Expression and Regulation of Telomerase in Arabidopsis thaliana

拟南芥端粒酶的表达与调控

• 批准号: 9982499
• 负责人: Dorothy Shippen
• 金额: $ 28.74万
• 依托单位: Texas A&M Research Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9982499&HistoricalAwards=false
• 关键词: Expression; Regulation; Telomerase; Arabidopsis; thaliana

9982499In this research, genetic and molecular approaches are used to elucidate the role of telomerase in genome maintenance and plant development, and to define the mechanism(s) that control telomerase expression in Arabidopsis thaliana. Preliminary data from the Shippen and McKnight labs indicated that telomerase expression in plants is developmentally controlled and tied to reproduction and proliferation. The gene encoding the catalytic subunit of telomerase, AtTERT, has been cloned and its expression correlates well with enzyme activity. A plant harboring a T-DNA insertion in the middle of this gene has no detectable telomerase activity. Telomerase null plants are viable for at least two generations, but their telomeres are shortening, implying they cannot survive indefinitely. The first objective is to define the role of telomerase in plant growth and development. The AtTERT gene will be used as a marker for telomerase activity to gauge the temporal and spatial patterns of enzyme expression during growth and development. The phenotype of telomerase null plants will be examined. Terminal restriction fragment analysis and cytogenetic studies will be performed with telomerase null plants in successive generations to correlate chromosome "uncapping" with phenotypic changes. Finally, experiments will be conducted to uncover alternative mechanisms for telomere maintenance in telomerase null mutants. Telomerase is inactive in most vegetative organs, but becomes reactivated during the transition from the vegetative phase to the reproductive phase. The second objective is to identify genes that control the developmental regulation of telomerase activity in transgenic activation-tagged lines of Arabidopsis. These lines carry random insertions of a T-DNA element linked to strong constitutive enhancers that can override endogenous transcriptional controls and activate normally quiescent genes near the site of insertion. Two mutant plants expressing telomerase inappropriately in leaves (tac1 and tac2) have been identified among the first 800 activation lines screened. The tac1 and tac2 genes will be cloned and the temporal and spatial expression profiles of these genes in normal plants will be compared with AtTERT gene expression to ask whether the TAC gene products might specifically activate telomerase or play a broader role in regulating gene expression. Because Arabidopsis is a multicellular eukaryote that displays developmentally programmed changes in telomerase expression, genetic investigations of the mechanisms involved in telomerase activation and repression will not only provide the first information about the role of telomerase in plants, but are also likely to yield insight into telomerase regulatory mechanisms operating in a broad range of higher eukaryotes.Telomeres are complex nucleoprotein structures that cap the ends of linear eukaryotic chromosomes and protect them from end to end fusions and degradation. The primary mechanism for generating and sustaining telomeric DNA is through the action of telomerase, an unusual ribonucleoprotein with reverse transcriptase activity. In mammals, telomerase is part of a biological clock that determines the capacity for cellular proliferation, as its expression is strongly linked to aging and tumorigenesis. Despite the fact that the essential functions of telomeres were first elucidated in plants by Barbara McClintock almost 60 years ago, relatively little is known about the role of telomeres and telomerase in plant growth and development. In this research, genetic and molecular approaches are used to elucidate the role of telomerase in genome maintenance and plant development, and to define the mechanism(s) that control telomerase expression in Arabidopsis thaliana. The advantages of working with Arabidopsis are numerous and include a short generation time, ease of transformation and a small genome whose entire sequence should be completed next year. Transgenic tools that permit both forward and reverse genetic approaches include a collection of T-DNA disruption lines and activation-tagged lines, both of which will be used throughout this work.

9982499在这项研究中，遗传和分子方法用于阐明端粒酶在基因组维持和植物发育中的作用，并定义控制拟南芥中端粒酶表达的机制。 来自Shippen和McKnight Labs的初步数据表明，植物中的端粒酶表达在发育中受到控制，并与生殖和增殖有关。编码端粒酶催化亚基Attert的基因已被克隆，其表达与酶活性很好地相关。 该基因中间携带T-DNA插入的植物没有可检测到的端粒酶活性。 端粒酶无效的植物至少可行两代，但是它们的端粒正在缩短，这意味着它们无法无限期地生存。 第一个目标是定义端粒酶在植物生长和发育中的作用。 Attert基因将用作端粒酶活性的标记，以评估生长和发育过程中酶表达的时间和空间模式。 将检查端粒酶无效植物的表型。末端限制片段分析和细胞遗传学研究将与端粒酶无效的世代进行，以将染色体“解蛋白”与表型变化相关联。 最后，将进行实验，以发现端粒无效突变体中端粒维持的替代机制。 端粒酶在大多数营养器官中都是不活跃的，但是在从植物阶段到生殖阶段的过渡过程中会重新激活。 第二个目标是确定控制拟南芥的转基因活化标记线中端粒酶活性发育调节的基因。 这些线随机插入与强构型增强子相关的T-DNA元件，这些元素可以覆盖内源性转录控制并激活插入部位附近的正常静态基因。 在筛选的前800个激活线中，已经确定了在叶片中不当表达端粒酶的两种突变植物（TAC1和TAC2）。 TAC1和TAC2基因将被克隆，并将这些基因在正常植物中的时间和空间表达谱与Attert基因表达进行比较，以询问TAC基因产物是否可以专门激活端粒酶或在调节基因表达中起更广泛的作用。 由于拟南芥是一种显示端粒酶表达中发育编程的变化的多细胞真核生物，因此对端粒酶激活和抑制作用机制的遗传研究不仅会提供有关端粒酶在植物中的作用的首次信息，而且还可以使远距离机理在较高的euk eukery sunders中产生洞察力的范围。线性真核染色体的膜，并保护它们免受末端融合和降解的影响。 产生和维持端粒DNA的主要机制是通过端粒酶的作用，端粒酶是一种具有逆转录酶活性的不寻常的核糖核蛋白。 在哺乳动物中，端粒酶是决定细胞增殖能力的生物钟的一部分，因为它的表达与衰老和肿瘤发生密切相关。 尽管端粒的基本功能首先是在芭芭拉·麦克林托克（Barbara McClintock）近60年前在植物中阐明的，但对端粒和端粒酶在植物生长和发育中的作用相对较少。 在这项研究中，遗传和分子方法用于阐明端粒酶在基因组维持和植物发育中的作用，并定义控制拟南芥中端粒酶表达的机制。 与拟南芥合作的优点很多，包括短期的时间，易于转化的易用性和一个小基因组，其整个序列应在明年完成。 允许前进和反向遗传方法的转基因工具包括T-DNA破坏线和激活标记的线的集合，这两种线将在整个工作中使用。