Carcinogens induce reversion of the mouse pink-eyed unstable mutation

Abstract

Deletions and other genome rearrangements are associated with carcinogenesis and inheritable diseases. The pink-eyed unstable (pun) mutation in the mouse is caused by duplication of a 70-kb internal fragment of the p gene. Spontaneous reversion events in homozygous pun/pun mice occur through deletion of a duplicated sequence. Reversion events in premelanocytes in the mouse embryo detected as black spots on the gray fur of the offspring were inducible by the carcinogen x-rays, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, benzo[a]pyrene, trichloroethylene, benzene, and sodium arsenate. The latter three carcinogens are not detectable with several in vitro or in vivo mutagenesis assays. We studied the molecular mechanism of the carcinogen-induced reversion events by cDNA analysis using reverse transcriptase-PCR method and identified the induced reversion events as deletions. DNA deletion assays may be sensitive indicators for carcinogen exposure.

Figure 1

p^un structure and possible mechanisms of intrachromosomal recombination resulting in deletions (according to refs. , , and 20). In the center the p^un structure is shown with exons 6–18 duplicated (21). (A) Intrachromatid crossing-over occurs after pairing of the two copies of the p^un duplication in a looped configuration (11). Crossing over results in deletion of one of the two copies giving rise to reversion to p⁺. (B) Single-strand annealing is initiated by a double-strand break between the duplicated exons (20, 22); DNA ends are degraded by a 5′–3′ single-strand specific exonuclease to expose the flanking homologous sequences. Annealing of the complementary single strands occurs, and the nonhomologous ends are removed followed by DNA synthesis and ligation. (C) Unequal sister chromatid exchange occurs as crossing over between one copy of the exon duplication on one sister chromatid and the other copy of the exon duplication on the other sister chromatid. Reciprocal products are the deletion of one copy of the exon 6–18 duplication resulting in reversion to p⁺ and the triplication of exons 6–18. (D) Sister chromatid conversion events can occur after unequal pairing of the homologous portions of both copies of the exon duplication on one sister chromatid with either one of the two copies on the sister chromatid having the duplicated sequence in a looped-out configuration (11). Double crossover or gene conversion may lead to a conversion event during which one of the two copies of exons 6–18 is lost. The other sister chromatid maintains its original configuration. This event may also be initiated by a double-strand break between the duplicated copies on one sister chromatid degradation with a single-strand exonuclease until to the region of homology after which invasion, D-loop formation, and repair synthesis might happen from the sister chromatid (11).

Figure 2

Detection of wt p mRNA in black spots on the gray coat of p^un/un mice. Black spots and similar gray areas were excised from 3- to 5-day-old mice delivered by treated and control dams. (A) The RT-PCR analysis was performed using primers spanning duplicated regions. The samples were run in 1% agarose gels and stained with ethidium bromide. (B) Photographs of stained gels were analyzed by scanning densitometry with a BioImage (Millipore). The relative intensity of the wt p mRNA was evaluated as the ratio of bands’ intensities corresponding to p and p^un. Lanes: M, 1kb DNA ladder; 1, wt black p/p mouse; 2, gray p^un/un mouse; 3 and 4, gray skin from x-ray-treated p^un/un mice; 5–7, black spots from x-ray-treated p^un/un mice; 8–10, black spots from EMS-treated p^un/un mice; 11 and 12, black spots from SOA-treated p^un/un mice; 13 and 14, black spots from BEN-treated p^un/un mice; 15, control RT-PCR without RNA.