characterization of a chinese hamster ovary cell mutant having a mutation in elongation factor-2描述中国仓鼠卵巢细胞突变体延伸因子2的突变.pdfVIP

Characterization of a Chinese Hamster Ovary Cell Mutant Having a Mutation in Elongation Factor-2 Pradeep K. Gupta, Shihui Liu, Stephen H. Leppla* Laboratory of Bacterial Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America Abstract Retroviral insertional mutagenesis provides an effective forward genetic method for identifying genes involved in essential cellular pathways. A Chinese hamster ovary cell line mutant resistant to several bacterial ADP-ribosylating was obtained by this approach. The toxins used catalyze ADP-ribosylation of eukaryotic elongation factor 2 (eEF-2), block protein synthesis, and cause cell death. Strikingly, in the CHO PR328 mutant cells, the eEF-2 substrate of these ADP-ribosylating toxins was found to be modified, but the cells remained viable. A systematic study of these cells revealed the presence of a structural mutation in one allele of the eEF-2 gene. This mutation, Gly717Arg, is close to His715, the residue that is modified to become diphthamide. This Arg substitution prevents diphthamide biosynthesis at His715, rendering the mutated eEF-2 non-responsive to ADP-ribosylating toxins, while having no apparent effect on protein synthesis. Thus, CHO PR328 cells are heterozygous, having wild type and mutant eEF-2 alleles, with the latter allowing the cells to survive even in the presence of ADP-ribosylating toxins. Here, we report the comprehensive characterization of these cells. Citation: Gupta PK, Liu S, Leppla SH (2010) Characterization of a Chinese Hamster Ovary Cell Mutant Having a Mutation in Elongation Factor-2. PLoS ONE 5(2): e9078. doi:10.1371/journal.pone.0009078 Editor: Dong-Yan Jin, University of Hong Kong, Hong Kong Received November 10, 2009; Accepted January 11, 2010; Published February 5, 2010 This is an open-access a