The goal of this proposal is to determine if the transcription level of a gene affects mutation at that locus. It is important to understand factors which contribute to mutation in somatic cells because of the causative role of mutation in the carcinogenic process.
The first aim of this project is to develop and characterize a human cell line which has an inducible thymidine kinase (tk) locus. The GalER induction system will be utilized. B-lymphoblastoid cells will be transfected stabley with a GalER fusion construct which transduces an external estrogen signal to yeast cis-acting Gal4 responsive UAS's (upstream activating sites). Then Gal4 responsive UAS's will be targeted to the promoter region of the endogenous tk gene using a gene replacement vector containing 8 kb of homology flanking the UAS's. The magnitude and time course of tk mRNA induction will be determined by northern analysis.
The second aim i s to measure tk mutant frequency (Mf) during low and high transcription levels using a standard human cell in vitro mutation assay. Both spontaneous and X-ray induced Mf's will be measured during low and high transcriptional activity. Lastly, the qualitative nature of mutations will be determined using Southern blot analysis with the human tk cDNA probe.
| Lippert, M J; Chen, Q; Liber, H L (1998) Increased transcription decreases the spontaneous mutation rate at the thymidine kinase locus in human cells. Mutat Res 401:1-10 |
