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 和细胞周期调节，以确保通过许多细胞的忠实遗传 分裂 对S. 在过去的5年里，酿酒厂有1。建立了其细胞学 分化与遗传模式2.导致了真空诱变， 特别是液泡遗传缺陷，和3.提供了一个在 体外反应，形成空泡衍生的管泡 突起，这忠实地反映了体内遗传过程。 这种体外反应需要半完整的细胞或分离的空泡， 细胞质，ATP，GTP，并在23至37摄氏度孵育，并取决于 微管蛋白和VAC1和VAC2蛋白。 计划中的研究将联合收割机 细胞学，遗传学和生物化学 新的，旧的，VAC变种人将 被收集，结合基因来探索相互关系，VAC 基因克隆和测序，并制备用于免疫定位的抗体 并有助于生物化学测定。 体外反应组分 将被分级分离和纯化，使用VAC提取物的互补， 核苷酸亲和基质和酵母的总分级分离 细胞质，测定支持小管囊泡所需的组分 阵 这种反应与蛋白质的关系， 控制细胞周期的细胞周期蛋白和激酶以及MAPS， 驱动基于微管细胞器运动的驱动蛋白和动力蛋白将被 探讨了 这些研究将提供关于一个重要的， 但很少有人研究细胞分裂。

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.