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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Comparison of the frequency of diphtheria toxin and thioguanine resistance induced by a series of carcinogens to analyze their mutational specificities in diploid human fibroblasts.

The mutagenic specificities of ethylnitrosourea (ENU), X-rays (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene (BPDE), ICR-191, and N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) were analyzed and compared in diploid human fibroblasts and Salmonella typhimurium. In the human fibroblasts, we compared the frequency of diphtheria toxin (DT)-resistant mutants, presumably induced in the gene coding for elongation factor-2, with the frequency of 6-thioguanine (TG) resistance induced by mutations in the gene coding for hypoxanthine(guanine)phosphoribosyltransferase (HPRT). Recovery of DT-resistant (DTr) cells requires that the mutant EF-2 retain the ability to carry on protein synthesis since the normal EF-2 will be inactivated by DT selection. Therefore, the DTr mutation cannot involve major changes in the gene. In contrast, cells can acquire TG resistance by any mechanism which eliminates HPRT activity, e.g., base substitution, frameshift, deletion, loss of chromosomes. Each agent was assessed by calculating the ratio of the slopes of the dose-response plots (induced variant frequency as a function of dose of the agent used) for the two markers (DTr/TGr variants.). In S. typhimurium we examined the reversion frequency in four histidine-requiring strains bearing forward mutations of the frameshift (TA1538, TA98) or missense (TA1535, TA100) type. ENU, which was predominantly a base substitution mutagen in the bacteria, gave a ratio of DTr to TGr variants of 1. 5. As expected of an agent inducing gross chromosomal changes, X-rays induced no revertants in bacteria and in human cells gave a ratio of 0. 1. ICR-191 which was predominantly a frameshift mutagen in bacteria gave a ratio of 0.15. In the set of bacterial strains containing the plasmid pKM101, BPDE reverted both frameshift and base substitution mutations. It did not cause reversions in the other set of strains. In human cells BPDE gave a response similar to ENU, i.e., a ratio of DTr/TGr variants of 1. 5. As reported by others, N-AcO-AAF was predominantly a frameshift mutagen in bacteria. However, in the human cells it gave a ratio of DTr/TGr variants of 1.5, similar to ENU and BPDE. These results suggest that in human cells, BPDE and N-AcO-AAF, like ENU, yield predominantly base substitutions, while ICR-191 and X-rays largely produce mutations by mechanisms which result in more extensive alterations in the gene.[1]


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