Expression and Regulation of Telomerase in Arabidopsis thaliana

9982499本研究采用遗传学和分子生物学方法，阐明了端粒酶在拟南芥基因组维持和植物发育中的作用，并确定了控制端粒酶表达的机制。 来自Shippen和McKnight实验室的初步数据表明，植物中的端粒酶表达受到发育控制，并与生殖和增殖有关。编码端粒酶催化亚单位AtTERT的基因已被克隆，其表达与酶活性密切相关。 在该基因中间插入T-DNA的植物没有检测到端粒酶活性。 端粒酶无效的植物可以存活至少两代，但它们的端粒正在缩短，这意味着它们不能无限期地存活。 第一个目的是明确端粒酶在植物生长发育中的作用。 AtTERT基因将被用作端粒酶活性的标志物，以测量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。末端限制性片段分析和细胞遗传学研究将在连续几代中用端粒酶无效植物进行，以将染色体“脱帽”与表型变化相关联。 最后，将进行实验，以揭示端粒酶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是无活性的，但在从营养期到生殖期的过渡期间被重新激活。 第二个目标是确定基因控制端粒酶活性的发育调控在转基因激活标记的拟南芥系。 这些线携带随机插入的T-DNA元件连接到强大的组成型增强子，可以覆盖内源性转录控制和激活正常静止的基因附近的插入位点。 在筛选的前800个激活系中，已经鉴定了两个在叶片中不适当地表达端粒酶的突变体植物（tac 1和tac 2）。 将克隆tac 1和tac 2基因，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基因产物是否可能特异性激活端粒酶或在调节基因表达中发挥更广泛的作用。 由于拟南芥是一种多细胞真核生物，在端粒酶表达中显示发育程序性变化，因此对端粒酶激活和抑制机制的遗传研究不仅将提供有关端粒酶在植物中作用的第一个信息，但也有可能产生深入了解端粒酶调控机制的运作在广泛的高等真核生物。端粒是复杂的核蛋白结构，末端的线性真核细胞染色体和保护他们从端到端融合和降解。 产生和维持端粒DNA的主要机制是通过端粒酶的作用，端粒酶是一种不寻常的具有逆转录酶活性的核糖核蛋白。 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。 尽管近60年前Barbara McClintock首次阐明了端粒在植物中的基本功能，但对端粒和端粒酶在植物生长和发育中的作用知之甚少。 本研究采用遗传学和分子生物学的方法来阐明端粒酶在拟南芥基因组维持和植物发育中的作用，并阐明控制端粒酶表达的机制。 与拟南芥合作的优势有很多，包括世代时间短、易于转化以及基因组小，其整个序列应在明年完成。 允许正向和反向遗传方法的转基因工具包括T-DNA破坏系和激活标记系的集合，这两者将在整个工作中使用。

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.