An Analysis of Spore Germination in Saccharomyces cerevisiae.

当条件不适合继续生长时，真核细胞停止分裂并进入一种特殊的静止状态，称为g0、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一个关键点。本项目将研究一种模式转变，即芽殖酵母的孢子萌发，酿酒酵母。这种出芽酵母为开展这些研究提供了有用的背景，因为这种有机体可以在实验室中相对容易地进行操作。此外，先前的研究表明，调节细胞生长和分裂的机制在所有真核生物中都是非常保守的。因此，从这些酵母研究中获得的见解应该是普遍适用的。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。遗传学、生物化学和细胞生物学方法的结合将用于实现这些目标。本项目的具体目的如下：1。描述孢子萌发的早期步骤所需要的成分。实验将阐明关键信号转导通路在孢子萌发早期阶段的作用。Ras蛋白是细胞分裂的重要调节因子，也是种子萌发所必需的。这一目的将测试人工激活该信号通路是否足以触发进入萌发程序。此外，实验将测试涉及Tor蛋白激酶的第二种途径对孢子萌发是否必要。Tor蛋白也是细胞生长所必需的，也是萌发过程的良好候选调节因子。最后，一个假设表明，发芽的早期步骤是控制在蛋白质合成水平将进行实验测试。2. 描述酵母突变体孢子萌发缺陷。要全面了解发芽过程，就需要了解参与这一途径的基因。本研究将采用两种新方法分离和鉴定酵母孢子萌发缺陷突变体。描述孢子脱壳所需的一种新酶。酵母孢子具有一层特殊的外壳，在发芽后的第一个子芽形成之前，必须将其部分去除。该项目的这一部分将涉及对这种脱壳过程至关重要的酶的纯化和表征，该过程在萌发程序的早期就开始了。了解这种酶的功能以及它是如何被调节的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.