Admin. supplement for equipment to Mechanisms underlying the Rlm1-dependent G1 checkpoint (NIH R15 GM135807)

(来自家长助学金) 当细胞分裂时，细胞分裂的不同细胞程序必须相互协调。为 例如，染色体的复制，这些复制的染色体的分离，以及 一个细胞分裂成两个细胞必须都发生在相对于彼此和其他细胞的精确时间 事件。为了实现这种协调，在细胞周期中有特殊的时间，称为检查点，在 细胞周期的哪个进程可以暂停。在这些检查点，准确/无损完成 在启动较晚的程序之前，必须先启动较早的程序。检查点之所以有效，是因为 细胞有专门的机器，由酶和其他蛋白质组成，由受损的或 不完整的细胞结构。一旦激活，这些检查点机器就会导致细胞的进展 循环停止，直到细胞结构修复完成。当检查点有缺陷时，单元 在整个周期中继续积累受损的结构并导致疾病状态。的确， 在癌细胞中，检查点经常被绕过，有缺陷的检查点会导致更高的癌症风险。我们 最近提出了一种新的检查点，这里被称为RLM1依赖的检查点，它延迟了通过 在特定的应激环境条件下通过细胞周期。顾名思义，这就是 R1m1转录因子编码基因的缺失突变揭示了检查点。RLM1为 已知对细胞壁应力的反应被激活，所以这个检查点可能对压力在 细胞表面。有趣的是，与绕过已知检查点不同，绕过RLM1检查点会导致 在细胞分裂的两种产物中只有一种导致细胞死亡。我们在这个项目中的长期目标是 描述依赖RLM1的检查点的功能和机制。我们的第一个具体目标是 描述此检查点运行的细胞周期中的位置，以及细胞周期 调节器和通过其发挥作用的其他细胞组件。我们的第二个具体目标是确定 此检查点响应的信号的性质以及累积的损害的性质 当检查站被绕过时。为了实现这些目标，我们将采用基因、 细胞学和分子生物学检测。例如，我们将同时培养普通酵母和突变酵母。 在无压力条件下RLM1检查点出现故障，然后突然将其释放到 压力很大的状况。然后我们将比较正常酵母和突变酵母随着时间的推移，已知的分子或 细胞周期中的细胞事件以及不同类型的细胞损伤。我们将测试特定的 通过检查已知存在缺陷的突变体，提出关于该检查点机制的假说 细胞维护和细胞周期控制的特定方面，以了解它们在这一检查点中的作用。 这种新型检查点的特征应该揭示细胞周期控制的重要新方面 在理解疾病状态的新方面可能会被证明是有用的。

(from parent grant) As cells divide the different cellular programs of cell division must be coordinated with one another. For example, the duplication of chromosomes, the separation of these duplicated chromosomes, and the division of one cell into two cells must all occur at precise times relative to one another and to other cellular events. To achieve this coordination, there are special times during the cell cycle, termed checkpoints, in which progression of the cell cycle can be paused. At these checkpoints, accurate/ undamaged completion of an earlier program is necessary before a later program will be initiated. Checkpoints work because the cell has specialized machines, made up of enzymes and other proteins, that are activated by damaged or incomplete cellular structures. Once activated these checkpoint machines cause the progression of the cell cycle to halt until the cellular structures are repaired and complete. When checkpoints are defective, cells continue through the cycle accumulating damaged structures and leading to disease states. Indeed, checkpoints are often bypassed in cancer cells, and defective checkpoints lead to higher risk of cancer. We recently proposed a novel checkpoint, here termed the “Rlm1-dependent checkpoint”, that delays passage through the cell cycle under a particular stressful environmental condition. As the name suggests, this checkpoint was revealed by a deletion mutant in the gene encoding the Rlm1 transcription factor. Rlm1 is known to be activated in response to cell-wall stress, so it may be that this checkpoint responds to stress at the cell surface. Interestingly, bypassing the Rlm1 checkpoint, unlike bypassing known checkpoints, leads to cell death in only one of the two products of cell division. Our long-term objective in this project is to characterize the function and mechanism of the Rlm1-dependent checkpoint. Our first specific aim is to characterize the position during the cell cycle at which this checkpoint operates, and the cell cycle regulators and additional cellular components through which it acts. Our second specific aim is to identify the nature of the signal to which this checkpoint responds and the nature of the damage that accumulates when the checkpoint is bypassed. To accomplish these aims we will employ a combination of genetic, cytological, and molecular biological assays. For example, we will grow both normal yeast and mutants defective in the Rlm1 checkpoint under non-stressful conditions and then release them suddenly into the stressful condition. We will then compare the normal and mutant yeast over time for known molecular or cellular events in the cell cycle as well as different types of cellular damage. We will test specific hypotheses regarding the mechanism of this checkpoint by examining mutants known to be defective in particular aspects of cell maintenance and cell cycle control for their role in this checkpoint. Characterization of this new type of checkpoint should reveal important new aspects of cell cycle control and may prove useful in understanding new aspects of disease states.