Induction of thymidine kinase in enzyme-deficient chinese hamster cells

Harris, Morgan

doi:10.1016/0092-8674(82)90165-9

Cited by 201 publications

(72 citation statements)

References 44 publications

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“…If mutations are scored while the gene is still in the mammalian cell, one faces the limitations associated with traditional somatic cell genetics: mutation selection is time consuming and subject to the ambiguities resulting from, for example, epigenetic effects (30), and detailed analysis of the mutations is difficult. For instance, a promising system based on the bacterial gpt gene embedded into the chromosomes of hamster cells has been devised in which mutations are analyzed by Southern blotting (31).…”

Section: Cactccagt Total 39mentioning

confidence: 99%

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

Lebkowski¹,

Clancy²,

Miller³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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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)....

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Section: Cactccagt Total 39mentioning

confidence: 99%

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

Lebkowski¹,

Clancy²,

Miller³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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“…Following treatment with azaCyd, RJK92 cells revert at a very high frequency to the TK+ phenotype (12), indicating that the TK gene in RJK92 cells is inactive but functionally intact and potentially expressible. Preliminary reports have suggested that carcinogen treatment of RJK92 and similar lines can induce aminopterin resistance (HAT') and TK expression (12,30).In this paper, we have characterized the changes in TK gene expression which follow treatment of RJK92 cells with the alkylating carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Furthermore, we have identified changes in hamster DNA associated with TK gene activation.…”

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confidence: 99%

Genomic hypomethylation and far-5' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.

Barr¹,

Rajagopalan²,

MacArthur³

et al. 1986

Mol. Cell. Biol.

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We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK-RJK92 Chinese hamster cells, aminopterin-resistant (HAT') colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high-abd low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity.Although numerous experimental models have demonstrated the ability of chemicals to initiate carcinogenesis (6), the genotypic changes and alterations in gene expression which constitute the initiated state remain unknown. As one approach to these problems, workers in many laboratories have used cell culture systems to investigate how a chemical carcinogen can alter gene expression. Some investigations focused on the ability of carcinogens to mutate an active gene and thereby inactivate gene expression or modify the gene product (10,18,26). Recent evidence, however, has expanded the repertoire of carcinogen-induced alterations to include amplification of active genes (34) and activation of quiescent genes (15,23,24).Our laboratory has been investigating carcinogen-induced activation of quiescent genes. To select model systems in which an inactive gene is functionally intact and potentially expressible, we have focused on the class of inactive genes which can be activated by 5-azacytidine (azaCyd), an agent which alters gene expression by epigenetic perturbations presumably related to inhibition of DNA methylation and not by a mutational mechanism (32). The general significance of this class of inactive genes is demonstrated by the occurrence of azaCyd-activatable genes and developmental programs in a variety of cell types (16, 23). One such set of inactive genes is the metallothionein family in mouse S49 T-lymphoma cells; we have previously reported that chemical carcinogens and UV radiation treatment of these cells will activate one or both metallothionein genes and thereby induce cadmi...

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“…One of the most dramatic 5-aza-CR-mediated gene inductions was a 10_-to 106-fold increase in thymidine kinase expression reported by Harris (96). Gene reactivation was observed in as many as 10 to 30% of the surviving cells, which is several orders of magnitude higher than that expected for a mutagenic agent.…”

Section: X-chromosome Reactivationmentioning

confidence: 86%

DNA Methylation and Differentiation

Michalowsky¹,

Jones²

1989

Environmental Health Perspectives

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The methylation of specific cytosine residues in DNA has been implicated in regulating gene expression and facilitating functional specialization of cellular phenotypes. Generally, the demethylation of certain CpG sites correlates with transcriptional activation of genes. 5-Azacytidine is an inhibitor of DNA methylation and has been widely used as a potent activator of suppressed genetic information. Treatment of cells with 5-azacytidine results in profound phenotypic alterations. The drug-induced hypomethylation of DNA apparently perturbs DNA-protein interactions that may consequently alter transcriptional activity and cell determination. The inhibitory effect of cytosine methylation may be exerted via altered DNA-protein interactions specifically or may be transduced by a change in the conformation of chromatin.Recent studies have demonstrated that cytosine methylation also plays a central role in parental imprinting, which in turn determines the differential expression of maternal and paternal genomes during embryogenesis. In other words, methylation is the mechanism whereby the embryo retains memory of the gametic origin of each component of genetic information. A memory of this type would probably persist during DNA replication and cell division as methylation patterns are stable and heritable.

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Induction of thymidine kinase in enzyme-deficient chinese hamster cells

Cited by 201 publications

References 44 publications

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

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

Genomic hypomethylation and far-5' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.

DNA Methylation and Differentiation

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