Host cell reactivation by human cells of DNA expression vectors damaged by ultraviolet radiation or by acid-heat treatment.
We utilized a plasmid vector host cell reactivation assay to probe the biological functioning of DNA expression vectors and their encoded genes. We studied the effect of ultraviolet radiation or acid-heat treatment on the transient expression of genes transfected into normal human cells and into DNA repair deficient (xeroderma pigmentosum) cells and modification of gene expression by sodium butyrate. U.v. inactivation of transient expression of the bacterial gpt gene contained in a non-replicating expression vector plasmid, pSV2catSVgpt, was much greater in three xeroderma pigmentosum lines than in the four other human cell lines tested. In contrast, treatment of pSV2catSVgpt with acid and heat to produce apurinic sites resulted in a similar slope of the inactivation curve of the bacterial cat gene in the repair deficient and repair proficient cells. Thus, u.v. damage of DNA expression vectors was subject to repair by the normal host cells, but acid-heat treatment resulted in damage (apurinic sites) that was handled in a similar manner by excision repair deficient and excision repair proficient human cells. In both normal and xeroderma pigmentosum cells sodium butyrate treatment of cells resulted in a greater stimulation of chloramphenicol acetyltransferase expression with u.v. damaged than with undamaged plasmid. This assay thus permits examination of the effects of defined types of DNA damage on plasmid expression and study of its modulation by cellular repair activities.
Protić-Sabljić
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Kraemer
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D000123:Acetyltransferases / Q000032:analysis; D000143:Acids; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase; D004249:DNA Damage; D004260:DNA Repair; D005822:Genetic Vectors; D006358:Hot Temperature; D006801:Humans; D010430:Pentosyltransferases / Q000032:analysis; D010957:Plasmids; D014162:Transfection; D014466:Ultraviolet Rays
D000143:Acids; D002087:Butyrates; D020148:Butyric Acid; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase