A mutation in the beta-globin gene detected in the progeny of a female mouse treated with ethylnitrosourea.

411

182

Transgenic nmice with a A shuttle vector containing a lacI target gene were generated for use as a shortterm, in vivo mutagenesis assay. The gene is recovered from the treated mice by exposing mouse genomic DNA to in vitro packaging extracts and plating the rescued phage on agar plates containing 5-bromo-4-chloro-3-indolyl (3-D-galactopyranoside (X-Gal). Phage with mutations in the lacl gene form blue plaques, whereas phage with a nonmutated lacl form colorless plaques. Spontaneous background mutant rates using this system range from 0.6 x 10-S to 1.7 x i0-0, depending upon tissue analyzed, with germ cells exhibiting less than one-third the background rate of somatic tissue. Treatment of the miceor cyclophosphamide caused an induction of mutations over background. Recovery of the lacI target for sequence analysis was performed by genetic excision of a plasmid from the phage using partial filamentous phage origins. The predominant mutations identified from untreated and treated populations were base substitutions. Although it has been shown by others that 70% of all spontaneous mutations within the laI gene, when replicated in Escherichia cohi, occur at a hot spot located at bases 620-632, only 1 of 21 spontaneous mutations has been identified in this region in the transgenic mouse system. In addition, 5 of 9 spontaneous transitions analyzed occur at CpG dinucleotides, whereas no transition mutations were identified at the prokaryotic deamination hot spots occurring at dcm sites (CCA/TGG) within the WaI gene. For EtNU, approximately equal amounts of transitions and transversions were observed, contrasting with B[a]P-induced mutations, in which only transversions were obtained. In addition, B[a]P mutagenesis showed a predominance ofmutations (81 %) involving cytosines and/or guanines, consistent with its known mode of action. The discovery of a spontaneous mutation spectrum different from that of bacterial assays, coupled with the concordance of EtNU and B[a]P base mutations with the known mechanisms of activity for these mutagens, suggests that this transgenic system will be useful as a short-term, in vivo system for mutagen assessment and analysis of mechanisms leading to mutations.The lacd gene has been used extensively as a target for the identification and analysis of spontaneous and induced mutations, due in part to the ease of using a calorimetric assay to rapidly screen for mutations. Genetic and sequence analysis of spontaneous and induced mutations detected in several systems utilizing lacd (1-5) has resulted in an extensive accumulation of data regarding the sequence specificity of spontaneous and induced mutations in lacd, which can be of considerable comparative value through the use of the lacd target gene in whole animal assay systems.To combine the cost-saving aspects of short-term assays with the predictive power of whole animal assays, we previously described the development of a system that depends upon efficient recovery of a A phage shuttle vector from transgenic mouse genomic DNA thro...

Section: Resultsmentioning

confidence: 99%

Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice.

Kohler¹,

Provost²,

Fieck³

et al. 1991

411

182

“…The resulting products are not efficiently repaired (28,29) by the suicidal methyltransferase (30) that reverts 06-alkylguanine residues to guanine. Thymine O-alkylation products can lead to transition mutations (T -* C) in vitro (31), and T -3 A transversions have been identified among mutations produced in mice by exposure to EtNU in vivo (32). It is thus possible that the initial event in chemical carcinogenesis by EtNU in the peripheral nervous system of the rat is mutation of the neu protooncogene by O-alkylation of a specific thymine residue.…”

Section: Discussionmentioning

confidence: 99%

Activated neu oncogene sequences in primary tumors of the peripheral nervous system induced in rats by transplacental exposure to ethylnitrosourea.

Perantoni¹,

Rice²,

Reed³

et al. 1987

Neurogenic tumors were selectively induced in high incidence in F344 rats by a single transplacental exposure to the direct-acting alkylating agent N-ethyl-N-nitrosourea (EtNU). We prepared DNA for transfection of NIH 3T3 cells from primary glial tumors of the brain and from schwannomas of the cranial and spinal nerves that developed in the transplacentally exposed offspring between 20 and 40 weeks after birth. DNA preparations from 6 of 13 schwannomas, but not from normal liver, kidney, or intestine of tumor-bearing rats, transformed NIH 3T3 cells. NIH 3T3 clones transformed by schwannoma DNA contained rat repetitive DNA sequences, and all isolates contained rat neu oncogene sequences. One schwannoma yielded a transformant with rat-specific sequences for both neu and N-ras. A point mutation in the transmembrane region of the putative protein product of neu was identified in all six transformants and in the primary tumors from which they were derived as well as in 5 of 6 schwannomas tested that did not transform NIH 3T3 cells. Of 59 gliomas, only one yielded transforming DNA, and an activated N-ras oncogene was identified. The normal cellular neu sequence for the transmembrane region, but not the mutated sequence, was identified in DNA from all 11 gliomas surveyed by oligonucleotide hybridization. Activation of the neu oncogene, originally identified [Schechter, A.L., Stern, D.F., Vaidyanathan, L., Decker, S.J., Drebin, J.A., Greene, M.I. & Weinberg, R.A. (1984) Nature (London) 312, 513-516] in cultured cell lines derived from EtNU-induced neurogenic tumors that by biochemical but not histologic criteria were thought to originate in the central nervous system in BD-IX rats, appears specifically associated with tumors of the peripheral nervous system in the F344 inbred strain.

“…The mutation spectra seen are consistent with proposed mechanisms of ENU mutagenesis and correlate with the in viva spectra seen in ENU studies by using transmissibiliy assays and the hpilgene. These findings represent significant progress toward dening the in vivo spectra of Ethylnitrosourea (ENU) induces a broad spectrum of promutagenic lesions in vivo (6, 7) and has been studied by using a variety of transmissibility assays (8)(9)(10)(11)(12)(13)(14)(15). Although ENU has been well characterized as a mouse germ cell mutagen, the spectrum of ENU-induced mutations in mouse germ cells remains largely undefined.…”

mentioning

confidence: 99%

Characterization of mutations induced by ethylnitrosourea in seminiferous tubule germ cells of transgenic B6C3F1 mice.

Provost¹,

Short²

1994

Transgenic B6C3F1 mice carrying a lacl target gene were exposed to acute and multiple doses of ethylnitrosourea (ENU), and germ cells from the seminiferous tubules were assayed for mutation 3 and 90 days after treatment. Relative to untreated controls, the mutation frequency increased 3.2-and 19.9-fold at 3 and 90 days after treatment, respectively. Mutant lacl genes recovered from untreated and treated groups were sequenced, and the spectra of mutations were determined. Eighty-flve percent (11/13) of the spontaneous mutatious resulted in G-C -* AT transitions, all ofwhich occurred at CpG dinucleotides. Fifteen of 22 sites (68%) found mutated 3 days after ENU treatment occurred at G-C base pairs, although some of these are expected to be spontaneous mutations. Ninety days after treatment, 13 of 19 sites (68%) found mutated occurred at APT base pairs. The mutation spectra seen are consistent with proposed mechanisms of ENU mutagenesis and correlate with the in viva spectra seen in ENU studies by using transmissibiliy assays and the hpilgene. These findings represent significant progress toward dening the in vivo spectra of Ethylnitrosourea (ENU) induces a broad spectrum of promutagenic lesions in vivo (6, 7) and has been studied by using a variety of transmissibility assays (8)(9)(10)(11)(12)(13)(14)(15). Although ENU has been well characterized as a mouse germ cell mutagen, the spectrum of ENU-induced mutations in mouse germ cells remains largely undefined. To date, only six germ-line mutations identified in ENU-treated mice offspring have been characterized at the nucleotide level and reported, all of which occur at either adenine or thymine residues (10-13, 16). The experiments described here characterize the mutagenic response oftransgenic mouse testicular germ cells to acute-and multiple-dose ENU exposure and outline the spectra of mutants recovered after these exposures. These findings represent progress toward defining the in vivo spectrum of ENU mutagenesis in germ cells and explore the relationship to the in vivo somatic cell spectrum in the endogenous hprt gene. MATERIALS AND METHODSTransgenic Mice. Transgenic B6C3F1 hybrid mice were generated from natural matings (performed at Taconic Farms) of inbred female transgenic hemizygous A/lacd C57BL/6 mice (2) and male nontransgenic C3H mice. DNA was prepared from tail tips of the resulting offspring and screened for the transgene by dot-blot DNA hybridization, as described (2). Animals were maintained on a 12-hr light schedule in cages containing heat-sterilized wood chips and were provided with F4 rodent block food (Harlan Bioproducts, Haslett, MI) and tap water ad libitum.Mutagen Treatment. ENU (Sigma, Lot no. 20H0369) was dissolved in Dulbecco's phosphate-buffered saline (pH 6.0) (GIBCO) and administered within 30 min of solution preparation. Four 8-to 10-week-old male transgenic B6C3F1 mice (Big Blue mouse, Stratagene) were given three i.p. injections of 100 mg (± 2%) of ENU per kg of body weight at 7-day intervals for a cumulative multiple dos...