Cellular senescence, the terminal non-dividing state of human cells in response to low level DNA damage, has long been postulated as a model for tissue and organ aging in humans. The Mortality Factor on Chromosome 4 (MORF4) gene product acts in the molecular pathway that leads to the induction of the senescence phenotype in cells. In addition, highly conserved MORF4 Related Genes (MRG) have been identified in numerous eukaryotes including two additional expressed family members in humans and one in the fruit fly Drosophila melanogaster. Currently there is no data on the cell's requirement for MORF4 and the MRG family. Double stranded RNA interference and small interfering RNAs targeted to MORF4, MRG 15 and MRGX in human HeLa cells and dmrg in Drosophila S2 cells will be used to induce degradation of their respective mRNAs. Effectiveness will be assayed by RT-PCR and Western immuno-blotting. The best conditions for creating protein nulls or near nulls will be determined in HeLA and S2 cells. These conditions will be used in several normal and immortal human cells as well as other Drosophila cell lines to determine their requirement for each MRG family member for cell senescence, cell cycle progression, and cell viability. The human telomerase reverse transcriptase (hTERT) will also be targeted by siRNA in hTERT positive immortal cell lines to asses their requirement for the hTERT protein. In addition, it has been demonstrated that RNAi leads to transcriptionally inhibitory methylation of CpG islands in the promoters of the targeted genes in plants. Methylation of the hTERT promoter will be determined in immortal cells before and after the introduction of hTERT as well as mock transfected cells. These experiments will lay the groundwork for further investigation into molecular and genetic interactions involving the MRG family and hTERT by providing an in vivo null system experimental system.
