detection of pigo-deficient cells using proaerolysin a valuable tool to investigate mechanisms of mutagenesis in the dt40 cell system检测使用proaerolysin pigo-deficient细胞的一个有价值的工具来调查的诱变机制dt40细胞系统.pdfVIP

Detection of PIGO-Deficient Cells Using Proaerolysin: A Valuable Tool to Investigate Mechanisms of Mutagenesis in the DT40 Cell System 1 1,2 1 3 1,4 Jun Nakamura *, Husamettin Gul , Xu Tian , Scott J. Bultman , James A. Swenberg 1 Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina, United States of America, 2 Department of Pharmacology and Toxicology, Gulhane Military Academy of Medicine, Etlik, Ankara, Turkey, 3 Department of Genetics, University of North Carolina, Chapel Hill, North Carolina, United States of America, 4 Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina, United States of America 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 mM, we identified that ,55% of the mutations are located at A:T sites with a high frequency of A to T transve