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. 1979 Oct 1;83(1):116–125. doi: 10.1083/jcb.83.1.116

Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

RK Draper, D Chin, D Eurey-Owens, IE Scheffler, MI Simon
PMCID: PMC2110445  PMID: 117016

Abstract

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boquet P., Pappenheimer A. M., Jr Interaction of diphtheria toxin with mammalian cell membranes. J Biol Chem. 1976 Sep 25;251(18):5770–5778. [PubMed] [Google Scholar]
  2. Chang T., Neville D. M., Jr Demonstration of diphtheria toxin receptors on surface membranes from both toxin-sensitive and toxin-resistant species. J Biol Chem. 1978 Oct 10;253(19):6866–6871. [PubMed] [Google Scholar]
  3. Chasin L. A. The effect of ploidy on chemical mutagenesis in cultured Chinese hamster cells. J Cell Physiol. 1973 Oct;82(2):299–307. doi: 10.1002/jcp.1040820218. [DOI] [PubMed] [Google Scholar]
  4. Collier R. J. Diphtheria toxin: mode of action and structure. Bacteriol Rev. 1975 Mar;39(1):54–85. doi: 10.1128/br.39.1.54-85.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collier R. J., Kandel J. Structure and activity of diphtheria toxin. I. Thiol-dependent dissociation of a fraction of toxin into enzymically active and inactive fragments. J Biol Chem. 1971 Mar 10;246(5):1496–1503. [PubMed] [Google Scholar]
  6. Creagan R. P., Chen S., Ruddle F. H. Genetic analysis of the cell surface: association of human chromosome 5 with sensitivity to diphtheria toxin in mouse-human somatic cell hybrids. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2237–2241. doi: 10.1073/pnas.72.6.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davidson R. L., O'Malley K. A., Wheeler T. B. Polyethylene glycol-induced mammalian cell hybridization: effect of polyethylene glycol molecular weight and concentration. Somatic Cell Genet. 1976 May;2(3):271–280. doi: 10.1007/BF01538965. [DOI] [PubMed] [Google Scholar]
  8. Dendy P. R., Harris H. Sensitivity to diphtheria toxin as a species-specific marker in hybrid cells. J Cell Sci. 1973 May;12(3):831–837. doi: 10.1242/jcs.12.3.831. [DOI] [PubMed] [Google Scholar]
  9. Draper R. K., Chin D., Simon M. I. Diphtheria toxin has the properties of a lectin. Proc Natl Acad Sci U S A. 1978 Jan;75(1):261–265. doi: 10.1073/pnas.75.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Draper R. K., Chin D., Stubbs L., Simon M. I. Studies of the diphtheria toxin receptor on Chinese hamster cells. J Supramol Struct. 1978;9(1):47–55. doi: 10.1002/jss.400090106. [DOI] [PubMed] [Google Scholar]
  11. FORD D. K., YERGANIAN G. Observations on the chromosomes of Chinese hamster cells in tissue culture. J Natl Cancer Inst. 1958 Aug;21(2):393–425. [PubMed] [Google Scholar]
  12. Gill D. M., Dinius L. L. The elongation factor 2 content of mammalian cells. Assay method and relation to ribosome number. J Biol Chem. 1973 Jan 25;248(2):654–658. [PubMed] [Google Scholar]
  13. Gupta R. S., Siminovitch L. Diphtheria-toxin-resistant mutants of CHO cells affected in protein synthesis: a novel phenotype. Somatic Cell Genet. 1978 Sep;4(5):553–571. doi: 10.1007/BF01542926. [DOI] [PubMed] [Google Scholar]
  14. Gupta R. S., Siminovitch L. Isolation and characterization of mutants of human diploid fibroblasts resistant to diphtheria toxin. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3337–3340. doi: 10.1073/pnas.75.7.3337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Honjo T., Nishizuka Y., Kato I., Hayaishi O. Adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis by diphtheria toxin. J Biol Chem. 1971 Jul 10;246(13):4251–4260. [PubMed] [Google Scholar]
  16. Iglewski B. H., Kabat D. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin,. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2284–2288. doi: 10.1073/pnas.72.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ittelson T. R., Gill D. M. Diphtheria toxin: specific competition for cell receptors. Nature. 1973 Mar 30;242(5396):330–332. doi: 10.1038/242330b0. [DOI] [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. Middlebrook J. L., Dorland R. B., Leppla S. H. Association of diphtheria toxin with Vero cells. Demonstration of a receptor. J Biol Chem. 1978 Oct 25;253(20):7325–7330. [PubMed] [Google Scholar]
  20. Moehring J. M., Moehring T. J. Comparison of diphtheria intoxication in human and nonhuman cell lines and their resistant variants. Infect Immun. 1976 Jan;13(1):221–228. doi: 10.1128/iai.13.1.221-228.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Moehring T. J., Moehring J. M. Response of cultured mammalian cells to diphtheria toxin. IV. Isolation of KB cells resistant to diphtheria toxin. Infect Immun. 1972 Oct;6(4):487–492. doi: 10.1128/iai.6.4.487-492.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moehring T. J., Moehring J. M. Selection and characterization of cells resistant to diphtheria toxin and pseudomonas exotoxin A: presumptive translational mutants. Cell. 1977 Jun;11(2):447–454. doi: 10.1016/0092-8674(77)90063-0. [DOI] [PubMed] [Google Scholar]
  23. Nicolson G. L. Ultrastructural analysis of toxin binding and entry into mammalian cells. Nature. 1974 Oct 18;251(5476):628–630. doi: 10.1038/251628a0. [DOI] [PubMed] [Google Scholar]
  24. Pappenheimer A. M., Jr Diphtheria toxin. Annu Rev Biochem. 1977;46:69–94. doi: 10.1146/annurev.bi.46.070177.000441. [DOI] [PubMed] [Google Scholar]
  25. Robinson E. A., Henriksen O., Maxwell E. S. Elongation factor 2. Amino acid sequence at the site of adenosine diphosphate ribosylation. J Biol Chem. 1974 Aug 25;249(16):5088–5093. [PubMed] [Google Scholar]
  26. Somers D. G., Pearson M. L., Ingles C. J. Regulation of RNA polymerase II activity in a mutant rat myoblast cell line resistant to alpha-amanitin. Nature. 1975 Jan 31;253(5490):372–374. doi: 10.1038/253372a0. [DOI] [PubMed] [Google Scholar]
  27. Uchida T., Pappenheimer A. M., Jr, Harper A. A. Reconstitution of diphtheria toxin from two nontoxic cross-reacting mutant proteins. Science. 1972 Feb 25;175(4024):901–903. doi: 10.1126/science.175.4024.901. [DOI] [PubMed] [Google Scholar]
  28. Yamaizumi M., Mekada E., Uchida T., Okada Y. One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell. 1978 Sep;15(1):245–250. doi: 10.1016/0092-8674(78)90099-5. [DOI] [PubMed] [Google Scholar]

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