Rapid detection of ultraviolet-induced reversion of an amber mutation in mouse L cells.
An amber codon (TAG) was introduced into the N-terminal coding region of the murine H-2Kb gene. The mutant gene was transfected into mouse L cells, and a clone containing a single unrearranged chromosomally integrated copy of the mutant gene was mutagenized with 254-nm UV radiation. Surviving cells were scored for surface expression of H-2Kb protein with in situ immunoperoxidase staining. Revertants were detected at a frequency of 3 X 10(-6) at a dose of 40 J/m2 (3-5% survival). Revertant genes, cloned by plasmid rescue, contained the expected thymine-to-cytosine transitions at the amber codon. These data show that revertants can be rapidly detected in mammalian cells without selection and provide a basis for the development of mammalian cell lines that could be used to study mutational phenomena. During this study the steady-state level of mRNA was reduced in L cells carrying the amber mutant H-2Kb gene compared with L cells containing a wild-type or revertant H-2Kb gene. This reduction was shown not to be due to transcriptional differences, suggesting that the amber mutation decreases stability of the H-2Kb mRNA.
Eccles
S J
SJ
Medical Research Council Collaborative Center, London, United Kingdom.
Vidal
M A
MA
Wrighton
C J
CJ
Grosveld
F G
FG
Burke
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006183:H-2 Antigens / Q000235:genetics*; D007739:L Cells / Q000201:enzymology / Q000528:radiation effects; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000528:radiation effects; D015183:Restriction Mapping; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic / Q000528:radiation effects; D014162:Transfection; D014466:Ultraviolet Rays
D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D012333:RNA, Messenger; D013937:Thymidine Kinase