S58 S-VIII: DNA recombination, transposition, amplification

Ip VIJI.91 Non-lnduced excision orthe lransposable element B104aner chemlcallreatmentln while Inserllonal mutants ofdrosophila rnelanogaster

Silvia Soriano, Antonia Velazquez, Ricardo Marcos, Oriol Cabri, NoelXamena. Grup de Muwgenesl, Departament de Gene tica I de Mlcrobinlogla,Unioersita: AutaflOma de Barcelona, Bellaterra; Spain

Several environmental agents have been described to induce genetic transpo­sition. In particular, we have previously shawn that alkylating agents inducesomatic revertants in three insertional mutants (wa4, wbf, wsp55), but notin the point mutant wa2.

The aim of this work was to obtain and characterize at a molecular level,chemically induced germ line mutants for the mutants wbf. wh and wspl ,carrying the transposable element B104 at different positions.

Red-eye revertants were obtained from wbf and wspl while only white­eyed revertants were obtained from who This agrees with the previouslydescribed lack of excision of the Doc element from the mutant white-one(wI), which is the original strain of who

The molecular analysis by Southern blot, of all the obtained revertants,indicates that they are not due to the excision of the BI04 element sinceno molecular differences seem apparent between original mutants and theirrevertants.

Finally, biochemical studies reveal that red-eyed revertants contain morered pigment than the original mutant, but less than a wild type strain, Asconsequence, they have been classified as partial revertants.

Recombination studies have shown the existence of modifier genes actingon the white gene, which are considered to be responsible of the partiallyrevertant phenotypes.

Keyword(s): Drosophila; melanogaster; Transposable clements; Alkylating

agents

Ip VIII.t 01 Aotlmetabolltes of DNA synlhesls are mutagenle aodrecomblnageolc as shown by the wing spot lest ofdrosophila

Hansjorg Frei, Friedrich E. Wiirgler. lnstitute of TOJ:it:n!ogy. ETH andUniversity ofZ",lch, CH·8603 Schwen:enbach, Sw itzerland

The wing spot lest of Drosophila is an In vivo Somatic Mutation and Recom­bination Test (SMART) and reveals induced loss of heterozygosity due tomitotic recombination, somatic mutation and deletion. Larvae heterozygousfor the recessive wing cell marker mutations multiple wing hairs (mwh) andflare (fir) arc exposed to the test compounds. Following metamorphosis,the wings of the nics are screened for the presence of mutant spots (mwhandlor fir) . More than 300 chemicals have been tested for genotoxicity in thistest. Of particular interest are compounds not covalently binding to DNA.Antimetabolites affecting the balance of the nucleotide pool are genotoxic,Hydroxyurea is clearly genotoxic and recombinagenic, but at exposure levelsfour orders of magnitude higher than the effective levels of the stronglygenotoxie ammopterin. The latter agent and triffuorothymidine produce wingspots mainly but not exclusively by mitotic crossing-over (-90-95%). With 5­azauraeil the contnbution of mechanisms other than recombination is morepronounced (-50%); in the SMART, this base analogue tS about twice aseffective than the nucleoside 5-azauridine. The antiviral guanosine analogueacyclovir (-70% recombination) is a rather potent genotoxin in contrast tothe structurally related ganciclovir which significantly induces wing spots atsubtoxic exposure levels, but at low frequencies.

Keyword(s): DNA·antimetabolites; Drosophila; Wing spot test (SMART)

Ip VIlLI t I H1l:b recomblnogealclty of aftatoxln III yeast may In­dicate a mechanism for &enetJe changes 10 aftaloxln­Induced hepatocartlnomas

Christian Sengstag l , Bea Weibel', Michael Fasulloz.JInst itute ojTOJ:icology,

Swiss Federal Institute a/Technology, CH-8603 Schwerunluu:h. Switzerland;

2Department of Biochemistry and Moteculor Biology, The Albany MediC41College. Albany. NY. USA

Anatoxin BI (AFBI) has previously been recognized as an extremely potentrodent and human carcinogen. Upon appropriate metabolism in a cytochrorneP45O-dependent reaction, the resulting epoxide binds to DNA and inducespoint mutations. In addition to that we have recently identified anotheractivity of AFBI in the lower eukaryote Saccharomyces cereuisiae: Whenintracellularly metabolized by human CYPIA enzymes. AFBI stronglyinduced mitotic recombination which was detected by a marked increasein gene conversion (Sengstag et al (1994) Mol. Carcinog. II : 227-235) andchromosomal translocation (Sengstag et al (1996) Cancer Res. 56: 5457­5465). By contrast, only a moderate mutagenic activity of metabolized AFBIwasdetected when forward mutanons were selected in the URA3 gene. Thus,in the lower ouJwyotic yeast S. cerevisiae, metabolized AFBI acts as a strongrecombinogen, but only as a weak mutagen.

Mitotic recombination is one possible mechanism for loss of heterozy_gosity (LOH), a phenomenon ollen observed during carcinogenesis. Inter­estingly, hepatocellular carcinomas isolated from patients living in AFB 1­contaminated areas exhibita higher frequency ofLOH than AFBI-unrelatedliver cancers. Thus, AFBI-mediated induction of mitotic recombination inhuman cells might explain the elevated frequency of LOH. We will presentvarious evidence from the literature arguing for a recombinogenic activityof AFB I in higher eukaryotic cells.

Keyword(s): Aftatoxin BI; mitotic recombination; loss of heterozygosity

Ip VIII.I21 Mlnlsatelllie rearrangement Induced by a tumor pro­moter, okadale acid

Minako Nagao, Shigeto KAneko, Hiroshi Irnai, Hirokazu Fulcuda, TakashiSugimura, Hitoshi Nakagama. Nat ional Cancer Center Res. Inst . Tokyo.Japan

Okadaic acid (OA), a tumor promoter in mouse skin carcinogenesis, is astrong inhibitor of protem phosphatase types 2A. 4 and 5. Although itshows no mutagenic activity ID S. typhimurium TAI00 or TA98, we havefound that it induces diphtheria-toxin resistant mutations, sister chromatidexchange and loss of amplified exogenous genes in mammalian cells . Inthe present study, induction of mimsatelhte rearrangement was detectedwhen NIH3T3 cells were cultured in the presence of 6 nglnl of OA for6 days. and then cloned. DNA extracted from each clone was examined formini~ellite r:earrangement by Southern biOI snalysis under low stringencyconditions usmg Pc-I as a probe. With OA treatment, rearrangement wasdetected in nine of 31 clones, in clear contrast to the one of 30 clonespositive without treatment. After inoculation of 3 x 106 cells treated withOA. Nude mice developed tumors at 7 of 20 sites, while only one tumorarose after inoculation of untreated-cells at 20 sites. The tumors deriVedfrom OA-treated NIH3T3 cells minisatellite sequence rearrangements . Theresults thus suggest that genetic rearrangement can be induced by changesin the phosphorylation status of proteins caused by OA, and further moreimply a role for minisatellite changes in malignant conversion.

Ip VIII.131 Mutagenicity of thermal decomposition products o(poly(vlnyl chloride) and polycyclic aromallc hydrocar_bons formalloD by Ihem

YoshiharuHisamatsu' , Satoshi Takaradaz, Hisao Hidakaz. JNationallnsliluteof Public Health. Shlro/canedal -4 chome, Minato -ku, T01cyo 108, Japan;2Meise l University. Hodokubo HlfIO. Tokyo 191. Japan

A laboratory-scale samples of a commercial type of poly(vinyl chloride)(PVC) were decomposed by using carefully controlled electric furnace inthe range of ISO"C to IOOO"C in nitrogen and air. Two fractions of thethermal decomposition products, gaseous products, which were adsorbed inTenax GR, and organic volatile-condensable products, which were adsorbedin quartz fiber wool, of PVC were collected to determine the mutagenicactivity in Salmonella cyphimuriumstrain TA98 and TAloo with and withOUt