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

1979 ◽  
Vol 83 (1) ◽  
pp. 116-125 ◽  
Author(s):  
RK Draper ◽  
D Chin ◽  
D Eurey-Owens ◽  
IE Scheffler ◽  
MI Simon
Keyword(s):  
Diphtheria Toxin ◽  
Elongation Factor ◽  
Cross Resistance ◽  
3T3 Cells ◽  
Toxin Resistance ◽  
Gene Coding ◽  
Recessive Phenotype ◽  
Mouse Cells ◽  
Pseudomonas Aeruginosa Exotoxin

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.

Binding and uptake of diphtheria toxin by toxin-resistant Chinese hamster ovary and mouse cells

1983 ◽  
Vol 3 (7) ◽  
pp. 1283-1294
Author(s):  
J R Didsbury ◽  
J M Moehring ◽  
T J Moehring
Keyword(s):  
Binding Sites ◽  
Diphtheria Toxin ◽  
Specific Binding ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Cross Resistance ◽  
Resistant Mouse ◽  
Pseudomonas Exotoxin A ◽  
Specific Binding Sites ◽  
Mouse Cells

We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally resistant mouse cells. All are resistant due to a defect in the process of internalization and delivery of toxin to its target in the cytosol, elongation factor 2. By cell hybridization studies, analysis of cross-resistance, and determination of specific binding sites for 125I-labeled diphtheria toxin, we showed that these cell strains fall into two distinct complementation groups. The Dipr group encompasses Chinese hamster strains that are resistant only to diphtheria toxin, as well as mouse LM cells. These strains possess a normal complement of high-affinity binding sites for diphtheria toxin, but these receptors are unable to deliver active toxin fragment A to the cytosol. Cells of the DPVr group have a broader spectrum of resistance, including Pseudomonas exotoxin A and several enveloped viruses as well as diphtheria toxin. In these studies, which investigate the resistance of these cells to diphtheria toxin, we demonstrate that they possess a reduced number of specific binding sites for this toxin and behave, phenotypically, like cells treated with the proton ionophore monensin. Their resistance is related to a defect in a mechanism required for release of active toxin from the endocytic vesicle.

scholarly journals Binding and uptake of diphtheria toxin by toxin-resistant Chinese hamster ovary and mouse cells.

1983 ◽  
Vol 3 (7) ◽  
pp. 1283-1294 ◽  
Author(s):  
J R Didsbury ◽  
J M Moehring ◽  
T J Moehring
Keyword(s):  
Binding Sites ◽  
Diphtheria Toxin ◽  
Specific Binding ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Cross Resistance ◽  
Resistant Mouse ◽  
Pseudomonas Exotoxin A ◽  
Specific Binding Sites ◽  
Mouse Cells

We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally resistant mouse cells. All are resistant due to a defect in the process of internalization and delivery of toxin to its target in the cytosol, elongation factor 2. By cell hybridization studies, analysis of cross-resistance, and determination of specific binding sites for 125I-labeled diphtheria toxin, we showed that these cell strains fall into two distinct complementation groups. The Dipr group encompasses Chinese hamster strains that are resistant only to diphtheria toxin, as well as mouse LM cells. These strains possess a normal complement of high-affinity binding sites for diphtheria toxin, but these receptors are unable to deliver active toxin fragment A to the cytosol. Cells of the DPVr group have a broader spectrum of resistance, including Pseudomonas exotoxin A and several enveloped viruses as well as diphtheria toxin. In these studies, which investigate the resistance of these cells to diphtheria toxin, we demonstrate that they possess a reduced number of specific binding sites for this toxin and behave, phenotypically, like cells treated with the proton ionophore monensin. Their resistance is related to a defect in a mechanism required for release of active toxin from the endocytic vesicle.

scholarly journals Translation Elongation Factor 2 Anticodon Mimicry Domain Mutants Affect Fidelity and Diphtheria Toxin Resistance

2006 ◽  
Vol 281 (43) ◽  
pp. 32639-32648 ◽  
Author(s):  
Pedro A. Ortiz ◽  
Rory Ulloque ◽  
George K. Kihara ◽  
Haiyan Zheng ◽  
Terri Goss Kinzy
Keyword(s):  
Diphtheria Toxin ◽  
Molecular Mimicry ◽  
Elongation Factor ◽  
Lethal Effect ◽  
Translational Machinery ◽  
Toxin Resistance ◽  
Protein Levels ◽  
Elongation Factor 2 ◽  
Eukaryotic Elongation Factor

Eukaryotic elongation factor 2 (eEF2) mediates translocation in protein synthesis. The molecular mimicry model proposes that the tip of domain IV mimics the anticodon loop of tRNA. His-699 in this region is post-translationally modified to diphthamide, the target for Corynebacterium diphtheriae and Pseudomonas aeruginosa toxins. ADP-ribosylation by these toxins inhibits eEF2 function causing cell death. Mutagenesis of the tip of domain IV was used to assess both functions. A H694A mutant strain was non-functional, whereas D696A, I698A, and H699N strains conferred conditional growth defects, sensitivity to translation inhibitors, and decreased total translation in vivo. These mutant strains and those lacking diphthamide modification enzymes showed increased -1 frameshifting. The effects are not due to reduced protein levels, ribosome binding, or GTP hydrolysis. Functional eEF2 forms substituted in domain IV confer dominant diphtheria toxin resistance, which correlates with an in vivo effect on translation-linked phenotypes. These results provide a new mechanism in which the translational machinery maintains the accurate production of proteins, establishes a role for the diphthamide modification, and provides evidence of the ability to suppress the lethal effect of a toxin targeted to eEF2.

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