Characterization of a Chinese hamster ovary cell mutant having a mutation in elongation factor-2.
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.
Laboratory of Bacterial Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
Pradeep K
PK
Gupta
Shihui
S
Liu
Stephen H
SH
Leppla
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D031541:Fluorescence Resonance Energy Transfer; D005838:Genotype; D006639:Histidine / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D020652:Peptide Elongation Factor 2 / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D017386:Sequence Homology, Amino Acid; D014118:Toxins, Biological / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology
D020652:Peptide Elongation Factor 2; D014118:Toxins, Biological; D000246:Adenosine Diphosphate Ribose; D006639:Histidine; C027527:diphthamide