STRUCTURE AND BEHAVIOR OF YEAST TELOMERES

酵母端粒对于染色体的维持至关重要， 至少部分是因为它们保护染色体不被降解。 端粒也是确保基因组完整复制所必需的。 染色体，并可能帮助定位染色体在细胞核内。 端粒结构蛋白可能影响这些功能中的每一个。 这项资助的目标是鉴定出与蛋白质相互作用的 与酵母端粒，并确定其在体内的功能。第一 目的是建立一个检测端粒蛋白的单杂交系统 交互.该系统将用于识别新的端粒 结构蛋白，并评估是否已知的基因产物，影响 端粒在体内通过直接与端粒DNA相互作用或 端粒染色质 第二个目标是鉴定末端特异性DNA 结合蛋白，即只与端粒结合的蛋白质，而不与端粒结合的蛋白质。 端粒序列的内部束。遗传学证据表明， 这些蛋白质影响端粒位置效应，ThE， 端粒抑制附近基因的转录。两个独立 提出了鉴定末端结合蛋白的方法。的 生物化学方法是基于这样的事实，即DNA末端在一些 生物体有短的G链尾巴。一种15 kDa的蛋白质， 体外单链TG 1 -3 DNA将被纯化，作为体外扩增的第一步。 分离并鉴定编码它的基因。第二种方法 基于额外的端粒减少反式ThE的观察， 推测是通过与染色体端粒竞争末端结合 蛋白质。将ThE恢复到携带额外ThE的细胞的文库质粒 端粒将被分离和表征。遗传证据表明 有一种因子与端粒的羧基末端结合 结合Raplp，其耗尽与端粒延长相关， 冷敏感（CS）生长表型。第三个目标是确定 通过选择文库质粒来逆转该因子的编码基因， cs和端粒长度表型，并确定这种蛋白质 限制了端粒长度。第四个目标是继续分析 TST（端粒稳定性）突变体的集合，其影响 端粒附近的基因，重点关注那些影响端粒的突变体 长度和。第五个目标是确定端粒是否定位 在细胞核的一个特定的亚区室，如果是这样， 国产化对TPE很重要。在酵母中，端粒长度， 端粒功能由许多基因的平衡活性控制， 一些促进端粒DNA缩短，另一些促进端粒DNA延长。在 端粒长度随着年龄的增长而减少。人们日益 推测与人类遗传不稳定性相关的 疾病，如癌症和衰老，可能是由失去 端粒DNA端粒区在结构和功能上 类似于酵母和人类。了解以下要求： 维持酵母端粒的结构和功能， 有助于了解遗传不稳定性的来源， 人类

Saccharomyces telomeres are essential for chromosome maintenance, at least in part because they protect chromosomes from degradation. Telomeres are also required to ensure the complete replication of the chromosome and possibly to help position chromosomes within the nucleus. Telomere structural proteins probably influence each of these functions. The goal of this grant is to identify structural proteins that interact with yeast telomeres and to determine their in vivo functions. The first aim is to develop a one-hybrid system for detecting telomere-protein interactions. This system will be used both to identify new telomere structural proteins and to assess whether known gene products that affect telomeres in vivo do so by interacting directly with telomeric DNA or telomeric chromatin. The second aim is to identify terminus-specific DNA binding proteins, that is proteins that bind only to telomeres, not to internal tracts of telomeric sequence. Genetic evidence suggests that such proteins influence telomere position effect, ThE, the ability of telomeres to repress transcription of nearby genes. Two independent approaches are proposed to identify terminus binding proteins. The biochemical approach is based on the fact that DNA termini in some organisms have short G-strand tails. A 15 kDa protein that binds to single-strand TG1-3 DNA in vitro will be purified, as the first step in isolating and characterizing the gene encoding it. The second approach is based on the observation that extra telomeres reduce ThE in trans, presumably by competing with chromosomal telomeres for terminus binding protein(s). Library plasmids that restore ThE to cells carrying extra telomeres will be isolated and characterized. Genetic evidence suggests that there is a factor that binds to the carboxyl terminus of telomere- bound Raplp whose depletion is associated with telomere lengthening and a cold sensitive (cs) growth phenotype. The third aim is to identify the gene encoding this factor by selecting library plasmids that reverse the cs and telomere length phenotypes and to determine how this protein limits telomere length. The fourth aim is to continue analysis of a collection of tst (telomere stability) mutants that affect expression of genes near telomeres, focusing on those mutants that affect telomere length and ThE. The fifth aim is to determine if telomeres are localized in a specific sub-compartment of the nucleus and, if so, if this localization is important for TPE. In yeast, telomere length and hence telomere function is controlled by the balanced activities of many genes, some that promote shortening and others lengthening of telomeric DNA. In humans, telomere length decreases with age. There is increasing speculation that the kinds of genetic instability associated with human disease, such as cancer and aging, could be triggered by the loss of telomeric DNA. Telomeric regions are structurally and functionally similar from yeast to humans. An understanding of the requirements for maintaining the structure and function of yeast telomeres is likely to be relevant to an understanding of the sources of genetic instability in humans.