Enhanced transforming activity of ultraviolet-irradiated pSV2-gpt is due to damage outside the gpt transcription unit.
We have shown that when pSV2-gpt is introduced into human cells by calcium phosphate coprecipitation, the yield of Gpt+ transformants is increased by irradiating the plasmid with 254 nm uv. To elucidate the mechanism underlying this response, we constructed pSV2-gpt molecules in which the uv damage was confined to a particular region: a 3.0-kb region containing the pBR322 sequences and simian virus 40 (SV40) sequences not required for expression of the gpt gene, or a 2.3-kb fragment containing the Escherichia coli gpt gene together with the SV40 early promoter and sequences needed for splicing and polyadenylation. The transforming activity of the plasmid was greatly enhanced by uv damage confined to the 3.0-kb pBR322 region and increased linearly with uv dose up to 1 kJ/m2, but remained relatively constant at doses between 2 and 8 kJ/m2. Positioning the damaged region upstream, or both upstream and downstream, from the gpt transcription unit increased the uv enhancement slightly compared to positioning the damaged region only downstream. In contrast, transforming activity was significantly decreased by damage in the 2.3-kb gpt transcription unit. These results suggest that uv damage outside a selectable marker gene in a plasmid can increase the probability of stable integration of the plasmid into the genome of recipient cells without inhibiting expression of of the gene.
Leadon
S A
SA
Department of Biological Sciences, Stanford University, California 94305-5020.
Ganesan
A K
AK
Hanawalt
P C
PC
D002460:Cell Line; D004249:DNA Damage; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005354:Fibrosarcoma; D005796:Genes / Q000528:radiation effects*; D005798:Genes, Bacterial / Q000528:radiation effects*; D006801:Humans; D010430:Pentosyltransferases / Q000235:genetics*; D010957:Plasmids / Q000528:radiation effects*; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014466:Ultraviolet Rays
D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D004262:DNA Restriction Enzymes