Expression and Regulation of Telomerase in Arabidopsis thaliana

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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.