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Yeast recombination assays for genotoxicity testing
168
DSB the question is whether the production of CA by RE can be taken as a model system for the production of CA by IR. Apart from other types of DNA lesions, IR lead to DSB by radical formation. RE produce DSB by splitting the phosphodiester bonds between the bases leading to 3'-OH and 5'-P ends, the bases are not changed in their molecular structure. This may cause different pathways in the production of CA. It can be expected that most RE-induced DSE are repaired simply by ligation of the 3'-OH and 5'-P ends. Another difficulty in comparing the CA-inducing capacities of IR and RE is that RE have to pass the cellular membrane and to enter the nucleus in order to induce DSB. This makes an exact determination of dose impossible at least as long as the mechanism of uptake is not known.
Fahrig, R., Department of Genetics, Fraunhofer Institute of Toxicology and Aerosol Research, Hannover (F.R.G.) How to detect and how to influence induction of recombination in mice in vivo The mammalian spot test using the mouse allows one to detect both induced mutations and reciprocal recombinations in vivo. In this method, mouse embryos, which are heterozygous for different recessive coat-color genes, are treated in utero between the 9th and l l t h days post conception. If this treatment leads to an alteration or loss of a specific wild-type allele in a pigment precursor cell, a spot of altered color may appear in the coat of the adult animal. A recombinational event may also result in a color spot. Gene mutations can be detected as genetic alterations at the c locus. It is also possible to detect reciprocal recombinations involving both the p and c loci which are located on the same chromosome, 14 Morgan units apart. Using tumor promoters in combination with a mutagenic carcinogen it is possible to enhance the recombinogenic effects of the carcinogen. On the other hand it is possible to suppress recombina-
tion induction by combining cocarcinogens with the mutagenic carcinogen.
7 Zimmermann, F.K., Institut fiir Mikrobiologie, Technische Hochschule, D-6100 Darmstadt (F.R.G.) Yeast recombination assays for genotoxicity testing The yeast Saccharomyces cerevisiae can be used to study the occurrence of different genetic rearrangements in mitotic cells. Non-reciprocal gene conversion occurs in diploid cells in G1 and at lower frequencies in G 2. It is detected by the generation of a functional allele from 2 mutant alleles with defects at different sites. It is screened by plating on a medium where only convertant cells can grow. Induction of mitotic gene conversion is by far the most frequently used test because of its technical ease, accuracy, sensitivity and reproducibility. Reciprocal mitotic crossingover in diploid cells can only be detected if it occurs in G 2. It is detected by the formation of twin-sectored colonies expressing 2 different recessive markers arranged in repulsion. Both types of mitotic recombination can be induced by all direct-acting mutagens and also promutagens after activation in vitro. Yeast strain D7 monitors induction of mitotic gene conversion detected by plating on a selective medium and reciprocal mitotic crossing-over by visual screening for 2 types of pigmented colonies. Intrachromosomal recombination mostly caused by sister-chromatid gene conversion also in haploids can be studied in special strains. Even though this test has not been widely used, it obviously responds to carcinogenic chemicals which do not induce interchromosomal conversion. Two systems have been designed to study transposition. Haploid yeast cells of heteroallelic strains are either of mating type a or a' according to the information at the mating-type locus. There are two additional silent genes which also contain mating-type information. Transposition of this information to the mating-type locus
169 can result in a change in mating type. Mating-type switching is a regular process in the germinating haploid meiospores of homothallic strains. Interestingly enough, heterothallic mating-type switching can be induced with sodium butyrate. Yeast cells contain retroviral genomes and a test has been developed to detect mutagen-induced transposition. Different repair gene functions are required for the different types of mitotic rearrangements so that a wide spectrum of cellular responses to induced genetic damage can be detected in diploid and some also in haploid yeast strains.
white~white + test, for instance, use of the excision repair-defective strain mus-201 did not result in hypermutability effects of the type found in germ cells. The fast and economic SMART systems also offer new perspectives for approaching questions related to metabolism of procarcinogens. This may be achieved by introducing proper marker genes (mwh, fir, w) into strains differing in their basic make-up and inducibility of the enzyme systems involved in the oxidative metabolism of xenobiotics.
9 Frei, H., Institute of Toxicology, ETH and University of Zurich, Schwerzenbach (Switzerland) Vogel, E.W., Department of Radiation Genetics and Chemical Mutagenesis, Leiden (The Netherlands) The future of SMART assays: emphasis on the underlying action principles? The conclusion from calibration studies on more than 350 chemicals, mostly genotoxins, is that both the wing spot test and the white~white + eye mosaic system monitor a broad spectrum of genetic damage, ranging from somatic mutations and deletions to mitotic recombination and structural chromosomal changes. However, MR between homologous chromosomes is by far the dominating event measured by these tests. Frequencies of MR between the 2 X-chromosomes can reach 10-2/cell after mutagen exposure of developing stages (larvae) of Drosophila. Major unresolved questions concern the relative distribution of MR between euchromatin and heterochromatin, which seems dose-dependent, and the contribution to somatic cell mutagenicity of gene conversion and unequal sister-strand recombination. In order to study MR at the molecular level, a feasible approach seems the use of PCR methodology in combination with alleles of the white or vermilion loci with known base-sequence changes. Little is also known about the role of different repair pathways in the fixation of different types of genetic damage in somatic cells of Drosophila. In the
Somatic mutation and (SMART) in Drosophila
recombination
tests
In Drosophila melanogaster, somatic test systems have been developed recently to identify genotoxic compounds with recombinogenic and mutagenic properties. In these tests, larvae heterozygous for 1 or 2 different recessive eye or wing-cell marker mutations are exposed to the test compounds. Genetic changes induced in the cells of the larval eye or wing primordia become manifested in the metamorphosed flies as clones exhibiting the mutant eye or wing-cell phenotype. The genetic changes are either due to mitotic recombination or to mutational events such as point mutations, deletions, or non-reciprocal translocations. Depending on the system, single spots a n d / o r twin spots can be found. Mitotic recombination may lead to twin spots which show the alternative mutant marker phenotypes in two adjacent clones. Depending on the system, not all recombination events can be identified in this way, and an indirect method is preferred to estimate at which proportions spot formation is due to recombination or to other mechanisms: in individuals made heterozygous for multiply inverted chromosomes, cross-over leads to inviable, chromosomally strongly defective cells; hence, somatic spots observed in inversion-heterozygous flies are due to mechanisms other than mitotic recombina-
DSB the question is whether the production of CA by RE can be taken as a model system for the production of CA by IR. Apart from other types of DNA lesions, IR lead to DSB by radical formation. RE produce DSB by splitting the phosphodiester bonds between the bases leading to 3'-OH and 5'-P ends, the bases are not changed in their molecular structure. This may cause different pathways in the production of CA. It can be expected that most RE-induced DSE are repaired simply by ligation of the 3'-OH and 5'-P ends. Another difficulty in comparing the CA-inducing capacities of IR and RE is that RE have to pass the cellular membrane and to enter the nucleus in order to induce DSB. This makes an exact determination of dose impossible at least as long as the mechanism of uptake is not known.
Fahrig, R., Department of Genetics, Fraunhofer Institute of Toxicology and Aerosol Research, Hannover (F.R.G.) How to detect and how to influence induction of recombination in mice in vivo The mammalian spot test using the mouse allows one to detect both induced mutations and reciprocal recombinations in vivo. In this method, mouse embryos, which are heterozygous for different recessive coat-color genes, are treated in utero between the 9th and l l t h days post conception. If this treatment leads to an alteration or loss of a specific wild-type allele in a pigment precursor cell, a spot of altered color may appear in the coat of the adult animal. A recombinational event may also result in a color spot. Gene mutations can be detected as genetic alterations at the c locus. It is also possible to detect reciprocal recombinations involving both the p and c loci which are located on the same chromosome, 14 Morgan units apart. Using tumor promoters in combination with a mutagenic carcinogen it is possible to enhance the recombinogenic effects of the carcinogen. On the other hand it is possible to suppress recombina-
tion induction by combining cocarcinogens with the mutagenic carcinogen.
7 Zimmermann, F.K., Institut fiir Mikrobiologie, Technische Hochschule, D-6100 Darmstadt (F.R.G.) Yeast recombination assays for genotoxicity testing The yeast Saccharomyces cerevisiae can be used to study the occurrence of different genetic rearrangements in mitotic cells. Non-reciprocal gene conversion occurs in diploid cells in G1 and at lower frequencies in G 2. It is detected by the generation of a functional allele from 2 mutant alleles with defects at different sites. It is screened by plating on a medium where only convertant cells can grow. Induction of mitotic gene conversion is by far the most frequently used test because of its technical ease, accuracy, sensitivity and reproducibility. Reciprocal mitotic crossingover in diploid cells can only be detected if it occurs in G 2. It is detected by the formation of twin-sectored colonies expressing 2 different recessive markers arranged in repulsion. Both types of mitotic recombination can be induced by all direct-acting mutagens and also promutagens after activation in vitro. Yeast strain D7 monitors induction of mitotic gene conversion detected by plating on a selective medium and reciprocal mitotic crossing-over by visual screening for 2 types of pigmented colonies. Intrachromosomal recombination mostly caused by sister-chromatid gene conversion also in haploids can be studied in special strains. Even though this test has not been widely used, it obviously responds to carcinogenic chemicals which do not induce interchromosomal conversion. Two systems have been designed to study transposition. Haploid yeast cells of heteroallelic strains are either of mating type a or a' according to the information at the mating-type locus. There are two additional silent genes which also contain mating-type information. Transposition of this information to the mating-type locus
169 can result in a change in mating type. Mating-type switching is a regular process in the germinating haploid meiospores of homothallic strains. Interestingly enough, heterothallic mating-type switching can be induced with sodium butyrate. Yeast cells contain retroviral genomes and a test has been developed to detect mutagen-induced transposition. Different repair gene functions are required for the different types of mitotic rearrangements so that a wide spectrum of cellular responses to induced genetic damage can be detected in diploid and some also in haploid yeast strains.
white~white + test, for instance, use of the excision repair-defective strain mus-201 did not result in hypermutability effects of the type found in germ cells. The fast and economic SMART systems also offer new perspectives for approaching questions related to metabolism of procarcinogens. This may be achieved by introducing proper marker genes (mwh, fir, w) into strains differing in their basic make-up and inducibility of the enzyme systems involved in the oxidative metabolism of xenobiotics.
9 Frei, H., Institute of Toxicology, ETH and University of Zurich, Schwerzenbach (Switzerland) Vogel, E.W., Department of Radiation Genetics and Chemical Mutagenesis, Leiden (The Netherlands) The future of SMART assays: emphasis on the underlying action principles? The conclusion from calibration studies on more than 350 chemicals, mostly genotoxins, is that both the wing spot test and the white~white + eye mosaic system monitor a broad spectrum of genetic damage, ranging from somatic mutations and deletions to mitotic recombination and structural chromosomal changes. However, MR between homologous chromosomes is by far the dominating event measured by these tests. Frequencies of MR between the 2 X-chromosomes can reach 10-2/cell after mutagen exposure of developing stages (larvae) of Drosophila. Major unresolved questions concern the relative distribution of MR between euchromatin and heterochromatin, which seems dose-dependent, and the contribution to somatic cell mutagenicity of gene conversion and unequal sister-strand recombination. In order to study MR at the molecular level, a feasible approach seems the use of PCR methodology in combination with alleles of the white or vermilion loci with known base-sequence changes. Little is also known about the role of different repair pathways in the fixation of different types of genetic damage in somatic cells of Drosophila. In the
Somatic mutation and (SMART) in Drosophila
recombination
tests
In Drosophila melanogaster, somatic test systems have been developed recently to identify genotoxic compounds with recombinogenic and mutagenic properties. In these tests, larvae heterozygous for 1 or 2 different recessive eye or wing-cell marker mutations are exposed to the test compounds. Genetic changes induced in the cells of the larval eye or wing primordia become manifested in the metamorphosed flies as clones exhibiting the mutant eye or wing-cell phenotype. The genetic changes are either due to mitotic recombination or to mutational events such as point mutations, deletions, or non-reciprocal translocations. Depending on the system, single spots a n d / o r twin spots can be found. Mitotic recombination may lead to twin spots which show the alternative mutant marker phenotypes in two adjacent clones. Depending on the system, not all recombination events can be identified in this way, and an indirect method is preferred to estimate at which proportions spot formation is due to recombination or to other mechanisms: in individuals made heterozygous for multiply inverted chromosomes, cross-over leads to inviable, chromosomally strongly defective cells; hence, somatic spots observed in inversion-heterozygous flies are due to mechanisms other than mitotic recombina-