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Isolation and characterisation of nuclear mutants with enhanced mitochondrial mutability in the fission yeast Schizosaccharomyces pombe
Microbiol. Res. (2002) 157, 197–200 (795) http://www.urbanfischer.de/journals/microbiolres
Isolation and characterisation of nuclear mutants with enhanced mitochondrial mutability in the fission yeast Schizosaccharomyces pombe Domenica Rita Massardo1, Luigi Del Giudice1, Angelica Del Giudice2, Klaus Wolf2 1 2
International Institute of Genetics and Biophysics, CNR, via G. Marconi 10, I-80125 Naples, Italy Department of Biology IV (Microbiology), Aachen University of Technology, Worringer Weg, D-52056 Aachen, Germany
Accepted: April 4, 2002
Abstract In this paper we report the isolation and preliminary characterisation of nuclear mutants with increased mitochondrial mutability in fission yeast. Screening of about 2000 clones after nitrosoguanidine mutagenesis led to the isolation of ten mutator mutants. For one of them (mut-1) we show that the mutation is chromosomally encoded. The activity of the mutator is restricted to the mitochondrial genome, since it increases the mutation rate to mitochondrially encoded drug resistance considerably, whereas the mutability of nuclear genes is not altered.
and Fox 1992). Mitochondrial mutants completely devoid of mitochondrial DNA can only be achieved after a mutational event in one or more nuclear gene(s) (Haffter and Fox 1992). In order to identify nuclear genes involved in mitochondrial genome stability, we started the isolation of nuclear mutants with an elevated mutation rate of the mitochondrial genome, without changing the mutability of nuclear genes.
Key words: mutator – mitochondria – fission yeast – drug resistance
Materials and methods Mutants. Mutant strains ade6-424 and ura4-D18h– were kindly provided by J. Kohli, University of Berne, Switzerland.
Introduction The fission yeast Schizosaccharomyces pombe harbors a small mitochondrial genome of only 15 to 20 kilobase pairs (depending on the strain used ; Merlos-Lange et al. 1987), which has been sequenced entirely (Sankoff et al. 1992). Unlike the mitochondrial genome of Saccharomyces cerevisiae it is very stable. Mitochondrial mutations are only obtained either in mutator strains carrying a mutation in the mitochondrially encoded rps3 gene (ribosomal protein of the small subunit of the ribosome; formerly urf a; Seitz-Mayr and Wolf 1982; Neu et al. 1998 ; Schäfer and Wolf in press) or after a mutation in a hitherto unknown nuclear gene (Haffter Corresponding author: L. Del Giudice e-mail: [email protected] 0944-5013/02/157/03-197
$15.00/0
Methods. Standard genetic methods were used according to the fission yeast handbook (http://bio.uva.n1/ pombe/handbook/). Media. Standard yeast media were were used as described in the fission yeast handbook.
Results and discussion For measuring mitochondrial mutation rates we used the resistance to the inhibitor diuron (diur) which acts by binding to cytochrome b and thus blocking electron transport. Mutants resistant to this drug carry mutations in the gene encoding apocytochrome b (cob) (Wolf and Del Giudice 1988). In addition we used an inhibitor of protein synthesis, erythromycin (Wolf 1995). Mutations Microbiol. Res. 157 (2002) 3
197
Fig. 1 198
Microbiol. Res. 157 (2002) 3
Fig. 2. Demonstration of mutator activity by drop-out. Dropping out of mutator mutant and wild type onto media containing diuron (A) and to erythromycin (B) The drop of wild type cells, which does not contain papillae, is indicated by the arrowheads. For further explanation see text.
leading to resistance to this drug (eryr) are located in the gene encoding the large ribosomal RNA (rns). Both genes are located distantly on the mitochondrial genome (Wolf and Del Giudice 1988). After mutagenesis with nitrosoguanidine (survival rate 5%) (Del Giudice and Puglisi 1974) cells were diluted and plated out onto glucose complete medium at 28 °C to form single colonies. After four days of incubation a small portion of the cell material of several hundred colonies was picked and each sample transferred to a well of microtiter plates filled with 0.2 ml of glucose complete medium. After incubation at 28 °C for four days the cultures reached a titer of approximately 107 cells/ml. Aliquots of 0.1 ml were spread an the surface of petri dishes containing glycerol complete medium supplemented with 0.05% glucose and 5 µg/ml of diuron (Fig. 1 A) or 400 µg/ml of erythromycin (Fig. 1 C). More than 400 colonies per plate, varying in size, were obtained after 14 days of incubation on diuron medium, whereas 15 – 40 colonies appeared an diuron plates when a wild type strain was plated out (Fig. 1 B). From the mutator strain also more than 400 colonies grew on erythromycin containing medium, but no colonies were observed from the wild type on erythromycin containing medium (Fig. 1D).
Genetic analysis revealed that the diuron resistant mutants originating from the wild type were of nuclear inheritance (K. Wolf, unpublished results) and could be deficient in the uptake of diuron. Since the next goal in this work is the identification of the nuclear mutator gene(s) and since the only selectable phenotype is the amount of colonies growing an drug media, we had to scale down the selection process. This was done by dropping out up to 100 spots per petri dish. An example that scaling down is possible, is given in Fig. 2A for diuron and Fig. 2B for erythromycin. Only for demonstration purposes the drop-out of only one mutator and one non-mutator is shown on the plate. The arrowheads mark the position of the non-mutator strain, where no papillae were visible. Drop-out of mutator strains gave between 20 and 50 papillae both, for diuron and erythromycin. The method also works with a metal device to directly replicate cells from a microtiter plate onto drug medium (about 100 spots per plate). The mitochondrial mutator encoded by the rps3 gene gives not only rise to drug resistant mutants, but also produces up to several percent of mitochondrial respira-
Fig. 1. Demonstration of mutator activity by plating. A. Colonies from a mutator mutant on diuron. B. Colonies from a wild-type strain on diuron. C. Colonies from a mutator strain on erythromycin. D. No visible colonies from the wild type on erythromycin. See text for further details. Microbiol. Res. 157 (2002) 3
199
tory deficient mutants. In order to test whether the nuclearly encoded mutator also produces respiratory deficient mutants, cells were plated onto petri dishes with glucose complete medium (about 10,000 cells altogether) and replica plated onto glycerol complete medium plus 0.05% of glucose without drugs. No respiratory deficient colony was identified among 10,000 colonies. Ten mutator mutants identified with the help of dropout technique (mut1–mut10) were purified three times and again tested for their mutator property by dropping out on drug plates. The percentage of drug resistant papillae was the same as in the preliminary studies. This demonstrates that the mutator is a stable heritable trait. In order to test the possible influence of the mutator an nuclear genes, we used a combined system to analyse both, forward and reverse mutation. The ade6 mutation leads to the accumulation of a red pigment on adenine limiting medium. By plating out 6 × 106 cells an plates containing complete medium with low amounts of yeast extract (0.5%), cells form a lawn which is red due to the adenine limitation. On top of this background white colonies appear which can either originate from a backmutation of the ade6 mutation to the wild genotype or from the mutation of any other gene which is epistatic to ade6. Both in the wild type and in the mutant, about 6 – 20 white colonies appeared among 4 × 107 cells plated altogether. In both cases half of them proved to be prototrophic (due to reversion of ade6 to ADE6 or to a suppressor mutation), the other half was still adenine requiring (very likely to a mutation in another gene in the adenine pathway). From these results it can be concluded that the mutator exclusively acts on mitochondrial DNA but not on nuclear genes. In order to prove that the mutator phenotype is due to a single nuclear mutation, we crossed the mutant mut1 (ade6 -424 ura4-D18 h–) with the strain leu2-120 h+. Analysis of 800 random spores showed a nearly 1:1 segregation of auxotrophic markers and the mating type (data not shown). Concerning the mutator property, 47.5% of the progeny was wild type and 52.5% showed a mutator phenotype which is identical to that observed in the parental mutant strain. This demonstrates that the mutator phenotype is due to a single nuclear mutation. In order to isolate the wild type allele(s) for the mutator trait, work is in progress to complement the mutant with a genomic library of the wild type. The transformants have to be analysed for the restoration of normal (low) mitochondrial mutation frequency by the technique outlined in this paper. The role of the gene product needed for maintaining the integrity of the mitochondrial genome could be direct or indirect. A direct role could be imagined for a subunit or a cofactor of the mitochondrial DNA polymerase. Alternatively the protein could also be engaged 200
Microbiol. Res. 157 (2002) 3
in repair processes of mitochondrial DNA, which so far have not been studied in fission yeast. The mutants described in this paper provide the basis for analysis of genes involved in the stable maintenance of mitochondrial DNA. Since all eukaryotic organisms are faced with the problem to conserve the integrity of their mitochondrial genomes, these genes were probably conserved during evolution. This would then allow the isolation of homologous genes in humans by functional complementation. This approach has already proved to be successful in the characterisation of genes involved in the regulation of cell cycle.
Acknowledgements The technical assistance of Mr. G. De Simone (Naples) is acknowledged. Research was supported by the Ministero delle Politiche Agricole e Forestali (MIPAF), Italy, special grant ‘Piano Nazionale Biotecnologie Vegetali’ to L. D. G. and by the Deutsche Forschungsgemeinschaft to K. W.
References Del Giudice, L., Puglisi, P. P. (1974): Induction of respiratorydeficient mutants in a “petite negative” yeast species Kluyveromyces lactis with N-methyl-N’-Nitro-N-nitrosoguanidine. Biochem. Biophys. Res. Commun. 59, 865–871. Haffter, P., Fox, T. (1992): Nuclear mutations in the petitenegative yeast Schizosaccharomyces pombe allow growth of cells lacking mitochondrial DNA. Genetics 131, 255–260. Merlos-Lange, A. M., Kanbay, F., Zimmer, M., Wolf, K. (1987): DNA splicing of mitochondrial group I and II introns in Schizosaccharomyces pombe. Mol. Gen. Genet. 206, 273–278. Neu, R., Goffart, S., Wolf, K., Schäfer, B. (1998): Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe. Molec. Gen. Genet. 258, 389–396. Sankoff, D., Leduc, G., Antoine, N., Paquin, B., Lang, B. F., Cedergren, R. (1992): Gene order comparison for phylogenetic inference: evolution of the mitochondrial genome. Proc. Natl. Acad. Sci. USA 89, 6575–6579. Schäfer, B., Wolf, K. (2002): Molecular Biology in Schizosaccharomyces pombe. Springer Verlag, Heidelberg, Berlin, New York, in press. Seitz-Mayr, G., Wolf, K. (1982): Extrachromosomal mutator inducing point mutations and deletions in mitochondrial genome of fission yeast. Proc. Natl. Acad. Sci. USA 79, 2618–2622. Wolf, K. (1995): Mitochondrial genetics of yeast. In: Kück, U. (ed) The Mycota, Vol II, Genetics and Biotechnology, Berlin Heidelberg: Springer, pp 75–91. Wolf, K., Del Giudice, L. (1988): The variable mitochondrial genome of ascomycetes: organization, mutations, alterations, and expression. Adv. Genet. 25, 186–308.
Isolation and characterisation of nuclear mutants with enhanced mitochondrial mutability in the fission yeast Schizosaccharomyces pombe Domenica Rita Massardo1, Luigi Del Giudice1, Angelica Del Giudice2, Klaus Wolf2 1 2
International Institute of Genetics and Biophysics, CNR, via G. Marconi 10, I-80125 Naples, Italy Department of Biology IV (Microbiology), Aachen University of Technology, Worringer Weg, D-52056 Aachen, Germany
Accepted: April 4, 2002
Abstract In this paper we report the isolation and preliminary characterisation of nuclear mutants with increased mitochondrial mutability in fission yeast. Screening of about 2000 clones after nitrosoguanidine mutagenesis led to the isolation of ten mutator mutants. For one of them (mut-1) we show that the mutation is chromosomally encoded. The activity of the mutator is restricted to the mitochondrial genome, since it increases the mutation rate to mitochondrially encoded drug resistance considerably, whereas the mutability of nuclear genes is not altered.
and Fox 1992). Mitochondrial mutants completely devoid of mitochondrial DNA can only be achieved after a mutational event in one or more nuclear gene(s) (Haffter and Fox 1992). In order to identify nuclear genes involved in mitochondrial genome stability, we started the isolation of nuclear mutants with an elevated mutation rate of the mitochondrial genome, without changing the mutability of nuclear genes.
Key words: mutator – mitochondria – fission yeast – drug resistance
Materials and methods Mutants. Mutant strains ade6-424 and ura4-D18h– were kindly provided by J. Kohli, University of Berne, Switzerland.
Introduction The fission yeast Schizosaccharomyces pombe harbors a small mitochondrial genome of only 15 to 20 kilobase pairs (depending on the strain used ; Merlos-Lange et al. 1987), which has been sequenced entirely (Sankoff et al. 1992). Unlike the mitochondrial genome of Saccharomyces cerevisiae it is very stable. Mitochondrial mutations are only obtained either in mutator strains carrying a mutation in the mitochondrially encoded rps3 gene (ribosomal protein of the small subunit of the ribosome; formerly urf a; Seitz-Mayr and Wolf 1982; Neu et al. 1998 ; Schäfer and Wolf in press) or after a mutation in a hitherto unknown nuclear gene (Haffter Corresponding author: L. Del Giudice e-mail: [email protected] 0944-5013/02/157/03-197
$15.00/0
Methods. Standard genetic methods were used according to the fission yeast handbook (http://bio.uva.n1/ pombe/handbook/). Media. Standard yeast media were were used as described in the fission yeast handbook.
Results and discussion For measuring mitochondrial mutation rates we used the resistance to the inhibitor diuron (diur) which acts by binding to cytochrome b and thus blocking electron transport. Mutants resistant to this drug carry mutations in the gene encoding apocytochrome b (cob) (Wolf and Del Giudice 1988). In addition we used an inhibitor of protein synthesis, erythromycin (Wolf 1995). Mutations Microbiol. Res. 157 (2002) 3
197
Fig. 1 198
Microbiol. Res. 157 (2002) 3
Fig. 2. Demonstration of mutator activity by drop-out. Dropping out of mutator mutant and wild type onto media containing diuron (A) and to erythromycin (B) The drop of wild type cells, which does not contain papillae, is indicated by the arrowheads. For further explanation see text.
leading to resistance to this drug (eryr) are located in the gene encoding the large ribosomal RNA (rns). Both genes are located distantly on the mitochondrial genome (Wolf and Del Giudice 1988). After mutagenesis with nitrosoguanidine (survival rate 5%) (Del Giudice and Puglisi 1974) cells were diluted and plated out onto glucose complete medium at 28 °C to form single colonies. After four days of incubation a small portion of the cell material of several hundred colonies was picked and each sample transferred to a well of microtiter plates filled with 0.2 ml of glucose complete medium. After incubation at 28 °C for four days the cultures reached a titer of approximately 107 cells/ml. Aliquots of 0.1 ml were spread an the surface of petri dishes containing glycerol complete medium supplemented with 0.05% glucose and 5 µg/ml of diuron (Fig. 1 A) or 400 µg/ml of erythromycin (Fig. 1 C). More than 400 colonies per plate, varying in size, were obtained after 14 days of incubation on diuron medium, whereas 15 – 40 colonies appeared an diuron plates when a wild type strain was plated out (Fig. 1 B). From the mutator strain also more than 400 colonies grew on erythromycin containing medium, but no colonies were observed from the wild type on erythromycin containing medium (Fig. 1D).
Genetic analysis revealed that the diuron resistant mutants originating from the wild type were of nuclear inheritance (K. Wolf, unpublished results) and could be deficient in the uptake of diuron. Since the next goal in this work is the identification of the nuclear mutator gene(s) and since the only selectable phenotype is the amount of colonies growing an drug media, we had to scale down the selection process. This was done by dropping out up to 100 spots per petri dish. An example that scaling down is possible, is given in Fig. 2A for diuron and Fig. 2B for erythromycin. Only for demonstration purposes the drop-out of only one mutator and one non-mutator is shown on the plate. The arrowheads mark the position of the non-mutator strain, where no papillae were visible. Drop-out of mutator strains gave between 20 and 50 papillae both, for diuron and erythromycin. The method also works with a metal device to directly replicate cells from a microtiter plate onto drug medium (about 100 spots per plate). The mitochondrial mutator encoded by the rps3 gene gives not only rise to drug resistant mutants, but also produces up to several percent of mitochondrial respira-
Fig. 1. Demonstration of mutator activity by plating. A. Colonies from a mutator mutant on diuron. B. Colonies from a wild-type strain on diuron. C. Colonies from a mutator strain on erythromycin. D. No visible colonies from the wild type on erythromycin. See text for further details. Microbiol. Res. 157 (2002) 3
199
tory deficient mutants. In order to test whether the nuclearly encoded mutator also produces respiratory deficient mutants, cells were plated onto petri dishes with glucose complete medium (about 10,000 cells altogether) and replica plated onto glycerol complete medium plus 0.05% of glucose without drugs. No respiratory deficient colony was identified among 10,000 colonies. Ten mutator mutants identified with the help of dropout technique (mut1–mut10) were purified three times and again tested for their mutator property by dropping out on drug plates. The percentage of drug resistant papillae was the same as in the preliminary studies. This demonstrates that the mutator is a stable heritable trait. In order to test the possible influence of the mutator an nuclear genes, we used a combined system to analyse both, forward and reverse mutation. The ade6 mutation leads to the accumulation of a red pigment on adenine limiting medium. By plating out 6 × 106 cells an plates containing complete medium with low amounts of yeast extract (0.5%), cells form a lawn which is red due to the adenine limitation. On top of this background white colonies appear which can either originate from a backmutation of the ade6 mutation to the wild genotype or from the mutation of any other gene which is epistatic to ade6. Both in the wild type and in the mutant, about 6 – 20 white colonies appeared among 4 × 107 cells plated altogether. In both cases half of them proved to be prototrophic (due to reversion of ade6 to ADE6 or to a suppressor mutation), the other half was still adenine requiring (very likely to a mutation in another gene in the adenine pathway). From these results it can be concluded that the mutator exclusively acts on mitochondrial DNA but not on nuclear genes. In order to prove that the mutator phenotype is due to a single nuclear mutation, we crossed the mutant mut1 (ade6 -424 ura4-D18 h–) with the strain leu2-120 h+. Analysis of 800 random spores showed a nearly 1:1 segregation of auxotrophic markers and the mating type (data not shown). Concerning the mutator property, 47.5% of the progeny was wild type and 52.5% showed a mutator phenotype which is identical to that observed in the parental mutant strain. This demonstrates that the mutator phenotype is due to a single nuclear mutation. In order to isolate the wild type allele(s) for the mutator trait, work is in progress to complement the mutant with a genomic library of the wild type. The transformants have to be analysed for the restoration of normal (low) mitochondrial mutation frequency by the technique outlined in this paper. The role of the gene product needed for maintaining the integrity of the mitochondrial genome could be direct or indirect. A direct role could be imagined for a subunit or a cofactor of the mitochondrial DNA polymerase. Alternatively the protein could also be engaged 200
Microbiol. Res. 157 (2002) 3
in repair processes of mitochondrial DNA, which so far have not been studied in fission yeast. The mutants described in this paper provide the basis for analysis of genes involved in the stable maintenance of mitochondrial DNA. Since all eukaryotic organisms are faced with the problem to conserve the integrity of their mitochondrial genomes, these genes were probably conserved during evolution. This would then allow the isolation of homologous genes in humans by functional complementation. This approach has already proved to be successful in the characterisation of genes involved in the regulation of cell cycle.
Acknowledgements The technical assistance of Mr. G. De Simone (Naples) is acknowledged. Research was supported by the Ministero delle Politiche Agricole e Forestali (MIPAF), Italy, special grant ‘Piano Nazionale Biotecnologie Vegetali’ to L. D. G. and by the Deutsche Forschungsgemeinschaft to K. W.
References Del Giudice, L., Puglisi, P. P. (1974): Induction of respiratorydeficient mutants in a “petite negative” yeast species Kluyveromyces lactis with N-methyl-N’-Nitro-N-nitrosoguanidine. Biochem. Biophys. Res. Commun. 59, 865–871. Haffter, P., Fox, T. (1992): Nuclear mutations in the petitenegative yeast Schizosaccharomyces pombe allow growth of cells lacking mitochondrial DNA. Genetics 131, 255–260. Merlos-Lange, A. M., Kanbay, F., Zimmer, M., Wolf, K. (1987): DNA splicing of mitochondrial group I and II introns in Schizosaccharomyces pombe. Mol. Gen. Genet. 206, 273–278. Neu, R., Goffart, S., Wolf, K., Schäfer, B. (1998): Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe. Molec. Gen. Genet. 258, 389–396. Sankoff, D., Leduc, G., Antoine, N., Paquin, B., Lang, B. F., Cedergren, R. (1992): Gene order comparison for phylogenetic inference: evolution of the mitochondrial genome. Proc. Natl. Acad. Sci. USA 89, 6575–6579. Schäfer, B., Wolf, K. (2002): Molecular Biology in Schizosaccharomyces pombe. Springer Verlag, Heidelberg, Berlin, New York, in press. Seitz-Mayr, G., Wolf, K. (1982): Extrachromosomal mutator inducing point mutations and deletions in mitochondrial genome of fission yeast. Proc. Natl. Acad. Sci. USA 79, 2618–2622. Wolf, K. (1995): Mitochondrial genetics of yeast. In: Kück, U. (ed) The Mycota, Vol II, Genetics and Biotechnology, Berlin Heidelberg: Springer, pp 75–91. Wolf, K., Del Giudice, L. (1988): The variable mitochondrial genome of ascomycetes: organization, mutations, alterations, and expression. Adv. Genet. 25, 186–308.