The lacI shuttle: rapid analysis of the mutagenic specificity of ultraviolet light in human cells.

Abstract

A system has been devised that allows the effect of mutagens acting in human cells to be readily analyzed at the DNA sequence level. The bacterial gene WIcI, carried on a shuttle vector, is introduced into human tissue culture cells by transfection and allowed to replicate in the cell nucleus.Twenty-four to 48 hr after transfection, the cells are exposed to a mutagen. After 1-2 days of further replication, vector DNA is purified and transfected back intoEscherichia coli for scoring and analysis of mutations in laW. The (1)(2)(3)(4). In these experiments, the target gene is carried on a shuttle vector, which can replicate in both mammalian and bacterial cells. Thus, the gene can be mutated in mammalian cells and then transferred to bacteria for analysis of mutations. The advantage of this scheme is that in bacteria mutations can be rapidly and unambiguously scored. Furthermore, if lad is used as the mutational target, sophisticated bacterial molecular genetics can be applied to characterize the mutations (5, 6). In lad, an extensive deletion map allows precise localization of mutations by using genetic techniques. Moreover, accurate DNA sequence assignments can be made for nonsense mutations by using simple genetic techniques.The use of shuttle vectors for determination of mutagenic specificity has been limited by a high spontaneous mutation frequency associated with transfection (1-4) and by an inability of the vectors to replicate efficiently in human cells. Recently, both of these problems were solved by the discovery that the human cell line 293 (7) replicates simian virus 40 (SV40)-based shuttle vectors extremely well with a relatively low spontaneous mutation frequency (4). When 293 cells containing shuttle vectors are exposed to mutagens, an increase in mutation frequency above the spontaneous background is readily obtained. Therefore, induced mutations can be rapidly isolated and characterized.We demonstrate here the operation ofthe lacd shuttle using UV light as the mutagen. The DNA sequence changes for 53 UV light-induced point mutations, as well as for 32 point mutations from unmutagenized cells, are reported. The mutagenic specificity of UV light thereby obtained in human cells has close parallels with that obtained previously for UV light treatment of Escherichia coli. This result suggests that human and bacterial cells may react to UV light damage in similar ways. It also argues that the lacd shuttle system is indeed detecting true UV light-induced mutations. Thus, it appears that this experimental system can rapidly elucidate the outcome of a mutagen's interaction with DNA in human cells at the most fundamental genetic level, the DNA sequence change. MATERIALS AND METHODSVector DNA. The previously described (4) plasmid pJYMib was used as the shuttle vector. pJYMib contains all of SV40, pML (a pBR322 derivative), the entire lacI gene, and the amino-terminal portion of lacZ. Plasmid DNA was prepared by the alkaline lysis procedure and purified on cesium chloride gradients as described (8)....

Determination of DNA sequence changes induced by ethyl methanesulfonate in human cells, using a shuttle vector system.

“…Operation of the shuttle system without external mutagenesis leads to a background lacI mutation frequency of 0.035% for plasmid pJYMib in cell line 293 (14,15). These spontaneous mutations are apparently incurred during transfection (1,14,15,20,21) and are discussed further below.…”

“…(14). Here we used the system to examine the mutagenic specificity of a member of another major group of mutagens, the alkylating agents.…”

“…Bacterial cells were spread on indicator plates containing 5-bromo-4-chloro-3-indolyl-f-D-galactoside, and lacI mutants were identified as blue colonies (see the legend to Fig. 1 and reference 14 for further details on the use of the lacI shuttle system).Operation of the shuttle system without external mutagenesis leads to a background lacI mutation frequency of 0.035% for plasmid pJYMib in cell line 293 (14, 15). These spontaneous mutations are apparently incurred during transfection (1,14,15,20,21) and are discussed further below.…”

“…Bacterial cells were spread on indicator plates containing 5-bromo-4-chloro-3-indolyl-f-D-galactoside, and lacI mutants were identified as blue colonies (see the legend to Fig. 1 and reference 14 for further details on the use of the lacI shuttle system).…”

Ionizing radiation-induced mutagenesis

“…Early such vectors, especially those related to simian virus 40 (SV40), were prone to high levels of spontaneous mutagenesis (Razzaque et al, 1983;Calos et al, 1983;Hauser et al, 1987). This has now been improved (Lebkowski et al, 1985;Glazer et al, 1986;MacGregor et al, 1987). In particular the new vectors based on EpsteinBarr virus are likely to be of value in human cell systems (DuBridge et al, 1987;Menck et al, 1987).…”