Analysis of Quiescent and Non-quiescent Cells in Stationary-phase Yeast Cultures

这项研究的长期目标是了解酵母细胞如何进入、存活和退出静止状态，并最终利用这些信息来了解其他生物的这一过程。静止或G0是地球上细胞最常见的状态，也是活细胞进化的最初状态之一。矛盾的是，这个基本重要的过程还没有得到很好的研究，但是基因组学的出现和辅助技术的发展最近使人们在理解这一状态方面取得了重大进展。在目前的项目中，这个研究小组研究了在静止期培养的细胞重新进入有丝分裂细胞周期时基因表达的变化。他们确定了30多个新基因，这些基因是细胞在静止期培养中长期存活所必需的。他们最近开发了一种方法，可以在静止期培养中分离静止和非静止细胞，并开发了一种快速采样器，使他们能够在短至10秒的间隔内可重复地收获培养中的细胞以进行基因组分析。该小组现在将从静止期酵母培养物中表征静止和非静止酵母细胞。绿色荧光蛋白结构和电子显微镜将用于检测细胞类型之间的形态学差异。将对两种细胞类型进行各种生化和基因组分析，作为培养时间的函数。非静止细胞将用几种方法评估，以确定表现出凋亡或衰老特征的细胞的百分比。如有必要，改良的分离方法将用于提高非静止细胞部分群体的纯度。这项工作的智力价值来自于能够首次研究静止和非静止细胞的纯群体，以及通过鉴定这些细胞类型分化的关键基因和途径所提供的新颖见解。这项工作具有广泛的相关性，因为基于先前对酵母的研究，这些分化过程可能在其他微生物和真核生物中保守。这项工作的更广泛的影响是来自代表性不足的群体的学生和教师的参与，生物学家，统计学家和工程师之间广泛的合作，对新开发技术的免费分发的承诺，以及共享基因组试剂。

The long-term goal of this research is to understand how yeast cells enter, survive in, and exit the quiescent state and, eventually, to use this information to understand this process in other organisms. Quiescence or G0 is the most common state of cells on earth and was surely one of the first states in the evolution of living cells. Paradoxically, this fundamentally important process has not been well studied, but the advent of genomics and the development of ancillary technologies have recently enabled significant progress in understanding this state. Leading up to the current project, this research group has studied the changes in gene expression as cells in stationary-phase cultures re-enter the mitotic cell cycle. They identified over 30 new genes required for long-term survival of cells in stationary-phase cultures. They recently developed a method that allows separation of quiescent and non-quiescent cells in stationary-phase cultures and developed a rapid sampler that allows them to reproducibly harvest cells in culture for genomic analysis at intervals as short as 10s. The group will now characterize quiescent and non-quiescent yeast cells from stationary-phase yeast cultures. GFP constructs and electron microscopy will be used to detect morphological differences between cell types. Various biochemical and genomic analyses will be carried out on both cell types as a function of time in culture. The non-quiescent cells will be evaluated using several assays to determine the percentage of cells that exhibit apoptotic or senescent characteristics. As necessary, modified separation methods will be used to increase the purity of populations from the non-quiescent-cell fraction. The intellectual merit of the work comes from the novel insight provided by being able to study, for the first time, pure populations of quiescent and non-quiescent cells and through the identification of genes and pathways critical for the differentiation of these cell types. This work has broad relevance, since, based on previous work with yeast, these differentiation processes are likely to be conserved in other microbes and eukaryotic organisms. The broader impacts of this work are in the involvement of students and faculty from underrepresented groups, the breadth of collaborations between biologists, statisticians and engineers, the commitment to the free distribution of newly developed technology, and sharing genomic reagents.