VACUOLE DIVISION/SEGREGATION DURING THE YEAST CELL CYCLE

细胞质细胞器的分裂和分离必须是空间上的。 并调节细胞周期，以确保通过多个细胞忠实遗传 组织。这一过程对S。 酿酒酵母在过去的5年中已经建立了其细胞学。 分裂和遗传模式2.导致了vac突变体 特别是在液泡遗传上有缺陷，以及3.提供了一种 体外反应，液泡衍生的小管泡的形成 突起，它真实地反映了体内的遗传过程。 这种体外反应需要半完整的细胞或孤立的空泡， 胞浆、ATP、GTP和23至37摄氏度的孵化，并取决于 微管蛋白和VAC1和VAC2蛋白。计划中的研究将结合 这是细胞学、遗传学和生物化学。新的和旧的吸尘器变种人将 被收集，从基因上结合起来探索相互关系，VAC 克隆和测序基因，并为免疫定位准备抗体 并协助进行生化分析。体外反应的组成成分 将利用VAc提取物的补充进行分级和提纯， 核苷酸亲和矩阵和酵母的总分级 细胞液，分析支持小管卵泡所需的组分 队形。这种反应与蛋白质的关系，如 控制细胞周期的细胞周期蛋白和激酶，以及 驱动基于微管的细胞器运动的动蛋白和动力蛋白 探索过了。这些研究将提供关于一个重要的、 然而，在细胞分裂方面的研究却很少。

The division and segregation of cytoplasmic organelles must be spatially and cell-cycle regulated to insure faithful inheritance through many cell divisions. Study of this process for the vacuole (lysosome) of S. cerevisiae during the last 5 years has 1. established the cytology of its division and inheritance pattern 2. led to vac mutants which are specifically defective in vacuole inheritance, and 3. provided an in vitro reaction, the formation of vacuole-derived tubulo-vesicular protuberances, which faithfully reflects the in vivo inheritance process. This in vitro reaction requires semi-intact cells or isolated vacuoles, cytosol, ATP, GTP, and incubation at 23 to 37 degrees C and depends on tubulin and the VAC1 and VAC2 proteins. Planned studies will combine this cytology, genetics and biochemistry. New, and old, vac mutants will be collected, combined genetically to explore interrelationships, VAC genes cloned and sequenced, and antibody prepared for immunolocalization and to aid in biochemical assay. The components of the in vitro reaction will be fractionated and purified, using complementation of vac extracts, nucleotide affinity matrices, and total fractionation of the yeast cytosol, assaying for the fractions required to support tubulovesicle formation. The relationships of this reaction to proteins such as cyclins and kinases which govern the cell cycle and to MAPS such as kinesin and dynein which drive microtubule-based organelle motion will be explored. These studies will provide basic information on an important, yet little studied, aspect of cell division.