STRUCTURE AND BEHAVIOR OF YEAST TELOMERES

端粒是真核细胞染色体的物理末端，是 线性染色体的稳定维持和完全复制。这个 端粒的特殊结构和归属于端粒的各种功能 它们表明，多个基因产物参与了它们的复制 和功能。 描述了遗传和生化方法以识别和 鉴定与酵母菌端粒相互作用的蛋白质 酿酒。编码端粒的两个候选基因Yalpha26和RAP1 结合交叉与一种已知结合的蛋白质的抗血清发生反应 特别是纤毛虫Oxytricha nova的端粒。 此外，Yalpha26的过量拷贝会影响酵母中的端粒长度。脱氧核糖核酸 测序将揭示Yalpha26是否真的与Oxytricha基因有关。 活性、端粒缺失和过表达等位基因的表型 长度和染色体稳定性也将被确定。RAP1，AN 必需基因，在体外对端粒序列也有很高的亲和力 至于各种基因的转录控制区。至 确定RAP1蛋白是否与体内的端粒相互作用， 表型(端粒长度、染色体稳定性、转录水平) 过表达或缺乏RAP1的菌株将被确定。 生化方法也将用于鉴定相互作用的蛋白质。 用端粒。染色体端粒的染色质结构将是 在正常细胞和在所示条件下生长的细胞中测定 先前影响端粒结构。亲和纯化方法 的端粒染色质将被开发出来，使我们能够 如果在端粒上发现Yalpha26(或RAP1)，并最终 鉴定其他端粒相关蛋白。最后，六个LP(线性 失去线性人工染色体的突变体 频率提高但以野生型比率丢失循环染色体将是 通过分子生物学和遗传学方法分析以确定是否和 他们的产品如何与酵母端粒相互作用。

Telomeres, the physical ends of eukaryotic chromosomes, are required for the stable maintenance and complete duplication of linear chromosomes. The special structure of telomeres and the variety of functions ascribed to them suggest that multiple gene products are involved in their replication and function. Genetic and biochemical approaches are described to identify and characterize proteins that interact with the telomeres of Saccharomyces cerevisiae. Yalpha26 and RAP1, two candidates for genes encoding telomere binding cross reacts with antiserum raised to a protein known to bind specifically to telomeres in the ciliated protozoan Oxytricha nova. Moreover, excess copies of Yalpha 26 affect telomere length in yeast. DNA sequencing will reveal if Yalpha26 is indeed related to the Oxytricha gene. The phenotype of null and overexpressing alleles on viability, telomere lengths, and chromosome stability will also be determined. RAP1, an essential gene, has high affinity in vitro for telomeric sequences as well as for the transcription control regions of a variety of genes. To determine if the RAP1 protein interacts with telomeres in vivo, the phenotype (telomere lengths, chromosome stability, transcription levels) of strains overexpressing or deficient in RAP1 will be determined. Biochemical methods will also be used to identify proteins that interact with telomeres. The chromatin structure of a chromosomal telomere will be determined in normal cells and in cell grown under conditions shown previously to affect telomere structure. Methods for affinity purification of telomeric chromatin will be developed that will enable us to ask directly if Yalpha26 (or RAP1) is found on telomeres, and, ultimately, to identify other telomere associated proteins. Finally, six lps (linear plasmid stability) mutants that lose linear artificial chromosomes at elevated frequency but lose circular chromosomes at wild-type rates will be analyzed by molecular biological and genetic methods to determine if and how their products interact with yeast telomeres.