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Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin (CROSBI ID 114017)

Prilog u časopisu | izvorni znanstveni rad | međunarodna recenzija

Ivanković, Milena ; Rubelj, Ivica ; Matulić, Maja ; Reich, Edward ; Brdar, Branko Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin // Mutation research. Genetic toxicology and environmental mutagenesis, 609 (2006), 1; 34-42. doi: 10.1016/j.mrgentox.2006.06.027

Podaci o odgovornosti

Ivanković, Milena ; Rubelj, Ivica ; Matulić, Maja ; Reich, Edward ; Brdar, Branko

engleski

Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin

Protein synthesis elongation factor-2 (EF-2) from eukaryots contains a conserved post-translationally modified histidine residue known as diphthamide. Diphthamide is a unique site of ADP-ribosylation by diphtheria toxin (DT) which is responsible for cell killing. In this report we describe the construction of DT-resistant HeLa cell lines by engineering the toxin– resistant form of its specific substrate, protein elongation factor-2. Using the site-specific mutagenesis of the histidine precursor of diphthamide, histidine 715 in human EF-2 was substituted with one of four amino acids: leucine, methionine, asparagine or glutamine. Mutant EF-2s were cloned into pCMVexSVneo expression vector, transfected into HeLa cells and DT-resistant cell clones isolated. The protective effect of mutant EF-2s against cell killing by DT, after exposing all four mutant strains derived from HeLa cells to different concentrations of the toxin was demonstrated by their: (1) normal morphological appearance ; (2) unaffected or slightly slower growth rates ; (3) undisturbed electrophoretic DNA profiles whose integrity was almost totally preserved. It was hence concluded that despite its strict conservation and unique modification the diphthamide histidine appears not to be essential to the function of human EF-2 in protein synthesis. In addition, DT-resistant HeLa cell clones may be useful as a host for various DT gene-containing vectors that express the toxin intracellularly.

EF-2; diphtheria toxin; site-specific mutagenesis

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Podaci o izdanju

609 (1)

2006.

34-42

objavljeno

1383-5718

10.1016/j.mrgentox.2006.06.027

Povezanost rada

Biologija

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