GENETICS OF NEGATIVE CELL CYCLE REGULATORS IN C ELEGANS

我们的长期目标是了解细胞的发育控制 循环递增。我们正在采取遗传方法，研究两个 线虫Cul-1和Lin-23中的突变体 多个组织。在这些突变体中，分裂的细胞忽略了发育 而且还在继续扩散。生成的额外单元格更小 比正常情况要好。我们已经克隆了CuL-1和Lin-23基因。CUL-1和 LIN-23与酿酒酵母基因CDC53和CDC4同源， 分别为G1期到S期细胞周期所需的基因 过渡。CDC53和CDC4是泛素介导的 细胞周期调节因子的降解，包括细胞周期蛋白和细胞周期蛋白- 依赖的激酶抑制剂。酵母中不能降解G1期细胞周期蛋白 产生较短的G1期、小细胞和连续细胞 在抑制外部信号存在的情况下增殖。这些 表型与在Cul-1和Lin-23突变体中观察到的相似。 一个引人注目的模型是，CUL-1和LIN-23是 关键的细胞周期调节因子在G1期的降解，特别是G1期 旋风队。这项拟议研究的目标是检验这一假设。 并确定CUL-1和LIN-23如何发挥负性调节细胞的作用 扩散。在三个目标的第一个目标中，我们将确定 CuL-1和LIN-23的表达模式。我们将检验我们的假设 CUL-1和LIN-23影响不同细胞周期的蛋白质水平 通过测定它们在野生型中的表达和耐受性来调节 和Cul-1或Lin-23突变株。此外，我们将确定是否 CuL-1和Lin-23突变株的G1期较短。在第二个 目的，我们将通过使用酵母来鉴定相互作用的蛋白质。 双混合屏幕。假定的交互克隆将通过以下方式进行分析 生化、细胞和遗传学方法来确定它们在体内的作用 关联性。在第三个目标中，我们将分离和分子表征 CuL-1和LIN-23的外源抑制因子。

Our long term goal is to understand the developmental controls on cell cycle progression. We are taking a genetic approach by studying two mutants in C. elegans, cul-1 and lin-23, that have hyperplasia in multiple tissues. In these mutants, dividing cells ignore developmental cues and continue to proliferate. The extra cells generated are smaller than normal. We have cloned both the cul-1 and lin-23 genes. cul-1 and lin-23 are homologous to the S. cerevisiae genes CDC53 and CDC4, respectively, which are required for the G1 to S phase cell cycle transition. CDC53 and CDC4 are required for the ubiquitin-mediated degradation of cell cycle regulators, including cyclins and cyclin- dependent kinase inhibitors. An inability to degrade G1 cyclins in yeast produces a shorter G1 phase, small cells, and continued cell proliferation in the presence of inhibitory external signals. These phenotypes are similar to what is observed in cul-1 and lin-23 mutants. A compelling model is that cul-1 and lin-23 are required for the degradation of key cell cycle regulators in G1 phase, in particular G1 cyclins. The goals of the proposed research are to test this hypothesis and determine how cul-1 and lin-23 function to negatively regulate cell proliferation. In the first of three aims, we will determine the expression pattern of cul-1 and lin-23. We will test our hypothesis that cul-1 and lin-23 affect the protein levels of various cell cycle regulators by determining their expression and perdurance in wild type and cul-1 or lin-23 mutant strains. Further, we will determine whether cul-1 and lin-23 mutant strains have a shorter G1 phase. In the second aim, we will identify interacting proteins through the use of a yeast two-hybrid screen. Putative interacting clones will be analyzed by biochemical, cellular, and genetic approaches to determine their in vivo relevance. In the third aim, we will isolate and molecularly characterize extragenic suppressors of cul-1 and lin-23.