title: Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to N-ethyl-N-nitrosourea.
abstract: We have determined the mutational spectrum of N-ethyl-N-nitrosourea (ENU) in exon 3 of the hypoxanthine (guanine) phosphoribosyltransferase gene (Hprt) in splenic T cells following in vivo exposure of male B6C3F1 mice (5-7 weeks old) to ENU. Hprt- mutants were isolated by culturing splenic T cells in microtiter dishes containing medium supplemented with interleukin 2, concanavalin A, and 6-thioguanine. DNA was extracted from 6-thioguanine-resistant colonies and amplified by the polymerase chain reaction (PCR) using primers flanking Hprt exon 3. Identification of mutant sequences and purification of mutant DNA from contaminating wild-type Hprt DNA was accomplished by denaturing-gradient gel electrophoresis. Purified mutant DNA was then sequenced. Treatment of mice with ENU at 40 mg/kg of body weight produced a Hprt- mutant frequency of 7.3 x 10(-5) in splenic T cells, approximately 35-fold above background levels. Sixty-nine of the 521 Hprt- mutants analyzed contained mutations in exon 3 (13%). Transversions and transitions at A.T base pairs dominated the spectrum; 62 of the 69 exon 3 mutations were at A.T base pairs (14 different sites). Thirteen of 14 thymine bases undergoing mutation (61 of 62 mutations at A.T bases) were located on the nontranscribed strand of exon 3. The majority of the remaining mutations (6 of 69) were transitions at a single G.C base pair. These results suggest the importance of thymidine alkylation in ENU-induced mutagenesis in vivo. The mouse Hprt- T-cell cloning/sequencing assay described here may represent a useful system for studying the molecular mechanism of chemically induced mutation occurring in vivo in an endogenous gene.
authors.affiliation: Department of Pathology, University of North Carolina, Chapel Hill 27599.
authors.forename: T R
authors.initials: TR
authors.lastname: Skopek
authors.forename: V E
authors.initials: VE
authors.lastname: Walker
authors.forename: J E
authors.initials: JE
authors.lastname: Cochrane
authors.forename: T R
authors.initials: TR
authors.lastname: Craft
authors.forename: N F
authors.initials: NF
authors.lastname: Cariello
mesh_terms: D000818:Animals; D001483:Base Sequence; D004249:DNA Damage; D004252:DNA Mutational Analysis; D005038:Ethylnitrosourea / Q000633:toxicity*; D005091:Exons; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000187:drug effects
chemical_list: D009838:Oligodeoxyribonucleotides; D007041:Hypoxanthine Phosphoribosyltransferase; D005038:Ethylnitrosourea