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Detection and analysis of UV-induced mutations in mammalian cell DNA using a lambda phage shuttle vector
Abstract:
In order to study mutagenesis in mammalian cells, stable mouse L-cell lines were established with multiple copies of a lambda phage vector that contains the supF gene of Escherichia coli as a target for mutagenesis. Rescue of viable phage from high molecular weight mouse cell DNA using lambda in vitro packaging extracts was efficient (5 phage per microgram of cell DNA per copy) and yielded a negligible background of mutant phage (0 out of 54,605). From mouse cells exposed to 254-nm ultraviolet light (12J/m2), 78,510 phage were rescued, of which 8 were found to have mutant supF genes. DNA sequence analysis of the mutants suggests that the primary site of UV mutagenesis in mammalian cells is at pyrimidine-cytosine (Y-C) sequences, and that the most frequent mutation at this site is a C----T transition.
Insights
Researchers studied mutagenesis in mammalian cells using a lambda phage vector. Ultraviolet light primarily caused mutations at pyrimidine-cytosine sequences, with most mutations being cytosine to thymine transitions.
Area of Science:
- Molecular Biology
- Genetics
- Mammalian Cell Mutagenesis
Background:
- Mammalian cell mutagenesis studies are crucial for understanding DNA damage and repair.
- A lambda phage vector containing the Escherichia coli supF gene provides a sensitive target for mutagenesis detection.
Purpose of the Study:
- To establish stable mouse L-cell lines for studying mutagenesis.
- To identify the primary mutation sites induced by ultraviolet (UV) light in mammalian cells.
Main Methods:
- Stable mouse L-cell lines containing multiple copies of a lambda phage vector with the supF gene were created.
- Lambda phage was rescued in vitro from high molecular weight mouse cell DNA.
- DNA sequence analysis was performed on rescued mutant phage.
Main Results:
- Efficient rescue of viable phage (5 per microgram of cell DNA) with negligible background mutants was achieved.
- Exposure to 254-nm UV light induced mutations in the supF gene.
- Sequence analysis revealed UV mutagenesis predominantly occurs at pyrimidine-cytosine (Y-C) sequences.
Conclusions:
- The study successfully established a system for mutagenesis studies in mammalian cells.
- UV mutagenesis in mammalian cells primarily targets Y-C sequences.
- The most frequent UV-induced mutation observed was a cytosine to thymine transition.