Site-Specific Mutagenesis in Human Cells by Bulky Exocyclic Amino-Substituted Guanine and Adenine Derivatives.
- Author:
Ki Young MOON
1
Author Information
1. Department of Clinical Pathology, and Bioindustry and Technology Research Institute, Kwangju Health College, Gwangju, Korea. kmoon@www.kjhc.ac.kr
- Publication Type:Original Article
- Keywords:
Mutagenesis;
Mutagen
- MeSH:
Adenine*;
Adenoviridae;
Ampicillin;
Bacteria;
Codon, Initiator;
DNA;
DNA Adducts;
Genetic Vectors;
Guanine*;
Humans*;
Isopropyl Thiogalactoside;
Kidney;
Mutagenesis;
Mutagenesis, Site-Directed*;
Nucleotides;
Plasmids
- From:Cancer Research and Treatment
2004;36(2):151-156
- CountryRepublic of Korea
- Language:English
-
Abstract:
PURPOSE: 7-Bromomethylbenz[a]anthracene is a well- known mutagen and carcinogen. The aim of this study is to determine the mutagenic potency of its two major DNA adducts [N2-(benz[a]anthracen-7-ylmethyl)-2'-deoxyguanosine (b[a]a2G) and N6-(benz[a]anthracen-7- ylmethyl)-2'-deoxyadenosine (b[a]a6A)] and the simpler benzylated analogs [N2-benzyl-2'-deoxyguanosine (bn2G) and N6-benzyl-2'-deoxyadenosine (bn6A)] in Ad293 human cells and to compare to their mutagenicity in human cells and E. coli. MATERIALS AND METHODS: The shuttle vector pGP50 is capable of replicating in E. coli and human cells. Modified nucleotides were positioned in the plasmid pGP50 in a manner similar to pGP10 as described (8). Adenovirus transformed human embryonic kidney cells (line 293) were transfected with a shuttle vector containing an adduct. Two days later, the plasmids were recovered and treated with DpnI to remove unreplicated DNA. DH10B E. coli were transformed with the plasmids. Bacteria were cultured with the media containing X-gal, IPTG and ampicillin. Bacteria transformed by the plasmid with the adduct-induced mutation in the initiation codon of lacZ' form white colonies whereas bacteria transformed by the plasmid without mutation form blue colonies. RESULTS: In the human cell site-specific mutagenesis system, bn2G exhibited weak mutagenicity and bn6A was not mutagenic, although b[a]a2G or b[a]a6A produced 8% and 7% mutant colonies, respectively. At the site of the adduct, b[a]a2G induced the G--> T transversion mutation while b[a]a6A produced the A--> G transition mutation. CONCLUSION: These data indicate that bulkier b[a]a2G and b[a]a6A exhibit significantly greater mutagenicity in human cells than in E. coli.
