Spontaneous and ultraviolet-induced mutations on a single-stranded shuttle vector transfected into monkey cells.
The shuttle vector plasmid PCF3A, carrying the supF target gene, can be transfected into monkey COS7 cells as single-stranded or double-stranded DNA. Single strand-derived plasmid progeny exhibited a 10-fold higher spontaneous mutation frequency than double strand-derived progeny. The location of spontaneous mutations obtained after transfection of the single-stranded vector shared similarities with that for double-stranded vectors. However, the nature of base changes was very different. Single-stranded PCF3A DNA was used to study ultraviolet-induced mutagenesis. An earlier report (Madzak and Sarasin, J. Mol. Biol., 218 (1991) 667-673) showed that single-stranded DNA exhibited a lower survival and a higher mutation frequency than double-stranded DNA after ultraviolet irradiation. In the present report, sequence analysis of mutant plasmids is presented. The use of a single-stranded vector allowed us to show the targeting of mutations at putative lesion sites and to determine the exact nature of the base implicated in each mutation. Frameshift mutations were more frequent after transfection of control or irradiated plasmid as single-stranded DNA than as double-stranded DNA. Multiple mutations, observed at a high frequency in the spontaneous and ultraviolet-induced mutation spectra following single-stranded DNA transfection, could be due to an error-prone polymerisation step acting on a single-stranded template.
Madzak
C
C
Laboratory of Molecular Genetics, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
Cabral-Neto
J B
JB
Menck
C F
CF
Sarasin
A
A
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000235:genetics*; D005822:Genetic Vectors; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000528:radiation effects*; D009152:Mutagenicity Tests; D014162:Transfection; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded