Stochastic models of cell cycle regulation in eukaryotes

 描述(申请人提供)：细胞周期是正在生长的细胞复制其基因组并在分裂时将每个染色体的两个副本分割为两个子细胞的过程。复制(DNA合成)和分裂(有丝分裂)的这些过程以极高的保真度进行，这对生命的永续至关重要。在真核细胞中，DNA合成(S期)和有丝分裂(M期)在时间上被两个间隙(G1和G2)分开。如果细胞的基因组完整性以任何方式受到损害，“检查点”就会阻止细胞周期的进展，从而强制S期和M期的适当交替。例如，如果DNA在G1期受损，检查点将阻止进入S阶段，直到损伤得到修复。如果复制的染色体在有丝分裂纺锤体上没有正确排列，不同的检查点会阻止进入后期(姐妹染色单体分离阶段)，直到所有姐妹染色单体正确地连接到纺锤体的相反两极。检查点能够通过向控制特定细胞周期转换的分子机制(G1-S、G2-M和M-G1)发送停止信号来阻止细胞周期的进展。支配每一种转变的分子机制都有一种特殊的性质，称为“双稳定性”。在生理条件下，控制机制可以无限期地持续在两种特征状态中的任何一种：关闭状态，对应于将细胞周期保持在过渡前阶段；以及开启状态，对应于将细胞周期推入过渡后阶段。检查点停止信号似乎通过将适当的双稳态开关稳定在其关断状态来起作用。由于这些检查点对于从一代细胞到下一代细胞保持有机体基因组的完整性至关重要，因此即使面对随机的分子波动，它们的可靠运行也是至关重要的，而随机分子波动在一个像酵母细胞这样的小细胞(30 Fl)中是不可避免的。基于控制细胞周期进程的分子机制的随机模型的计算表明，在野生型发芽酵母细胞中，检查点功能确实是强大的，但在携带特定检查点基因突变的菌株中，它们可能会受到影响。这项建议的目的是提供所需的数学模型和实验数据，以了解细胞周期检查点如何在野生型酵母细胞中可靠地运行，以及它们如何在突变细胞中失败。为了实现这一目标，需要在随机建模和测量单个酵母细胞中的mRNA和蛋白质分子的技术方面取得新的进展。要测试这些模型，需要专业知识来构建 并表征了由该模型预测的萌芽酵母特定突变株的表型，这些突变株在酵母mRNAs和蛋白质的随机波动面前表现出检查点阻止的脆弱性。由于所有真核生物似乎都使用相同的基本分子机制来控制细胞分裂周期的进展，因此对酵母细胞中检查点操作的理解将转化为对其他类型细胞(尤其是人类细胞)中检查点功能和故障的更好理解。

 DESCRIPTION (provided by applicant): The cell cycle is the process by which a growing cell replicates its genome and partitions the two copies of each chromosome to two daughter cells at division. It is of utmost importance to the perpetuation of life that these processes of replication (DNA synthesis) and partitioning (mitosis) be carried out with great fidelity. In eukaryotic cells, DNA synthesis (S phase) and mitosis (M phase) are separated in time by two gaps (G1 and G2). Proper alternation of S phase and M phase is enforced by `checkpoints' that block progression through the cell cycle if the genomic integrity of the cell is compromised in any way. For example, if DNA is damaged in G1 phase a checkpoint blocks progression into S phase until the damage can be repaired. If replicated chromosomes are not properly aligned on the mitotic spindle, a different checkpoint blocks progression into anaphase (the phase of sister chromatid separation) until all sister chromatids are properly attached to opposite poles of the spindle. Checkpoints are able to block cell cycle progression by sending a STOP signal to the molecular mechanisms that govern specific cell-cycle transitions (G1-S, G2-M, and M-G1). The molecular mechanisms that govern each of these transitions have a peculiar property called `bistability.' Under physiological conditions, the control mechanism can persist indefinitely in either of two characteristic states: the OFF state, which corresponds to holding the cell cycle in the pre-transition phase; and the ON state, which corresponds to pushing the cell cycle into the post-transition phase. Checkpoint STOP signals seem to act by stabilizing the appropriate bistable switches in its OFF state. Because these checkpoints are crucial to maintaining the integrity of an organism's genome from one generation of cells to the next, it is vital that they function reliably even in the face of random molecular fluctuations that are inevitable in a cell a small as a yeast cell (30 fL). Calculations based on stochastic models of the molecular mechanisms governing cell cycle progression suggest that checkpoint functions are indeed robust in wild-type budding yeast cells, but they may be compromised in strains carrying mutations of specific checkpoint genes. The purpose of this proposal is to provide the mathematical models and experimental data needed to understand how cell cycle checkpoints operate reliably in wild-type yeast cells and how they fail in mutant cells. To reach this goal wil require new advances in stochastic modeling and in the technology of measuring mRNA and protein molecules in single yeast cells. To test the models will require the expertise to construct and characterize the phenotypes of specific mutant strains of budding yeast that are predicted by the model to exhibit fragility of checkpoint arrest in the face of random fluctuations in yeast mRNAs and proteins. Because all eukaryotic organisms seem to employ the same fundamental molecular machinery that governs progression through the cell division cycle, the understanding of checkpoint operations in yeast cells will translate into a better understanding of checkpoint functions and failures in other types of cells, most notably human cells.