An Analysis of Spore Germination in Saccharomyces cerevisiae.

当条件不是持续生长的最佳条件时，真核细胞停止分裂并进入一种特殊的静止状态，称为G零，静止期或孢子状态。 处于静止状态的细胞通常对环境压力具有抵抗力，并且可以在很长一段时间内保持活力，尽管不分裂。 静止期和有丝分裂周期之间的过渡已被证明是增殖控制的关键点。本计画将探讨一种模式转换，即芽殖酵母（Saccharomycescerevisiae）中孢子萌发的模式转换。 这种芽殖酵母提供了一个有用的背景，在其中进行这些研究，因为这种生物体可以在实验室中操作相对容易。 此外，先前的工作表明，调节细胞生长和分裂的机制在所有真核生物中是非常保守的。 因此，从这些酵母研究中获得的见解应该普遍适用。 这些实验将进一步加深我们对启动发芽的调控机制和进行这一多步骤过程所需的分子的理解。 遗传、生物化学和细胞生物学方法的组合将用于实现这些目标。 本项目的具体目标如下：1. 表征孢子萌发早期步骤所需的组分。 实验将阐明孢子萌发早期关键信号转导途径的作用。 Ras蛋白是细胞分裂的重要调节剂，并且已知是发芽所需的。 这一目标将测试这种信号传导途径的人工激活是否足以触发进入萌发程序。 此外，这些实验将测试涉及Tor蛋白激酶的第二种途径是否是孢子萌发所必需的。 Tor蛋白也是细胞生长所必需的，并且是萌发过程调节剂的良好候选物。 最后，一个假设表明，发芽的早期步骤控制在蛋白质合成的水平将进行实验测试。 2. 鉴定孢子萌发缺陷的酵母突变体。 要全面了解发芽需要了解参与这一途径的基因。 本研究将采用两种新的策略来分离和鉴定孢子萌发缺陷的酵母突变体. 鉴定一种新的芽孢脱壳酶。酵母孢子具有一种特殊的外壳，在萌发后形成第一个子芽之前必须部分去除。 该项目的这一部分将涉及酶的纯化和表征，这是必不可少的脱壳过程，这是在发芽计划的早期开始。 了解这种酶的功能及其调节方式将为孢子萌发的控制提供重要的见解。

When conditions are not optimal for continued growth, eukaryotic cells stop dividing and enter into a specialized resting state known variously as G zero, the stationary phase, or the spore state. Cells in a quiescent state are often resistant to environmental stress and may remain viable, although not dividing, for very long periods of time. The transition between the period of quiescence and the mitotic cycle has been shown to be a key point of proliferative control. This project will examine a model transition, that of spore germination in the budding yeast, Saccharomyces cerevisiae. This budding yeast provides a useful background in which to carry out these studies because of the relative ease with which this organism can be manipulated in the laboratory. In addition, previous work has shown that the mechanisms regulating cell growth and division are remarkably conserved in all eukaryotes. Therefore, insights gained from these studies with yeast should be generally applicable. The experiments will further our understanding of both the regulatory mechanisms responsible for initiating germination and the molecules required for carrying out this multi-step process. A combination of genetic, biochemical and cell biological approaches will be used to achieve these goals. The specific aims of this project are as follows:1. To characterize components required for the early steps of spore germination. Experiments will clarify the role of key signal transduction pathways in the early steps of spore germination. The Ras proteins are important regulators of cell division and are known to be required for germination. This aim will test whether an artificial activation of this signaling pathway is sufficient to trigger entry into the germination program. In addition, the experiments will test whether a second pathway, involving the Tor protein kinases, is necessary for spore germination. The Tor proteins are also essential for cell growth and good candidates for regulators of the germination process. Finally, a hypothesis suggesting that early steps in germination are controlled at the level of protein synthesis will be tested experimentally. 2. To characterize yeast mutants defective in spore germination. A full understanding of germination requires knowledge of the genes involved in this pathway. This aim will use two novel strategies to isolate and characterize yeast mutants defective in spore germination.3. To characterize a novel enzyme required for spore uncoating. Yeast spores possess a specialized coat that must be partially removed prior to the formation of the first daughter bud following germination. This part of the project will involve the purification and characterization of an enzyme that is essential for this uncoating process, which is initiated very early in the germination program. Understanding the function of this enzyme and how it is regulated will provide important insights into the control of spore germination.