Copyright 0 1989 by the Genetics Society of America Sequence Analysis of N-Ethyl-N-Nitrosourea-Inducedvermilion Mutations in Drosophila melanogaster Albert Pastink,*.+ Cees Vreeken,*’+ MadeleineJ. M. Nivard,* Lillie L. Searles* and Ekkehart W. Vogel* *Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands, 7.A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection, The Netherlands, and $Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27514 Manuscript received February 2 1, 1989 Accepted for publicationMay 8, 1989 ABSTRACT The mutational specificity of N-ethyl-N-nitrosourea (ENU) was determined in Drosophila melano- gaster using the vermilion locus as a target gene. 25 mutants (16F1 and 9 F2 mutants) were cloned and sequenced. Only base-pair changes were observed; three of the mutants represented double base substitutions. Transition mutations were the most prominent sequence change:61% were GC+AT and 18%AT4C substitutions. Both sequence changes can be explainedby the miscoding properties of the modified guanine and thymine bases.A strong bias of neighboring bases on the occurrence of the GC+AT transitions or a strand preferenceof both types of transition mutationswas not observed. The spectrum of ENU mutations in D. melanogaster includes a significant fraction(2 1 %) of transversion mutations. Our dataindicate that like in other prokaryoticand eukaryotic systems also in I). melanogaster the0‘“ethylguanine adduct is the most prominent premutational lesion after ENU treatment. The strong contribution of the 06-ethylguanine adduct to the mutagenicity of ENU possibly explains the absenceof distinct differences between the typeof mutations observedin the F1 and F:! mutants. Althoughthe latter arise laterduring development, the spectrumof mosaic mutations is also dominated by GC+AT transition mutations. OST alkylating agents (AAs) are known to in- and OSGOOD1986; BATZERet al. 1988; PASTINK, M duce mutations, chromosomal aberrations and VREEKENand VOGEL 1988). The objective ofthis tumors in a variety of prokaryoticand eukaryotic study has been to characterize by sequence analysis organisms. Treatment ofDNA in vivo or in vitro with alkylation-induced mutations and togain information these agents resultsin a diverseset of adducts (SINGER on the contribution of specific adducts to the type of and GRUNBERCER 1983).The relative distribution of mutations finally recovered. these adductsin the DNA is dependent on thenucleo- Against this background, we selected a small group philic selectivity of the compound used and has an of mono-functional alkylating carcinogens, differing effect on the type of mutations recovered.Previously, in their Swain-Scott constant’s(SWAIN and SCOTT it was shown that thereis a strong correlation between 1953), for sequenceanalysis of alkylation-induced mu- the relative extent of alkylation at the base oxygens tations. This paper reports the analysis of ENU-in- and the inductionof mutations (measuredas recessive duced mutations atthe vermilion locus. ENU is a lethal mutations). With compounds acting more ex- potent carcinogen and has the ability to ethylate var- tensively at the ring-nitrogen atoms in the DNA, a ious sites in the DNA. The major alkylation sites are relatively high chromosome breakage effect was ob- the O6 and N-7 positions of guanine, the O2 positions served in addition to mutations (VOCELand NATA- of cytosine and thymine, the N-3 position of adenine RAJAN 1979, 1982; NATARAJANet al. 1984). Analysis and thephosphodiester groups in the DNA backbone. of ethyl methanesulfonate (EMS)- and N-ethyl-N-ni- One of the minor alkylation sites is the O4 position of trosourea (ENU)-induced mutations at the adh, white, thymine(BERANEK, WEIS and SWENSON1980, and RpII.215 and ry loci in Drosophila melanogaster by blot- referencescited therein). ENU represents the first hybridization, indicated that most of the mutations chemical carcinogen of a whole series of monofunc- induced by thesemonofunctional ethylating agents tional AAs whose mutational spectra are analyzed by are caused by base-pair changes or deletions smaller DNA sequencecharacterization in our laboratory. than 50-1 00 bp (COT&et al. 1986; LACY,EISENBERG Comparative studies of this type may eventually con- tribute to a better understanding of the sequence of The publication costs of this article were partly defrayed by the payment of page charges. This articlemust therefore be hereby marked“advertisement” events leading from the initial DNA adduct to the in accordance with 18 U.S.C. $1734 solely to indicate this fact. final genetic alteration. Genetics 123: 123-129 (September, 1989) 124 A. Pastink et al. The wild-type eye color of D. melanogaster is a vermilion phenotype to the next generation,strains 40 homo- composite of red and brown pigments. Mutations at zygous for vermilion could be built up (Table 1). In addition one mutant (#156), which was female sterile, was kept over the vermilion locus result in a bright red eye color. a C(1)DX chromosome. The vermilion locus has been studied extensively at DNA isolation: About 0.5-1 g flies were frozen in liquid the genetic and biochemical level (LINDSLEYand nitrogen and grounded toa fine powder in a mortar in the GRELL 1968; O'BRIEN and MACINTYRE 1978). This presence of liquid nitrogen. The powder was resuspended gene codes forthe enzyme tryptophan oxygenase in 15 ml of ice-cold homogenization buffer (0.1 M NaCI; 30 mM Tris-HCI (pH 8.0); 10 mM EDTA; 10 mM P-mercapto- (BAGLIONI1960; BAILLIEand CHOVNICK197 l),which ethanol; 15 mM spermine; 15 mM spermidine; 0.5% Triton catalyzes the first step in the synthesis of the brown X-100) and homogenized with a glass pestle. The homoge- eye pigment of the fly. Because tryptophan oxygenase nate was centrifuged for 10min at 4000 X g and the pellet is not cell autonomous, i,e.,diffuses through thewhole resuspended in 15 ml extraction buffer (0.1 M NaCI; 0.1 M body, most mosaic mutations will result in a wild-type Tris-HCI (pH 8.4); 20 mM EDTA) and centrifuged again. The pellet was resuspended in 10 ml extractionbuffer, eye color. The gene has been cloned and the nucleo- proteinase K and SDS were added tofinal concentrations of tide sequence is also known (SEARLESand VOELKER 100 Pg/ml and 1%, respectively, and incubated at 50" for 1986; WALKER, HOWELLS andTEARLE 1986; L. L. 1 hr. The mixture was cooled to room temperature and0.2 SEARLES,unpublished results). The vermilion gene has volume of 8 M potassium acetate was added. After 30 min a size of about 2 kb and codes for a mRNA of 1.3 kb on ice the DNA solution was extracted twice with chloro- form. Nucleic acidswere precipitated with 1volume of (see Figure 1 for further details). Due to its small size ethanol and dissolved in 10 mM Tris-HCI (pH 7.5); 1 mM the vermilion locus is very suitable for the sequence EDTA (TEbuffer). The DNA samples were treated succes- analysis of mutations due to base-pair changes and sively with pancreatic RNase (20 rg/ml) and proteinase K small deletions. A potential disadvantagemay be seen (50 rg/ml) and further purified by fenol-chloroform-iso- in the fact that many large multilocus deletions are amylalcohol and chloroform-isoamylalcohol extractions. After ethanol precipitation the DNA was dissolved in TE not recovered due to the proximity of a haplo-insuf- buffer. ficient female fertility gene to the rightof the vermilion Cloning of vermilion alleles: The rapid cloning and gene (LEFEVRE 1967, 1969). sequencing procedure used, is based on the recombination The mutational specificity of ENU has been deter- screening method, originally developed byB. SEED(SEED mined in Escherichia coli and in human cells using 1983; LUTZet al. 1987). Fly DNA was digested with HindIII and XbaI, producing a 5.2-kb HindIII fragment containing episomal genes as targets (RICHARDSONet al. 1987; the vermilion gene (Figure 1). Digestion with XbaI, which ECKERTet al. 1988).Only base-pair changes were does not cut inside the 5.2-kb fragment, was included to observed, the majority being transitionmutations. enhance the enrichment after fractionation on a 0.8% aga- Our results obtained by germ cell analysis using an rose gel. DNA fragments ranging in size between 4.4 kb endogenous chromosomal locus as a target gene, are and 6.7 kb were eluted from thegel and ligated into PDJl 1, an A""B"" lambda vector (P. DE JONG,unpublished results). in good agreement with the other studies. About 100 ng fly DNA and 800 ng PDJl 1 DNA, digested with HindIII and treated with calf intestine phosphatase, MATERIALSAND METHODS were ligated in a volume of 10 rl. After packaging in vitro theprimary library was amplified by plate lysis. About Bacterial strains and media: All strains are E. coli K-12 15,000-20,000recombinant phages were used to infect derivatives.XS127 isA(1ac-ProXZZZ), gyrA, thi, rpoB argE, XS127 harboring the probe plasmid pMv2. In addition to [F', laciqZAh415,proAB, traD361, [p3, kan, ampam, tet."], vermilion sequences a supF gene is located on this plasmid, LG75 issup', lacZ"" (LEVINSON,SILVER and SEED 1984; allowing the suppression of the amber mutations of the SHENand HUANC 1986).LC medium contains per liter 10 lambdavector (Figure 2). Plating cells were grown in g bactotryptone, 5g yeast extractand 8 g NaCI. The NZCYM medium containing 0.2% maltose, ampicillin and constitutionof NZCYM medium has beendescribed by tetracycline. After infection, the cells were plated on 10-cm MANIATIS, FRITSCHand SAMBROOK(1 982). NZCYM plates in soft agar containing ampicillin. Basic Isolation of mutants: Forthe inductionof vermilion phage techniques were performed as described by MANIA- mutants, bw males (derived from wild-type Berlin K) were TIS, FRITSCHand SAMBROOK(1 982). placed in glass vials containing 8 layers of glass microfiber Homologous recombination between phages containing paper (WhatmannGF/A) soaked with 0.9 ml of a 1mM ENU the 5.2-kb vermilion fragment results in the incorporation solution in 33 mM sodium phosphate (pH 6.0); 5% sucrose.