Enhanced transforming activity of pSV2 plasmids in human cells depends upon the type of damage introduced into the plasmid.
When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt+ or Neo+) is increased by irradiating the respective plasmid DNA in vitro with UV (254 nm). To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.
Spivak
G
G
Department of Biological Sciences, Stanford University, CA 94305.
Leadon
S A
SA
Vos
J M
JM
Meade
S
S
Hanawalt
P C
PC
Ganesan
A K
AK
D002460:Cell Line; D004249:DNA Damage; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000187:drug effects / Q000528:radiation effects*; D011740:Pyrimidine Dimers / Q000528:radiation effects; D013539:Simian virus 40 / Q000235:genetics*; D013941:Thymine / Q000031:analogs & derivatives / Q000494:pharmacology; D014170:Transformation, Genetic / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays
D011740:Pyrimidine Dimers; C029389:thymine glycol; D013941:Thymine