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.
Gupta
Pradeep K
PK
Laboratory of Bacterial Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
Liu
Shihui
S
Leppla
Stephen H
SH
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