TELOMERE STRUCTURE AND FUNCTION

端粒结构和功能

• 批准号: 3306317
• 负责人: BIK-KWOON TYE
• 金额: $ 13.51万
• 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/3306317
• 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.

染色体的端粒或末端包含使用的简单重复序列 作为缓冲，保留编码遗传信息的完整性 染色体。 最近的几份报告与端粒的缩短有关 衰老的长度（自然345：458;单元61：193）。 但是，尚不清楚 端粒缩短是衰老的原因还是影响。 诸如综合重复序列和重组等活动 不平等的交流可能有助于端粒的延长。 滞后的女儿链和核解核酸的复制不完整 降解导致端粒缩短。保持最佳 在这些发生的许多代谢过程之间的平衡 端粒，许多蛋白质必须直接与端粒序列相互作用。 它已在人类和四膜虫中显示出端粒的复制 由专门的逆转录酶Termer酶进行。 四膜膜中端粒酶活性的改变导致细胞 表现出异常的形态，并变得衰老。 在酵母中，尚未确定端粒酶活性。 我们的实验室 部分纯化了两种新型的酵母端粒结合活动， tbfalpha和tbfbeta。 tbfalpha与 亚电体X序列和polyC（1-3）克隆的端粒序列 酵母端粒。 在某些条件下，tbfalpha也与 染色体的结尾，特别是与GT链 终点。 对已知X序列的连接处的检查表明 它们都包含一个或多个重复ccctaa，一个序列是 在脊椎动物端粒中重复。 这种异源端粒序列， 位于线性末端的几百个碱基对 分子，允许从 体内的非晶状体末端。 我们建议tbfalpha用作 通过与保守的连接结合来为酵母端粒酶的锚定 距终点的距离的序列，以允许添加不规则 在染色体末端重复对富含GT的链的序列。 我们将 通过测定与 tbfalpha。 我们还将尝试克隆编码tbfalpha的基因和 通过筛选酵母基因的表达库，用DNA筛选TBFBETA tbfalpha和tbfbeta的底物，或针对这些抗体提出的抗体 蛋白质。 TBFALPHA和TBFBETA的进一步生化特征 再加上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 在酵母中。