Discovery of genes that extend yeast lifespan: Aging in immobilized cell reactors

描述(由申请人提供)-我们对衰老生物学的许多最深刻的见解是使用简单的真核生物酿酒酵母获得的。利用酵母进行的基础研究发现了沉默信息调节蛋白Sir2在调节复制寿命中所起的作用，以及Ras2、Cyr1和Sch9在调节时间序列寿命中所起的作用。这些基因中的每一个在包括哺乳动物在内的高等真核生物中都有功能相似的同源基因。这些发现使人们对延长人类人口中老年人口的寿命和提高生活质量的前景感到兴奋。到目前为止，对酵母中延长时间寿命的遗传机制的所有研究都集中在静止相批量培养中不生长的浮游细胞的生存能力。在那里，细胞进入一种生理状态，新陈代谢被重新编程，以有效地利用存储的资源来维持细胞。不足为奇的是，筛选在这些条件下改变时间寿命的突变体已经产生了一系列影响抗逆性的基因。但酵母种群在另一个非常不同的环境环境中基本上停止了繁殖。连续或半连续供料的生物反应器由固定化在半固体珠子中的酵母填充，可维持数周甚至数月。这些反应器继续生产接近理论产量的乙醇，但与底物投入相比，生物质产量非常少。在这里，我们提供了人口统计学、生理学和基因组数据，这些数据阐明了固定化状态的特征，我们认为这些特征使该系统非常适合于时间老化的研究。我们建议在这些结果的基础上进行一项机制的研究(S)，该机制使酵母能够在热量不受限制的环境中长时间存活，在这种环境中，代谢流量似乎是以牺牲细胞生长和繁殖为代价的。这项研究的具体目的有三个；我们将：(1)在野生型酵母中确定长期固定化是否改变了与老化浮游培养物相关的时间寿命和全球基因表达；(2)相对于野生型，建立已知的改变浮游细胞复制和时间寿命的突变是否会在老化的固定化细胞中产生类似的变化。(3)对在固定化和浮游态培养的单基因缺失酵母菌株进行大规模的遗传筛选(A)发现以未知途径发挥作用的新类别的长寿基因，以及(B)确定在卡路里限制和非卡路里限制的条件下是否存在共同的时间长寿决定因素。项目说明-我们对衰老生物学的许多最深刻的见解都是使用简单的微生物面包师酵母获得的。单个酵母细胞的寿命有限，它们以两种方式老化：母细胞在经历连续几轮细胞分裂时老化；未分裂的细胞也老化并最终死亡。卡路里限制似乎可以延长所有被研究物种的寿命，包括酵母。但关于限制卡路里摄入是长寿的近因还是终极原因这一问题存在争议。到目前为止，所有关于未分裂细胞的遗传学研究都限制了卡路里，使得这个变量不可能被梳理出来。酵母可以被限制在半固体的海藻酸盐珠中，并暴露在过量的营养中。在那里，它们以极高的速率将糖转化为乙醇，但这样做时几乎没有细胞分裂。我们将利用这个系统作为一种工具来发现在卡路里非限制性条件下是否有可能延长寿命，如果是的话，我们的目标是发现潜在的遗传决定因素。

DESCRIPTION (provided by applicant): - Many of our deepest insights into the biology of aging have been gained using the simple Eukaryote Saccharomyces cerevisiae. Foundational studies using yeast have led to the discovery of the role played by the silent information regulator protein Sir2 in modulating replicative lifespan, and the roles played by Ras2, Cyr1 and Sch9 in modulating chronological lifespan. Each of these genes has a homologue with similar function in higher Eukaryotes, including mammals. These discoveries have generated excitement about the prospects for extending lifespan and improving quality of life for older members of the human population. To date, all investigations into the genetic mechanisms that extend chronological lifespan in yeast have focused on the viability of non-growing planktonic cells in stationary-phase batch culture. There, cells enter a physiological state where metabolism is reprogrammed to efficiently use stored resources for the purpose of cell maintenance. Not surprisingly, screens for mutants which alter chronological lifespan under these conditions have produced a collection of genes whose activities influence resistance to stress. But yeast populations largely cease to reproduce in another, very different environmental context. Continuously or semi-continuously fed bioreactors populated by yeast immobilized in semi-solid beads can be maintained for weeks, or even months. These reactors continue to produce close-to-theoretical yields of ethanol but very little biomass, relative to substrate input. Herein, we present demographic, physiological and genomic data that illuminate features of the immobilized state, features that we contend make this system ideally suited for the study of chronological aging. We propose to follow on from these results with an investigation into the mechanism(s) that enable yeast to live for extended periods of time in a calorically un-restricted environment wherein metabolic flux appears to be maximized at the expense of cell growth and reproduction. The Specific Aims of this investigation are threefold; we will: (1) Determine in wild-type yeast whether long-term immobilization alters chronological lifespan and global gene expression relative to aging planktonic cultures; (2) Establish, relative to wild-type, whether mutations known to alter both replicative and chronological lifespan in planktonic cells produce similar alterations in aging immobilized cells. (3) Conduct a large-scale genetic screen of single-gene-deletion yeast strains cultured in the immobilized and planktonic states (a) to discover new classes of longevity genes that act in undescribed pathways, and (b) to establish whether there are common determinants of chronological longevity under calorically-restricted and non-calorically-restricted conditions. PROJECT NARRATIVE - Many of our deepest insights into the biology of aging have been gained using the simple microbe, Bakers yeast. Individual yeast cells have a finite lifespan, and they age in two ways: mother cells age as they undergo successive rounds of cell division; non-dividing cells also age and eventually die. Caloric restriction appears to extend lifespan in all species studied, including yeast. But controversy surrounds the issue of whether caloric restriction is a proximal or ultimate cause of longevity. To date, all studies on the genetics on non-dividing cells have imposed caloric restriction, making this variable impossible to tease out. Yeast can be confined within semi-solid alginate beads and exposed to excess nutrients. There, they convert sugar to ethanol at extremely high rates, but do so with little or no cell division. We will exploit this system as a tool to discover whether lifespan extension is possible under calorically non- restrictive conditions, and if so, we aim to discover underlying genetic determinants.