Rapid detection of ultraviolet-induced reversion of an amber mutation in mouse L cells.

Abstract

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.

Self major histocompatibility complex class I antigens expressed solely in lymphoid cells do not induce tolerance in the CD4+ T cell compartment.

“…lff' thymocytes were stained with biotinylated MV3 (anti-H-2K b) antibody (14) and developed with streptavidin-PE (Biogenesis, Poole, UK). Thymocytes were gated, and selected events were displayed as histograms using FACSplot software (NIMR Computing Division) using a FACScan | (Becton Dickinson & Co., Mountain View, CA) cytofluorimeter.…”