Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6667-6671, September 1986 Applied Biology Genetic-molecular basis for a simple Drosophila melanogaster somatic system that detects environmental mutagens (mutagen testing/somatic mutation/deletion induction) M. M. GREEN*+, TAKESHI TODO*, HARUKO RYo*, AND KAZUO FUJIKAWAt *Department of Fundamental Radiology, Faculty of Medicine, Osaka University, Kita-ku, Osaka 530 Japan; tDepartment of Genetics, University of California, Davis, CA 95616; and tCentral Research Division, Takeda Chemical Industries, Ltd., Osaka 532 Japan Contributed by M. M. Green, May 27, 1986 ABSTRACT We have developed a simple, objectively irradiation (5-7). In the case of somatic reversions, these are scorable test for the mutagenicity of chemical compounds readily scored as spots or clones of red, probably w', eye which can be fed Drosophila melanogaster. The test depends facets on a background of ivory, wi, facets. The frequency of upon the somatic reversion of the X chromosome, recessive eye both somatic and germinal reversions increases after x- color mutation, white-ivory (w') to wild type (w'). Reversions irradiation of larvae or adults, respectively. Second, the are scored as clones of w+ facets in the w'eyes ofeclosing adults. nature of the wi mutant and its germinal w+ reversions have To increase the sensitivity, a tandem quadruplication contain- been defined molecularly: wi is a 2.9-kilobase (kb) tandem ing four w' mutations was synthesized. Thus, in homozygous duplication of w+ DNA appended directly onto a normal w+ females eight w' mutations are potentially revertible. Six gene (8). Reversion of wi to w+, spontaneous and x-ray-in- mutagenic compounds, all alkylating agents, all gave positive duced, is associated with the more or less clean loss of the results at several concentrations tested. Molecular analysis appended 2.9 kb, thereby restoring an intact w+ gene. Thus, demonstrates that the induced reversions, germinal and so- any agent, physical or chemical, that increases the reversion matic, are associated with the loss of 2.9-kilobase DNA dupli- frequency of wi to w+ does so presumably by deleting the cated in the w' mutation. appended 2.9 kb and is, therefore, a deletion-making agent. Although in homozygous females and males the somatic The need for a simple, utilitarian genetic test for environ- and germinal reversion frequency of wi, spontaneous or mental mutagenic compounds was dramatically demonstrat- x-ray-induced, is high compared to the vast majority of ed by the finding of McCann et al. (1), employing the Ames spontaneous D. melanogaster mutations, it is too low to be (2) Salmonella test, that most carcinogenic compounds are used as a basis for a routine test for mutagenic compounds. also mutagenic. The elegance and utility ofthe Ames test and If, however, the number of copies of the wi mutation in the its more recent improvement (3) cannot be denied. Nonethe- fly's genome can be increased, it follows that the number of less, there are cogent reasons for supplementing or comple- "targets" for reversion to w+ also increases. An increased menting the Ames test with a comparable eukaryote test "target" number translates into an increased likelihood of system. There are fundamental differences in how the reversion and concomitantly into an increased sensitivity to genomic DNA of prokaryotes and eukaryotes is organized, environmental mutagens capable of generating DNA dele- which could conceivably influence the outcome of the Ames tions. In the narrative that follows we will document the test. Similarly, there are real differences in prokaryote and synthesis of a tandem quadruplication of the w' mutation. We eukaryote metabolism that do affect the outcome ofthe Ames will demonstrate that when chemical mutagens are fed to test-e.g., compounds that in eukaryotes are metabolically quadruplication larvae the frequency of somatic reversions of converted to mutagens are not so converted in prokaryotes. w' to w+ is increased significantly. Finally, we will establish On hand are a number of genetic and cytological tests for at the molecular level that chemical-mutagen-induced rever- mutagens that employ intact eukaryote organisms or cultured sions of wi result from the deletion of the appended 2.9 kb. cells. There is no need to review these test systems except to note that each suffers from one or another limitation. By and MATERIALS AND METHODS large, most tests are not simple to execute or the nature ofthe genetic damage is unclear. Historically, the eukaryote orga- Synthesis of a Tandem Quadruplication of w' [Dp(1:1:1:1)- nism and mutagen test of choice have been Drosophila w'. The wi mutation is X chromosome linked, mapping to melanogaster and X chromosome lethal mutations-the CIB position 1.5. The synthesis of the quadruplication was envi- test and later improvements. Yet, even this comparatively sioned to be a two-step process. In the first step a tandem simple test is wanting, because when the results are positive duplication of wi [Dp(1:J)w'] would be generated by x- the precise nature of the induced lethal mutations is unclear. irradiating homozygous wi females. Subsequently, unequal In fact, a review of the pertinent D. melanogaster mutagen crossing-over in homozygous Dp(J:J)wi females would be test literature (4) demonstrates that in general such tests, exploited to generate a tandem wi triplication [Dp(J:1:J)w'] whether somatic or germinal, are ambiguous when the issue and in homozygous triplication females to generate a tandem of the nature of the mutagen-induced genetic lesion is raised. wi quadruplication [Dp(J :1:1 :J)w']. The synthesis of We, therefore, undertook the development of a D. Dp(J:I)w' took advantage ofthe fact that tandem duplications melanogaster somatic mutagen test based on the reversion of of the w locus can be objectively identified phenotypically the X-linked eye color mutation white-ivory (wi) to wild type (9). Thus, females possessing one wild-type X chromosome (w+). Our choice of wi stems from two observations. First, it and one bearing a tandem duplication of the X chromosome has been known for more than 25 years that wi reverts to w+ recessive eye color mutation zeste [Dp(J :I)z] are wild type in both somatically and germinally, spontaneously and after x eye color, but females possessing one X chromosome bearing The publication costs of this article were defrayed in part by page charge Abbreviations: kb, kilobase(s); EMS, ethyl methanesulfonate; ENU, payment. This article must therefore be hereby marked "advertisement" ethyl nitrosourea; DEB, diepoxybutane; TEM, triethylene mela- in accordance with 18 U.S.C. §1734 solely to indicate this fact. mine. 6667 Downloaded by guest on September 25, 2021 6668 Applied Biology: Green et al. Proc. Natl. Acad. Sci. USA 83 (1986) a tandem duplication of the w+ locus and the homologous X medium in either 190-ml milk bottles or 30-ml vials. Females bearing Dp(1]:)z exhibit a variegated zeste eye color pheno- were removed and eggs were allowed to hatch during the type. ensuing 24 hr. An aqueous solution of a chemical mutagen in In practice, homozygous y2w1 females (y2 = yellow-2 body appropriate concentration was then pipetted onto the surface color at position 0.0 on the X chromosome) were irradiated of the medium, 1 ml to the bottles, 0.2 ml to the vials. with -4000 rads (1 rad = 0.01 gray) of x rays and crossed to Development proceeded at 250C to eclosion of adults, which Dp(J:1)z males. In each of two independent experiments a were then scored. single female was found of the zeste-variegated eye color. Where chemical-mutagen-induced germinal reversions Retesting the presumptive Dp(1 :1)w1 chromosomes with were sought, males that had been fed the mutagen as larvae Dp(J :J)z confirmed that each generated the zeste-variegated were crossed either to females homozygous for a small white eye color. In addition, the eye color of Dp(1 :J)w1 males is locus deficiency (w-) or to double X females [C(1)DX] distinctly darker than that of wi males, and similarly the eye homozygous for the recessive mutants yellow body (y) and color of homozygous Dp(J:1)w' females is distinctly darker forked bristles (f). In the first cross the female progeny were than that of homozygous wi females. These eye color phe- scored for reversions; in the second cross the male progeny notypes are consistent with increased dosage of the wi were scored. mutation. Finally, salivary gland polytene chromosome cy- Nucleic Acid Procedures. We analyzed the molecular status tology confirmed each Dp(1 :J)w' to be a tandem duplication of germinal reversions of wi to w+ induced by chemical involving a short interval of the X chromosome extending mutagens as follows. High molecular weight DNA was from section 3A to section 3C, thereby including wi. prepared from adult flies as described in ref. 10. One- To synthesize wi triplications, homozygous y2 Dp(J:1)wi microgram samples of each DNA were digested with appro- females were crossed to Dp(1 :J)z males and female progeny priate amounts of restriction enzymes under their optimal near zeste in phenotype were sought. Three independent conditions. The digests were subjected to electrophoresis in Dp(1:1:I)wi chromosomes were recovered. As expected, the Sigma type I agarose gels and then transferred to nitrocellu- eye color ofDp(J :1 :J)wi males is darker than that ofDp(J .:)w1 lose filters as described in ref. 11. Hybridization was carried males and the eye color of homozygous Dp(1:J :1)w1 females out with nick-translated (12) gel-purified fragments derived is darker than that of homozygous Dp(J:J)w1 females.