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Bisulfite-inflicted damage to nucleic acids
234
I s t INTERNATIONALCONFERENCE, ASILOMAR
exhibited structural anomalies of the chromosome type: deletions, acentrics, dicentrics, rings and translocations. Numerical aneuploidy in structurally normal karyotypes or chromatid-type aberrations were not found.
45 SHAHIN, M. M., AND F. J. DE SERRES, Mutagenesis Branch, NIEHS-NIH, Research Triangle Park, N.C. 27709 (U.S.A.).
The effect of p H on hycanthone methanesulfonate-induced inactivation and mitotic recombination in D5, a new diploid strain of
Saccharomyces cerevisiae Recent papers by others have shown that hycanthone methanesulfonate, a drug used for the treatment of bilharziasis (schistosomiasis)-induced gene mutations in T4 bacteriophage, bacteria, yeast, Drosophila and mammalian cells in culture. The present experiments were designed to explore the effects of this compound on a diploid strain of Saccharomyces cerevisiae (D5) developed by ZIMMERMANN (Mutation Res., in the press) for the detection of mitotic recombination. The strain is heterozygous for two phenotypically distinguishable ade2 alleles which complement each other fully on minimal medium so that the diploid heterozygote forms white colonies. One allele, ade2-ii9, is leaky so the homozygous diploid forms pink colonies, whereas the other allele, ade2-4o is non-leaky and forms red colonies. In D5, the appearance of pink and red sectored colonies provides evidence for mitotic recombination. At pH 5.5 and 7.2, hycanthone was found to induce sectored colonies at a higher frequency than that which was found in the controls. However, a 5o-fold difference was found between the number of sectored colonies observed after treatment at pH 5.5 (0.63%) and pH 7.2 (o.o13 %). In addition, hycanthone methanesulfonate was found to be less toxic at pH 5.5 than pH 7.2. For example, using 400/zg hycanthone/ml and an incubation time of 48 h the frequency of survivors increased from o.oo12% at pH 7.2 to 4.5% at pH 5-5-
46 SHAPIRO,ROBERT,Department of Chemistry, New York University, New York, N. Y. (U.S.A.).
Bisulfite-inflicted damage to nucleic acids Bisulfite ion is the aqueous form of the air pollutant, sulfur dioxide. It is also used widely as an additive to foods and pharmaceuticals. Our prediction of its mutagenicity (J. Am. Chem. Soc. 92, (197o) 422) has been confirmed in four microorganisms. The mutagenic reaction, deamination of cytosine to uracil, takes place in single-stranded, but not double-stranded DNA. A poly(I)- poly(C) double helix also fails to react. The inhibition of the reaction takes place at the first step in the deamination sequence, addition of bisulfite to the 5-6 double bond of cytosine. The optimal conditions for deamination of cytidine and deoxycytidine are at pH 5, in the presence of a high concentration of bisulfite and buffer anions. The rate of deamination at pH 7 is approx. I % of that observed at pH 5.
THE AMERICAN ENVIRONMENTAL MUTAGEN SOCIETY
235
Bisulfite also reacts with the uracil residues of RNA. The optimal p H for this reaction is 7. At a lO -3 M concentration of bisulfite, 2% saturation of polyuridylic acid is obtained, which is sufficient to reduce its activity as a messenger in the cell-free protein-synthesizing system of E. coli by almost 500/0 .It is plausible that reduction of protein synthesis due to m R N A inactivation is a cause of the adverse physiological effects produced in lung tissues b y sulfur dioxide.
47 SHERMAN, FRED, AND JOHN W. STEWART, Department Radiation Biology and Bio-
physics, University of Rochester School of Medicine, Rochester, N.Y. 14642 (U.S.A.).
Frameshift mutants of iso-l-cytochrome c in yeast Altered iso-I-cytochromes c from intragenic revertants established that the mutant, cyi-I83, contained an insertion of an AT base-pair within or adjacent to the codon of lysine at residue position IO. Analyses were performed on iso-i-cytochromes c from 56 revertants that were obtained spontaneously or induced with UV, X-rays, a-particles, nitrous acid, nitrogen mustard, or methyl methanesulfonate. The 56 proteins comprised 20 different sequences which were the result of mainly single base-pair deletions, and less frequently of deletions of small sequences, tandem duplications, and single base-pair deletions with accompanying nearby base substitutions. One m u t a n t protein contained a replacement of the 9 amino terminal residues and a blocked amino terminus. The following nucleotide sequence of the first 44 bases of the m R N A could be deduced from the amino acid changes in the revertant proteins; i 2 3 4 5 6 7 8 9 IO i i 12 13 14 Thr-Glu-Phe-Lys-Ala-Gly-Ser-Ala-Lys-Lys-Gly-Ala-Thr-LeuAUG ACU GAA UUC AAG GCC GGU UCU GCU AAG AAA GGU GCU ACA CUN
None of the chemical agents was shown to unambiguously delete single AT base-pairs, while UV and ionizing radiation induced deletions of both AT and GC base-pairs. Supported by N I H (GMI27O2) and AEC (USAEC Report No. UR-349o-314).
48 SIMONS, J . W . J . M., A. A. VAN ZEELAND AND M. C. C. VAN DIGGELEN, Department
of Radiation Genetics and Chemical Mutagenesis, University of Leiden (The Netherlands).
The role of 8-azaguanine in the selection of hypoxanthine guanine phosphoribosyl transferase (I-IGPRT)-deficient mutants from diploid human cells To test whether HGPRT-defieient mutants arise independently of the selective agent, mutants were selected at concentrations of 1.2 and 5 Pg AG per ml. If AG only selects for mutants, the frequency of m u t a n t s without detectable enzyme activity is expected to be the same at both concentrations, whereas the overall m u t a n t frequency
I s t INTERNATIONALCONFERENCE, ASILOMAR
exhibited structural anomalies of the chromosome type: deletions, acentrics, dicentrics, rings and translocations. Numerical aneuploidy in structurally normal karyotypes or chromatid-type aberrations were not found.
45 SHAHIN, M. M., AND F. J. DE SERRES, Mutagenesis Branch, NIEHS-NIH, Research Triangle Park, N.C. 27709 (U.S.A.).
The effect of p H on hycanthone methanesulfonate-induced inactivation and mitotic recombination in D5, a new diploid strain of
Saccharomyces cerevisiae Recent papers by others have shown that hycanthone methanesulfonate, a drug used for the treatment of bilharziasis (schistosomiasis)-induced gene mutations in T4 bacteriophage, bacteria, yeast, Drosophila and mammalian cells in culture. The present experiments were designed to explore the effects of this compound on a diploid strain of Saccharomyces cerevisiae (D5) developed by ZIMMERMANN (Mutation Res., in the press) for the detection of mitotic recombination. The strain is heterozygous for two phenotypically distinguishable ade2 alleles which complement each other fully on minimal medium so that the diploid heterozygote forms white colonies. One allele, ade2-ii9, is leaky so the homozygous diploid forms pink colonies, whereas the other allele, ade2-4o is non-leaky and forms red colonies. In D5, the appearance of pink and red sectored colonies provides evidence for mitotic recombination. At pH 5.5 and 7.2, hycanthone was found to induce sectored colonies at a higher frequency than that which was found in the controls. However, a 5o-fold difference was found between the number of sectored colonies observed after treatment at pH 5.5 (0.63%) and pH 7.2 (o.o13 %). In addition, hycanthone methanesulfonate was found to be less toxic at pH 5.5 than pH 7.2. For example, using 400/zg hycanthone/ml and an incubation time of 48 h the frequency of survivors increased from o.oo12% at pH 7.2 to 4.5% at pH 5-5-
46 SHAPIRO,ROBERT,Department of Chemistry, New York University, New York, N. Y. (U.S.A.).
Bisulfite-inflicted damage to nucleic acids Bisulfite ion is the aqueous form of the air pollutant, sulfur dioxide. It is also used widely as an additive to foods and pharmaceuticals. Our prediction of its mutagenicity (J. Am. Chem. Soc. 92, (197o) 422) has been confirmed in four microorganisms. The mutagenic reaction, deamination of cytosine to uracil, takes place in single-stranded, but not double-stranded DNA. A poly(I)- poly(C) double helix also fails to react. The inhibition of the reaction takes place at the first step in the deamination sequence, addition of bisulfite to the 5-6 double bond of cytosine. The optimal conditions for deamination of cytidine and deoxycytidine are at pH 5, in the presence of a high concentration of bisulfite and buffer anions. The rate of deamination at pH 7 is approx. I % of that observed at pH 5.
THE AMERICAN ENVIRONMENTAL MUTAGEN SOCIETY
235
Bisulfite also reacts with the uracil residues of RNA. The optimal p H for this reaction is 7. At a lO -3 M concentration of bisulfite, 2% saturation of polyuridylic acid is obtained, which is sufficient to reduce its activity as a messenger in the cell-free protein-synthesizing system of E. coli by almost 500/0 .It is plausible that reduction of protein synthesis due to m R N A inactivation is a cause of the adverse physiological effects produced in lung tissues b y sulfur dioxide.
47 SHERMAN, FRED, AND JOHN W. STEWART, Department Radiation Biology and Bio-
physics, University of Rochester School of Medicine, Rochester, N.Y. 14642 (U.S.A.).
Frameshift mutants of iso-l-cytochrome c in yeast Altered iso-I-cytochromes c from intragenic revertants established that the mutant, cyi-I83, contained an insertion of an AT base-pair within or adjacent to the codon of lysine at residue position IO. Analyses were performed on iso-i-cytochromes c from 56 revertants that were obtained spontaneously or induced with UV, X-rays, a-particles, nitrous acid, nitrogen mustard, or methyl methanesulfonate. The 56 proteins comprised 20 different sequences which were the result of mainly single base-pair deletions, and less frequently of deletions of small sequences, tandem duplications, and single base-pair deletions with accompanying nearby base substitutions. One m u t a n t protein contained a replacement of the 9 amino terminal residues and a blocked amino terminus. The following nucleotide sequence of the first 44 bases of the m R N A could be deduced from the amino acid changes in the revertant proteins; i 2 3 4 5 6 7 8 9 IO i i 12 13 14 Thr-Glu-Phe-Lys-Ala-Gly-Ser-Ala-Lys-Lys-Gly-Ala-Thr-LeuAUG ACU GAA UUC AAG GCC GGU UCU GCU AAG AAA GGU GCU ACA CUN
None of the chemical agents was shown to unambiguously delete single AT base-pairs, while UV and ionizing radiation induced deletions of both AT and GC base-pairs. Supported by N I H (GMI27O2) and AEC (USAEC Report No. UR-349o-314).
48 SIMONS, J . W . J . M., A. A. VAN ZEELAND AND M. C. C. VAN DIGGELEN, Department
of Radiation Genetics and Chemical Mutagenesis, University of Leiden (The Netherlands).
The role of 8-azaguanine in the selection of hypoxanthine guanine phosphoribosyl transferase (I-IGPRT)-deficient mutants from diploid human cells To test whether HGPRT-defieient mutants arise independently of the selective agent, mutants were selected at concentrations of 1.2 and 5 Pg AG per ml. If AG only selects for mutants, the frequency of m u t a n t s without detectable enzyme activity is expected to be the same at both concentrations, whereas the overall m u t a n t frequency