TELOMERE STRUCTURE AND FUNCTION

端粒结构和功能

• 批准号: 3306318
• 负责人: BIK-KWOON TYE
• 金额: $ 12.18万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1995-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306318
• 关键词: DNA binding protein; DNA footprinting; aging; chromosomes; fungal genetics; molecular cloning; nucleic acid repetitive sequence; protein structure function; telomere; yeasts

Telomeres or ends of chromosomes contain simple repeat sequences that serve as buffer to, preserve integrity of genetic information coded in the chromosome. Several recent reports linked the shortening of telomere lengths to aging (Nature 345:458; Cell 61:193). However, it is not clear whether telomere shortening is the cause or the effect of aging. Activities such as synthesis of repeat sequences and recombination by unequal exchanges can contribute to the lengthening of telomeres. Incomplete replication of the lagging daughter strand and exonucleolytic degradation result in shortening of telomeres. To maintain an optimal balance between these many metabolic processes that take place at telomeres, many proteins must interact directly with telomeric sequences. It has been shown in human and in Tetrahymena that replication of telomeres is carried out by a specialized reverse transcriptase-termed telomerase. Alteration of telomerase activity in Tetrahymena resulted in cells that exhibited abnormal morphology and that became senescent. In yeast, telomerase activity has not yet been identified. Our laboratory has partially purified two novel yeast telomere-binding activities, TBFalpha and TBFbeta. TBFalpha binds to the junction between the subtelomeric X sequence and the polyC(1-3)A telomeric sequence in a cloned yeast telomere. Under certain conditions, TBFalpha also interacts with the end of the chromosome, and specifically, with the GT strand of the terminus. Examination of the junctions of known X sequences indicate that they all contain one or more repeats of CCCTAA, a sequence which is repeated in vertebrate telomeres. Such heterologous telomeric sequences, positioned as far as several hundred base pairs from the termini of linear molecules, allow the addition of yeast telomeric sequences from the nontelomeric termini in vivo. We propose that TBFalpha serves as an anchor for the yeast telomerase by binding to the conserved junction sequence at a distance from the terminus to allow addition of an irregular repeating sequence to the GT-rich strand at the chromosome end. We will test this hypothesis by assaying for telomerase activity associated with TBFalpha. We will also try to clone the genes that encode TBFalpha and TBFbeta by screening an expression library of yeast genes, either with DNA substrates of TBFalpha and TBFbeta, or with antibodies raised against these proteins. Further biochemical characterizations of TBFalpha and TBFbeta coupled with genetic analysis of the genes that encode these proteins should reveal the biological functions of these telomere-binding proteins in yeast.

端粒或染色体末端包含简单的重复序列，用于 作为缓冲，保护编码在 染色体。最近的几篇报道将端粒的缩短与 衰老的长度(自然345：458；细胞61：193)。然而，目前还不清楚 端粒缩短是衰老的原因还是结果。 重复序列的合成和重组等活动 不平等的交换会导致端粒的延长。 滞后子链的不完全复制和核酸外切 降解会导致端粒缩短。要保持最佳状态 在这些代谢过程之间的平衡发生在 端粒，许多蛋白质必须直接与端粒序列相互作用。 已经在人类和四膜虫中证明了端粒的复制 是由一种被称为端粒酶的特殊逆转录酶执行的。 四膜虫端粒酶活性的改变导致细胞 表现出异常的形态，并开始衰老。 在酵母中，端粒酶活性尚未被鉴定。我们的实验室 部分纯化了两种新的酵母端粒结合活性， TbFAlpha和TbFbeta。TbFAlpha绑定到 克隆的亚端粒X序列和PolyC(1-3)A端粒序列 酵母端粒。在某些条件下，TBFAlpha还与 染色体的末端，具体地说，与GT链 终点站。对已知X序列的连接的检查表明 它们都含有CCCTAA的一个或多个重复序列，该序列是 在脊椎动物的端粒中重复。这种异源端粒序列， 距离LINARY基因末端几百个碱基对的位置 分子，允许添加酵母端粒序列 活体内的非端粒末端。我们建议将TBFpha作为一个 酵母端粒酶通过与保守的连接处结合来锚定 在距离末端一定距离处的序列，以允许添加不规则的 重复序列到染色体末端的富含GT的链上。我们会 通过分析与端粒酶相关的端粒酶活性来检验这一假设 TBFAlpha。我们还将尝试克隆编码tbfα和tbf的基因。 通过筛选酵母基因的表达文库，或者用DNA TBFpha和TBFbeta的底物，或与针对这些的抗体一起产生 蛋白质。转铁蛋白α和转铁蛋白β的进一步生化性质 再加上对编码这些蛋白质的基因的遗传分析 应该揭示这些端粒结合蛋白的生物学功能 放在酵母里。