STRUCTURE AND BEHAVIOR OF YEAST TELOMERES

酵母端粒对于染色体的维持是必不可少的，在 至少在一定程度上是因为它们保护了染色体不会退化。 端粒也是必需的，以确保完全复制 这可能有助于将染色体定位在细胞核内。 端粒结构蛋白可能影响这些功能中的每一个。 这项资助的目标是识别相互作用的结构蛋白。 与酵母端粒结合并确定其在体内的功能。第一 目的是开发一种检测端粒蛋白的单杂交系统 互动。该系统将用于识别新的端粒 结构蛋白和评估是否已知的基因产物影响 体内的端粒是通过直接与端粒DNA或 端粒染色质。第二个目标是识别特定于末端的DNA 结合蛋白，即只与端粒结合而不与端粒结合的蛋白质 端粒序列的内部束。基因证据表明 这样的蛋白质会影响端粒的位置效应，影响端粒的这个能力 端粒来抑制附近基因的转录。两个独立的 提出了识别末端结合蛋白的方法。这个 生化方法基于这样一个事实，即在某些情况下，DNA末端 生物体有短的G链尾巴。一种15 kDa的结合蛋白 将在体外纯化单链TG1-3DNA，作为研究的第一步 分离和鉴定编码它的基因。第二种方法 是基于观察到额外的端粒减少了反式， 推测是通过与染色体端粒竞争末端结合 蛋白质(S)。修复携带额外基因的细胞的库质粒 端粒将被分离和鉴定。基因证据表明 有一种因子与端粒的羧基末端结合- 结合的RAPLP的耗竭与端粒延长和 一种冷敏感(Cs)生长表型。第三个目标是确定 通过选择逆转该因子的文库质粒来编码该因子的基因 CS和端粒长度表型并确定该蛋白质如何 限制端粒长度。第四个目标是继续分析 影响TST(端粒稳定性)表达的突变体的收集 端粒附近的基因，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 在核的特定亚室中，如果是这样的话，如果这 本地化对TPE很重要。在酵母中，端粒长度，因此 端粒的功能由许多基因的平衡活动控制， 一些促进端粒DNA的缩短，另一些促进端粒DNA的延长。在……里面 人类的端粒长度随着年龄的增长而减少。有越来越多的 推测与人类有关的遗传不稳定 疾病，如癌症和衰老，可能由丢失的 端粒DNA。端粒区域在结构和功能上都是 从酵母到人类都很相似。了解以下方面的要求 维持酵母端粒的结构和功能可能会 与了解人类遗传不稳定性的来源有关 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.