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Analysis of in vivo mutation induced by N ‐ethyl‐ N ‐nitrosourea in the hprt gene of rat lymphocytes
Author(s) -
Mittelstaedt Roberta A.,
Smith Beverly A.,
Heflich Robert H.
Publication year - 1995
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.2850260402
Subject(s) - heteroduplex , microbiology and biotechnology , exon , biology , hypoxanthine guanine phosphoribosyltransferase , mutation , mutant , in vivo , gene , genetics , nitrosourea , dna , mutagen , ethyl methanesulfonate , chemotherapy
The rat lymphocyte hprt assay measures in vivo mutagenicity by quantifying the frequency of 6‐thioguanine‐resistant (TG') spleen lymphocytes cultured in vitro. In this study we have examined the types of mutations induced in the hprt gene of TG' lymphocyte clones from female Fischer 344 rats exposed to 100 mg/kg N ‐ethyl‐ N ‐nitrosourea (ENU). Hprt exons 3 and 8 were amplified from DNA extracted from each of 249 clones, and the resulting products were screened for mutant:wild‐type heteroduplex formation by denaturing gradient gel electrophoresis. The analysis revealed 59 clones with mutations in exon 3, and 20 clones with mutations in exon 8. DNA sequence analysis of the heteroduplexes identified 84 mutations: all of the mutations were base pair substitutions, and 88% were mutations of A:T base pairs. At least 82% were induced independently. These results suggest that the mutations found in TG' rat lymphocytes from ENU‐treated rats were due mainly to ethylthymidine adducts. In addition, a comparison of these results with previously reported in vivo ENU mutational profiles indicates that the types of mutation detected by heteroduplex screening of rat hprt exons 3 and 8 are representative of mutation in the entire protein coding sequence. © 1995 Wiley‐Liss, Inc. This article is a US Government work and, as such, i s in the public domain in the United States of America.