title: Detection of PIGO-deficient cells using proaerolysin: a valuable tool to investigate mechanisms of mutagenesis in the DT40 cell system.
abstract: While isogenic DT40 cell lines deficient in DNA repair pathways are a great tool to understand the DNA damage response to genotoxic agents by a comparison of cell toxicity in mutants and parental DT40 cells, no convenient mutation assay for mutagens currently exists for this reverse-genetic system. Here we establish a proaerolysin (PA) selection-based mutation assay in DT40 cells to identify glycosylphosphatidylinositol (GPI)-anchor deficient cells. Using PA, we detected an increase in the number of PA-resistant DT40 cells exposed to MMS for 24 hours followed by a 5-day period of phenotype expression. GPI anchor synthesis is catalyzed by a series of phosphatidylinositol glycan complementation groups (PIGs). The PIG-O gene is on the sex chromosome (Chromosome Z) in chicken cells and is critical for GPI anchor synthesis at the intermediate step. Among all the mutations detected in the sequence levels observed in DT40 cells exposed to MMS at 100 µM, we identified that ∼55% of the mutations are located at A:T sites with a high frequency of A to T transversion mutations. In contrast, we observed no transition mutations out of 18 mutations. This novel assay for DT40 cells provides a valuable tool to investigate the mode of action of mutations caused by reactive agents using a series of isogenic mutant DT40 cells.
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina, United States of America. ynakamur@email.unc.edu
authors.forename: Jun
authors.initials: J
authors.lastname: Nakamura
authors.forename: Husamettin
authors.initials: H
authors.lastname: Gul
authors.forename: Xu
authors.initials: X
authors.lastname: Tian
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Bultman
authors.forename: James A
authors.initials: JA
authors.lastname: Swenberg
mesh_terms: D020856:AT Rich Sequence; D000704:Analysis of Variance; D000818:Animals; D001427:Bacterial Toxins / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival; D002645:Chickens; D004260:DNA Repair / Q000502:physiology*; D005434:Flow Cytometry; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis / Q000172:deficiency*; D008741:Methyl Methanesulfonate; D016296:Mutagenesis / Q000502:physiology*; D009152:Mutagenicity Tests / Q000379:methods*; D052899:Pore Forming Cytotoxic Proteins / Q000633:toxicity; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001427:Bacterial Toxins; D017261:Glycosylphosphatidylinositols; D052899:Pore Forming Cytotoxic Proteins; C107096:proaerolysin; D008741:Methyl Methanesulfonate