The structure of the telomere is fundamental to chromosomal integrity and, by extension, to the state of the cell. Chromosome ends are maintained by the ribonucleoprotein telomerase in a regulated manner. When telomerase is absent and cells divide, telomeres erode; when telomeres reach a critical length, the cells will either enter a non-dividing state or will undergo programmed cell death. Telomere length regulation in the presence of telomerase, as well as telomere structural state, can be perturbed by manipulating proteins that bind the telomere directly; in mammals, these proteins include TRF1, TRF2, and POT1. Such manipulations have been generally limited to overexpression or expression of dominant negative forms in cultured human or mouse cell lines. With the exception of the insights gained from the telomerase-knockout mouse and the Arabidopsis plant, little is known about the effects of telomere dysfunction in vivo. In this project the amphibian Xenopus will be used to address the hypotheses that TRF1 is a negative regulator of telomere length in vivo, and that telomere dysfunction can be initiated by disrupting the telomere complex, which will result in phenotypic and chromosome disturbances. The project will identify and clone telomere binding protein genes from Xenopus laevis and the true diploid Xenopus tropicalis, using EST database mining and molecular techniques to amplify the genes from messenger RNA. Cloned gene products will be tested for telomere binding capability. Finally, the function of these genes will be disrupted in vivo, using the technique of short interfering RNA (siRNA). Short double-stranded RNA reflecting the sequence of the target mRNA will be injected into early Xenopus embryos, which results in the destruction of the mRNA and the resulting "knockdown" of the target protein. The experimental and control embryos will then be analyzed for effects on telomere length as well as developmental phenotype.
Chromosome ends, or telomeres, are critical to the cell, signalling whether the cell can continue to divide or if it should die. Though much is known about telomere function in cultured cells, this project will investigate the role of telomere state in whole organisms. Furthermore, the project will provide the framework and resources for undergraduate students to pursue independent research as part of their educational experience, and prepare them more rigorously for their roles as educated citizens and future scientists.
