对分离的、专门的位点。分裂是一种内在过程，染色体配对的同阶段，质粒复制。随着质粒成对结合，一种分裂结构将每对质粒中的每一个移向相对的子细胞中。这种分裂结构的性质仍难于想象，我们仍有必要解释细胞是如何处理多类型质粒的分裂。图没有质粒的存在并合成长效毒素和短效解毒剂，细菌就不能存活。 Figure A common segregation system consists of genes parA and parB and the target site parS.

Extrinsic structures to which the plasmids must bind are limiting. The model for a single-copy plasmid is the same as illustrated previously in Figure The cell must contain only two sites, and each copy of the plasmid binds to one. The sites could be membranebound or could simply constitute regions of the bacterial chromosome itself. A disadvantage of this type of model is that many different types of plasmids are found in bacterial populations, which makes it necessary to suppose that there are separate and exclusive pairs of sites for each type of plasmid. Figure Attachment of bacterial DNA to the membrane could provide a mechanism for segregation.

12.15 Plasmid incompatibility is connected with copy number
Compatibility group: Compatibility group of plasmids contains members unable to coexist in the same bacterial cell.

Figure Two plasmids are incompatible (they belong to the same compatibility group) if their origins cannot be distinguished at the stage of initiation. The same model could apply to segregation. The introduction of a new origin in the form of a second plasmid of the same compatibility group mimics the result of replication of the resident plasmid; two origins now are present. So any further replication is prevented until after the two plasmids have been segregated to different cells to create the correct prereplication copy number as illustrated in Figure 图如果两质粒的复制启始点在复制起始不被区分，他们是不相的(他们属于同一相组)。该模式还可用于分裂。质粒细胞循环不相容质粒的细胞循环质粒不相容性和拷贝数有关质粒不相容现象和质粒拷贝数和分裂的调控有关。质粒相类组的定义：一套质粒其成员不能在同一细菌细胞中共存。他们不相类的原因是：他们在质粒维持的一些重要阶段彼此不都区分。这适用于DNA复制和分裂的阶段。质粒不相类的消极控制模式遵循：拷贝数通过抑制子合成而得到控制的思想，该抑制子控制复制起点的浓度。(这同调控细菌染色体复制的滴定模式相同) 同一相类组中第二个质粒的形成引进了一个新的复制起点，这同原有质粒的复制相模仿，现在有两个复制起点了。如图14.33所示，在两个质粒被分裂到不同的细胞，进一步产生正确的拷贝数前，任何进一步的复制都受抑制。如果分裂产物到子细胞时，系统不能区别两个质粒，将产生类似的效果。例如，如果两质粒有同样的顺式作用分裂位点，它们间的竞争将确保而质粒被分裂到不同的细胞，因而不能在同一细胞中存活。从目的性来看，质粒是自私的。已经有质粒的细菌，现有质粒将设法阻止同一类型的其它质粒取得居留权。这些领土权力的建立主要通过质粒的不相容性取得。某一质粒在一个相容组中的存在并不直接影响该质粒在不同相容组的存活。给定的一个相容组(单拷贝质粒)中仅有一个复制子能在细菌中维持，但不能与别的相容组中的复制子相互反应。(尽管在限定条件下他们为lebensraum而竞争)。

The best characterized copy number and incompatibility system is that of the plasmid ColE1, a multicopy plasmid that is maintained at a steady level of ~20 copies per E. coli cell. The system for maintaining the copy number depends on the mechanism for initiating replication at the ColE1 origin, as illustrated in Figure 图 colE1 DNA的重制通过切割引物RNA产生3-OH末端启始。引物RNA与启始区形成牢固的杂交。质粒colEl是目前描绘最好的拷贝数和不相系统。colE1质粒是每个E.col之细胞维持约20个拷贝数的多拷贝质粒。维持拷贝数目的系统依赖于在colE1起始点用于复制启始的结构。在图14.34中阐明了相关问题。复制以复制起点上游555bp的RNA的转录启始为开始。能录继续通过复制起点。RNaseH(RNA酶H)其名称反应了分离RNA和DNA杂交链的特异性)在起点切断转录。这产生了一个引发DNA合成启始的3-OH末端引物。(引物的作用在15章有详细讨论)与前体RNA和DNA形成牢固的杂交。RNA和DNA的配对发生在约-20的上游启始点和更上游的-265启始点。互补链的合成；另一个是邻近位点蛋白质的编码。两个调控系统对RNA前体施加了影响：一个是RNA前体。 Figure Replication of ColE1 DNA is initiated by cleaving the primer RNA to generate a 3-OH end. The primer forms a persistent hybrid in the origin region.

The regulatory species RNA I is a molecule of ~108 bases, coded by the opposite strand from that specifying primer RNA. The relationship between the primer RNA and RNA I is illustrated in Figure The RNA I molecule is initiated within the primer region and terminates close to the site where the primer RNA initiates. So RNA I is complementary to the 5terminal region of the primer RNA. Base pairing between the two RNAs controls the availability of the primer RNA to initiate a cycle of replication (Tomizawa and Itoh, 1981; Masukata and Tomizawa, 1990). 图14.35 RNAI序列同引物RNA 5区互补 RNAI调控子是一个约108碱基的分子，被前体RNA的反链编码。图14.35阐明引物RNA和RNAI间的关系。RNAI分子启始于primer区终止于接近引物RNA启始的位点，因此RNAI分子与引物RNA的5，末端区互补。两RNA间的碱基配对控制引物RNA启始复制循环的可行性。象RNAI那样和在同一区域被编码的RNA有互补功能，这种功能叫逆转录。这种结构类型是仅意RNA用途的另一例(参看12章)。减少或根除质粒间不相容性的突变能通过同组共存质粒的选择获得。ColE1图谱的不相容突变位于RNAI和引物RNA间的部分重叠区。由于该区在两个不同RNA中得到再现，所以它们中的一个或两个将受到影响。当把RNAI加入一个colE1 DNA复制在体外系统，它抑制有活性引物RNA的形成，但RNAI的存在并不抑制引物RNA合成的启始或延伸。这表明RNAI阻止RNA酶H产生引物RNA的3末端。这种影响的基础是RNAI和引物RNA间的碱基配对。这个区域两RNA分子都有同样的潜在二级结构，有三个二重发卡终止于单链环。降低不相容性的突变位于这些单链环上。这表明RNAI和引物RNA碱基配对的启始步骤是与未配对的单链环相接触的。 Figure The sequence of RNA I is complementary to the 5 region of primer RNA.

How does pairing with RNA I prevent cleavage to form primer RNA? A model is illustrated in Figure In the absence of RNA I, the primer RNA forms its own secondary structure (involving loops and stems). But when RNA I is present, the two molecules pair, and become completely doublestranded for the entire length of RNA I. The new secondary structure prevents the formation of the primer, probably by affecting the ability of the RNA to form the persistent hybrid. 和RNAI的配对是如何阻止引物RNA形成的呢?图14.36阐明了一种模式。缺乏RNAI时，引物RNA形成了自己的二级结构。(包括环和杆)。当RNAI存在，两分子配对，使RNAI分子的全长都变成完全双链。新的二级结构或许是通过影响RNA形成牢固杂交链的能力，来阻止引物RNA的形成转录的弱化结构模式同上，这种结构中，RNA序列的交替配对允许或阻止二级结构的形式，RNA聚合酶终止时需要这种二级结构。(参看12章)。RNAI通过其影响引物RNA远区域前体的能力而被检验。形式上，该模式同包括两个RNA样品的假定控制循环相同。一个巨大的RNA引物前导是正调控子，同于复制启始。小的RNAI是负调控子，能抑制正调控子的活动。在作用于存在于细胞中的任何质粒的能力上，RNAI提供了抑制子阻止新引入的DNA发生作用，这类似于入源性抑制子的作用(参看13章)。一条RNA结合新合成的前导和RNA引物，而不是抑制子蛋白结合新的DNA。在RNAI与引物RNA之间的连结能被Rom蛋白影响，该蛋白是由位于起始点下游的一个基因编码的。Rom蛋白增强了RNAI与多于200个碱基的RNA引物转录物的结合，结果抑制了引物的形成。 Figure Base pairing with RNA I may change the secondary structure of the primer RNA sequence and thus prevent cleavage from generating a 3-OH end.

Figure Mutations in the region coding for RNA I and the primer precursor need not affect their ability to pair; but they may prevent pairing with the complementary RNA coded by a different plasmid. How do mutations in the RNAs affect incompatibility? Figure shows the situation when a cell contains two types of RNA I/primer RNA sequence. The RNA I and primer RNA made from each type of genome can interact, but RNA I from one genome does not interact with primer RNA from the other genome. This situation would arise when a mutation in the region that is common to RNA I and primer RNA occurred at a location that is involved in the base pairing between them. Each RNA I would continue to pair with the primer RNA coded by the same plasmid, but might be unable to pair with the primer RNA coded by the other plasmid. This would cause the original and the mutant plasmids to behave as members of different compatibility groups. RNA中的突变是如何影响不相溶性的呢?图14.37表示了细胞内含有两类RNAI/引物RNA序列的情形。从各类基因组得到的RNAI与引物RNA能相互作用，但来自两个不同基因组的RNAI与RNA引物不能作用。如果RNAI和引物RNA共同的区域在涉及二者之间碱基配对的位点处发生突变，上述情形将更为显著。每个RNAI与同一质粒编码的引物RNA都能继续配对，但可能不与另一质粒所编码的引物RNA配对，这可能导致起始及突变的质粒表现为属于不同的相溶性类别。

Summary 1. The entire chromosome is replicated once for every cell division cycle. 2. Eukaryotic replication is (at least) an order of magnitude slower than bacterial replication. 3. The minimal E. coli origin consists of ~245 bp and initiates bidirectional replication. 4. The rolling circle is an alternative form of replication for circular DNA molecules in which an origin is nicked to provide a priming end. 5. Rolling circles are used to replicate some phages. 提要每个细胞进行分裂的周期，整个染色体都复制一次。复制起始阶段将细胞定型为一个分裂周期，复制完成后，将为实际分离过程提供引物，细菌染色体含有一个单独的复制子，但真核染色体分为许多复制子，它们在S相的伸展期起作用。线形复制子末端的复制问题有许多方法来解决，许多把复制子转换成环状形式。一些病毒有特异蛋白来识别末端，真核染色体遇到的问题是末端复制子。真核复制的重要性至少要次于细菌复制。起始点负责双向复制，可能用于S相的固定顺序。唯一在序列水平上作鉴定的真核起始点是SCerevisiae,其核心序列含有11个碱基对，绝大多数是A·T。 E. coli最小的起始点含有245个碱基对，它起动双向复制。任何含有此序列的DNA分子均能在Ecoli中复制。两个复制叉离开起始点，绕染色体移动，直到二者相遇，尽管已识别了能导致复制叉在相遇后终止的ter序列。转录单位组织起来，以便转录通常与复制在同一方向上进行。滚动环是环状DNA分子复制的形式之一。一个起始点上打开缺口而形成引物端，DNA的一条链由此端合成，取代了原先的伴随链并像尾巴一样挤出来，环继续滚动，将形成多基因组。滚动环用于复制一些阶段，X174起始点上空缺的蛋质A有反作用的特殊功能，它在DNA上自身被合成的部位开始作用，它维持与被取代链的连接，直到合成一条完整的链，然而再次在起始点上打开缺口，释放被取代的链，开始另一循环的复制。

Summary 6. Rolling circles also are involved in bacterial conjugation, when an F plasmid is transferred from a donor to a recipient cell, following the initiation of contact between the cells by means of the F-pili. 7. A fixed time of 40 minutes is required to replicate the E. coli chromosome and a further 20 minutes is required before the cell can divide. 8. Segregation involves additional sequences that have not yet been characterized. 9. Plasmids have a variety of systems that ensure or assist partition, and an individual plasmid may carry systems of several types. 滚动环也用于细菌的结合作用，此时一个F质粒从供体上传递到一个受体细胞中，之后便以F-Pili的方式开始细胞间接触，一个游离的F质粒以同样方式浸染其他细胞；一个整合的F因子产生可传递染色体DNA的Hfr菌株，在偶联情形下，通过复制、补充物合成留在供体中的单链及传送到受体中的单链，但不提供动力。复制EColi染色体需要40分钟，细胞分离前又需要20分钟，当不到60分钟即迅速进行细胞分裂时，在前面的解链循环结束前即开始了复制循环，由此产生了多复制叉的染色体， “起始”活动依赖于细胞物质的滴定，这可能要通过某种起始蛋白的积累。由于起始后的一小段时间内起始点与细胞膜相连，起始可能是在细胞膜上发生。分离涉及到尚未鉴定出的其它序列。大多数妨碍正确分离的活动取决于辅助功能，例如需要酶来解开两条姊妹染色体。目前发现的唯一似乎直接影响的分离活动可能鉴别了一种将染色体与外膜相连接的动力或蛋白质。分隔细胞的隔片所生长的位置受限于先前存在的周隔环，此基因座位置有三个基因密码，其产物调节细胞中的周隔环或者原先的环产生的极性位置是否用于形成隔片。未形成隔片会导致多核的丝状体；隔片形成过多，会导致无核小细胞。质粒有许多系统来保证或协助分隔，一个独立的质粒可能携带几种类型的系统，一个质粒的拷贝数描绘出它是处于与微生物染色体同一水平(每个单位细胞1个)还是数量更大。质粒不相溶性的结果可能由于涉及到复制或是分隔(对于单拷贝质粒)的机制。共享同一复制调控系统的两个质粒不相溶。因为复制次数确保每一细菌基因组只有一个质粒。

Chapter 12 The replicon.

Similar presentations

Presentation on theme: "Chapter 12 The replicon."— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

Chapter 12 The replicon.

Similar presentations

Presentation on theme: "Chapter 12 The replicon."— Presentation transcript:

Similar presentations

About project

Feedback