Protocol

Advanced Protocols in Oxidative Stress I

Volume 477 of the series Methods In Molecular Biology pp 331-342

A Functional Genomics Approach to Identify and Characterize Oxidation Resistance Genes

  • Michael R. VolkertAffiliated withDepartment of Molecular Genetics and Microbiology, University of Massachusetts Medical School
  • , Jen-Yeu WangAffiliated withInstitute of Molecular Biology, Academica Sinica
  • , Nathan A. ElliottAffiliated withNanoString Technologies

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Abstract

In order to develop a more complete understanding of the genes required for resistance to oxidative DNA damage, we devised methods to identify genes that can prevent or repair oxidative DNA damage. These methods use the oxidative mutator phenotype of a repair deficient E. coli strain to measure the antimutator effect resulting from the expression of human cDNAs. The method can be adapted to characterize the function, and to determine the active site domains, of putative antimutator genes. Since bacteria do not contain subcellular compartments, genes that function in mitochondria, the cytoplasm, or the nucleus can be identified. Methods to determine the localization of genes in their normal host organism are also described.

Key words

Oxidative DNA damage DNA repair Mutagenesis Mitochondrial localization Fluorescence microscopy Functional gene cloning 8-oxoguanine