- Email: [email protected]
Genetic effects of 5-azacytidine in Saccharomyces cerevisiae
Mutation Research, 139 (1984) 21-24
21
Elsevier MRLett 0510
Genetic effects o f 5-azacytidine in Saccharomyces cerevisiae Friedrich K. Zimmermann and I. Scheel Institut fiir Mikrobiologie, Technische Hochschule, D-6100 Darmstadt (Federal Republic of Germany) (Accepted 30 September 1983)
Summary The base analog 5-azacytidine induced a variety of genetic and epigenetic effects in differentl organisms. It was tested in two diploid strains of the yeast Saccharomyces cerevisiae to study the induction of point mutation, mitotic reciprocal crossing-over, mitotic gene conversion (strain D7) and mitotic aneuploidy (strain D61.M). It was used on cells growing in its presence for 4-5 generations. There was a strong induction of both types of mitotic recombination and point mutation. However, there was no induction of mitotic chromosomal malsegregation under the same conditions.
Most mutagens induce genetic alterations via primary chemical changes of DNA. Only few mutagens act more indirectly by being incorporated into DNA or through interference with DNA metabolism. 5-Azacytidine (CAS No. 320-67-2) seems to be a particularly interesting chemical. It induces chromosome decondensation, sister-chromatid exchanges, endoreduplications and activation of silent genes in heterochromatic regions (Hori, 1983). It inhibits methylation of cytosine in Escherichia coli and is mutagenic in a Salmonella reverse mutation system using a trpE8 mutation (Podger, 1983). The yeast Saccharomyces cerevisiae provides the possibility to test chemicals for their ability to induce a wide spectrum of genetic effects ranging from simple point mutations and mitotic recom-
Research sponsored by Contract No. NO 1-ES-1-5005, Development of a Yeast Aneuploidy System, from the National Institute of Environmental Health Sciences, Research Triangle Park, NC (U.S.A.) 0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
bination to chromosomal malSegregation. Therefore, it was interesting to investigate 5-azacytidine in respect to the range of genetic alterations it can induce in a eukaryote. Material and methods
Strains. The diploid strain D7 has been described by Zimmermann et al. (1975). It carries two non-complementing alleles, trp5-12 and trp5-27, which cause a requirement for trypt0phan. Prototrophy can be restored by intragenic recombination, mostly non-reciprocal gene conversion, with an admixture of reciprocal intragenic crossing-over and reverse mutation. There is a pair of complementing alleles, ade2-40 (causing an Iadenine requirement and deep-red colonies) arid ade2-119 (not causing a stringent requirement for adenine but causing a pink pigmentation of colonies). Both alleles are recessive, and D7 forms white colonies. However, mitotic crossing-over, mitotic gene conversion and rarer events of point mutation,
22 chromosomal deletion or loss of an entire chromosome can lead to the expression of one of the two alleles. This can readily be detected by the appearance of pigmented colonies. Only reciprocal mitotic crossing-over can be recognized as such by the appearance o f colonies with simultaneously occurring red and pink sectors. All other types of colony can by caused by any of the above-mentioned genetic events. Therefore, all pigmented colonies are combined into one category which is said to reflect mitotic segregation. There is also a homozygous condition for allele ilvl-92 which causes a requirement for isoleucine. Restoration o f prototrophy can be brought about by mutation of the mutant allele itself or in some suppressor genes. Strain D61 .M is similar to strain D6 described by Parry and Zimmermann (1976). It can be used to monitor mitotic chromosome loss. The relevant gen0type is:
cyh2 + leul ade6 ade2 iivl-92 M A L his centromere + trp5 + + ade2 ilvl-92 M A L + The marker cyh2, a recessive resistance to 2 ppm cycloheximide, is not expressed in strain D61.M, and this strain is sensitive. Resistance to cycloheximide can be caused by the expression of the recessive cyh2 allele by a number of genetic events:
mitotic crossing-over, mitotic gene conversion, mutation in the dominant wild-type allele, loss of a chromosomal fragment or loss of the entire chromosome. The latter genetic effect can be distinguished from all others by the simultaneous expression of the recessive marker ade6. Normally, D61.M forms red colonies because of the homozygous condition of ade2. When ade6 is expressed, colonies are white. White resistant colonies can be caused by a single event of mitotic chromosome loss or by two simultaneous events of mitotic recombination or mutation. Mitotic chromosome loss is indicated when white resistant colonies express centromere-linked leul (Parry and Zimmermann, 1976). Media and culturing. The composition of synthetic and rich growth media used in mutagenicity testing with yeast have been described by Zimmermann (1975). Cultures were started with an inoculum of about 200 cells per 5 ml yeast-extractpeptone-glucose medium in test tubes and grown to about 5 x 107 - 1 x 10a cells/ml. At this point, 0.1-ml samples were removed and plated onto a medium without tryptophan for D7 or on a yeastextract-peptone-glucose medium with 1.5 ppm cycloheximide for D61.M. The plates were incubated until they could be scored for prototrophic or resistant ceils, respectively. During this time, the test tubes were stored in a refrigerator and were us-
TABLE 1 THE EFFECTS OF 5-AZACYTIDINE ON THE INDUCTION OF MITOTIC SEGREGATION AND CHROMOSOMAL MALSEGREGATIONIN YEAST STRAIN D61.M mg/ml
Titer ( x 106)
Resistant colonies Direct
1:5
Resistants (x l0 -5)
Monosomics (x l0 -6)
0.0 0.4 0.8
18.9 29.5 30.0
1381 (1) 1399 (0) 1445 (2)
289 (0) 334 (0)
24.31 16.35 18.57
0.18 0.22
1.5 2.5 5.0
28.6 15.2 20.4
1939 (2) (6) (2)
411 (0) 695 (2) 1244 (0)
25.67 76.38 101.80
0.23 1.31 0.33
4.8
328 (15)
29.52
10.34
Bavistan 20 ppm Cells were plated, without a washing, directly or after a 1: 5 dilution, onto the selective medium with cycloheximide.The actual colony counts from three petri dishes are given under column heading 'Resistant colonies'. White and leucine-requiring colonies are listed in parentheses. Cell titers were computed from colony counts on a non-selective medium seeded with appropriate dilutions. Total exposure time: 17 h.
23
ed within 2 weeks. In this way it was possible always to use cultures with low spontaneous backgrounds.
TABLE 2
Mutagenic treatments. 5-Azacytidine (Serva, Heidelberg) was dissolved in the synthetic growth medium. The initial cell titers at the start of the treatment were about 1 x 106 ceils per ml. After an overnight incubation of 16-17 h on a reciprocal shaker at 28°C, the cultures were placed in ice and plated without washing, directly or after appropriate dilution, on the selective media and a non-selective medium to determine viable titers.
mg/ml
Results and discussion
Cells were plated, without washing or dilution~ onto the synthetic media lacking tryptophan to select for convertants or isoleucine to select for reverse mutants. Cell titers were computed from colony counts on a non-selective medium seeded with appropriate dilution. The colony counts ate from 3 petri dishes in each category. Total exposure time: 17 h.
The effects of 5-azacytidine on strain D61 .M are shown in Table 1. There was a distinct increase in the frequencies of red resistant colonies in cultures with higher concentrations o f the analog. This is indicative o f the induction o f mitotic recombination or mutation. However, there was no increase in white or leucine-requiring resistant colonies which would have signaled induction of mitotic c h r o m o s o m e loss. Bavistan is known to induce mitotic c h r o m o s o m e loss in yeast (Wood, 1982) and was used as a positive control agent. It induced monosomics in the experiment reported showing that the test was functioning properly. The genetic effects induced by 5-azacytidine in
I N D U C T I O N OF MITOTIC GENE CONVERSION AND REVERSE M U T A T I O N W I T H 5-AZACYTIDiNE IN YEAST STRAIN D7 Titer
Convertants
Revertants
( × 106) Colonies
( × 10 -~)
Colonies
( × 10 -6 )
0.0 1.0 2.0
27.5 25.7 27.3
15 218 273
0.18 1.69 2.00
• 48 130
1.27 3.73 9.52
3.0 4.0 5.0
25.2 24.6 25.2
395 525 710
3.13 4.27 5.64
200 292 439
15.86 23.76 34.87
7.5 10.0
28.6 29.2
1061 9009
7.41 6.23
657 774
45.88 53.09
strain D7 are shown in Table 2. There was a strong induction of both mitotic gene conversion and reverse mutation. It is important to note that the spontaneous frequency of mitotic convertants in the control was about one-fifth of the normal level. Th~s suggested that the culture used contained a high percentage of cells that were homoallelic for one of the trp5 alleles. 29 colonies from the control plating on the
TABLE 3 I N D U C T I O N OF M I T O T I C S E G R E G A T I O N W I T H 5-AZACYTIDINE IN YEAST STRAIN D7 mg/ml
Unselected colonies Total
Aberrant
Percent
Aberrant convertant colonies
Percent
Aberrant revertant colonies
Percent
0.0 1.0 2.0
826 1287 1365
0 0 4
0,29
0 2 1
0.92 0.37
0 0 0
-
3.0 4.0 5.0
1261 1229 1259
5 6 5
0.40 0.49 0.40
1 2 5
0.25 0.38 0,70
1 0 4
0.50 0.91
7.5 10.0
1432 1458
10 7
0.70 0.48
8 9
0,75 0.99
6 10
0.91 1.29
The data shown here are from the experiment described in Table 2. Mitotic segregation was followed among unselected (~olumns 2-4), convertant and reverse mutant colonies. The total numbers of convertant and reverse mutant colonies are listed in Table 2 (columns 3 and 5) and are not repeated here.
24 non-selective medium were tested for heteroallelic instability. Only 7 o f those single-colony clones showed the typical heteroallelic instability. This clearly indicated that continued selection for cultures with a low spontaneous background in convertants had resulted in cultures that consisted mostly of homoallelic and stable cells which were unable to signal mitotic gene conversion at the trp5 locus. Mitotic segregation was observed among colonies on all media: the non-selective medium where all viable cells could grow as well as on the media selective for mitotic convertants and reverse mutants (Table 3). This unequivocally showed that 5-azacytidine did not select cycloheximide-resistant cells or cells prototrophic for tryptophan or isoleucine but induced genetic effects. 5-Azacytidine has to be considered as a strong mutagen in yeast. It induces genetic effects at the D N A level, but it does not induce chromosomal malsegregation. Apparently, its primary effects are directed at D N A integrity itself possibly through incorporation into D N A or interference with D N A metabolism. As reviewed by Hori (1983), it has various effects on chromosome condensation, ac-
tivation of genes in heterochromatic regions and cell differentiation. Yeast has become a prime organism in the study of eukaryotic molecular genetics, and 5-azacytidine may become a powerful research tool in this organism.
References Hori, T.-A. (1983) Induction of chromosomedecondensation, sister-chromatid exchanges and endoreduplication by 5-azacytidine, an inhibitor of DNA-methylation, Mutation Res., 121, 47-52. Parry, J.M., and F.K. Zimmermann (1976) The detection of monosomic colonies by mitotic non-disjunction in the yeast Saccharomyces cerevisiae, Mutation Res., 36, 49-66. Podger, D.M. (1983) Mutagenicity of 5-azacytidine in Salmonella typhimurium, Mutation Res., 121, 1-6. Wood, J.S. (1982) Genetic effects of methylbenzimidazole-2-ylcarbamate on Saccharomyces cerevisiae, Mol. Cell Biol., 2, 1069-1074. Zimmermann, F.K. (1975) Procedures used in the induction of mitotic recombination and mutation in the yeast Saccharomyces cerevisiae, Mutation Res., 31, 71-86. Zimmermann, F.K., R. Kern and H. Rasenberger (1975) A yeast strain for the simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381-388.
21
Elsevier MRLett 0510
Genetic effects o f 5-azacytidine in Saccharomyces cerevisiae Friedrich K. Zimmermann and I. Scheel Institut fiir Mikrobiologie, Technische Hochschule, D-6100 Darmstadt (Federal Republic of Germany) (Accepted 30 September 1983)
Summary The base analog 5-azacytidine induced a variety of genetic and epigenetic effects in differentl organisms. It was tested in two diploid strains of the yeast Saccharomyces cerevisiae to study the induction of point mutation, mitotic reciprocal crossing-over, mitotic gene conversion (strain D7) and mitotic aneuploidy (strain D61.M). It was used on cells growing in its presence for 4-5 generations. There was a strong induction of both types of mitotic recombination and point mutation. However, there was no induction of mitotic chromosomal malsegregation under the same conditions.
Most mutagens induce genetic alterations via primary chemical changes of DNA. Only few mutagens act more indirectly by being incorporated into DNA or through interference with DNA metabolism. 5-Azacytidine (CAS No. 320-67-2) seems to be a particularly interesting chemical. It induces chromosome decondensation, sister-chromatid exchanges, endoreduplications and activation of silent genes in heterochromatic regions (Hori, 1983). It inhibits methylation of cytosine in Escherichia coli and is mutagenic in a Salmonella reverse mutation system using a trpE8 mutation (Podger, 1983). The yeast Saccharomyces cerevisiae provides the possibility to test chemicals for their ability to induce a wide spectrum of genetic effects ranging from simple point mutations and mitotic recom-
Research sponsored by Contract No. NO 1-ES-1-5005, Development of a Yeast Aneuploidy System, from the National Institute of Environmental Health Sciences, Research Triangle Park, NC (U.S.A.) 0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
bination to chromosomal malSegregation. Therefore, it was interesting to investigate 5-azacytidine in respect to the range of genetic alterations it can induce in a eukaryote. Material and methods
Strains. The diploid strain D7 has been described by Zimmermann et al. (1975). It carries two non-complementing alleles, trp5-12 and trp5-27, which cause a requirement for trypt0phan. Prototrophy can be restored by intragenic recombination, mostly non-reciprocal gene conversion, with an admixture of reciprocal intragenic crossing-over and reverse mutation. There is a pair of complementing alleles, ade2-40 (causing an Iadenine requirement and deep-red colonies) arid ade2-119 (not causing a stringent requirement for adenine but causing a pink pigmentation of colonies). Both alleles are recessive, and D7 forms white colonies. However, mitotic crossing-over, mitotic gene conversion and rarer events of point mutation,
22 chromosomal deletion or loss of an entire chromosome can lead to the expression of one of the two alleles. This can readily be detected by the appearance of pigmented colonies. Only reciprocal mitotic crossing-over can be recognized as such by the appearance o f colonies with simultaneously occurring red and pink sectors. All other types of colony can by caused by any of the above-mentioned genetic events. Therefore, all pigmented colonies are combined into one category which is said to reflect mitotic segregation. There is also a homozygous condition for allele ilvl-92 which causes a requirement for isoleucine. Restoration o f prototrophy can be brought about by mutation of the mutant allele itself or in some suppressor genes. Strain D61 .M is similar to strain D6 described by Parry and Zimmermann (1976). It can be used to monitor mitotic chromosome loss. The relevant gen0type is:
cyh2 + leul ade6 ade2 iivl-92 M A L his centromere + trp5 + + ade2 ilvl-92 M A L + The marker cyh2, a recessive resistance to 2 ppm cycloheximide, is not expressed in strain D61.M, and this strain is sensitive. Resistance to cycloheximide can be caused by the expression of the recessive cyh2 allele by a number of genetic events:
mitotic crossing-over, mitotic gene conversion, mutation in the dominant wild-type allele, loss of a chromosomal fragment or loss of the entire chromosome. The latter genetic effect can be distinguished from all others by the simultaneous expression of the recessive marker ade6. Normally, D61.M forms red colonies because of the homozygous condition of ade2. When ade6 is expressed, colonies are white. White resistant colonies can be caused by a single event of mitotic chromosome loss or by two simultaneous events of mitotic recombination or mutation. Mitotic chromosome loss is indicated when white resistant colonies express centromere-linked leul (Parry and Zimmermann, 1976). Media and culturing. The composition of synthetic and rich growth media used in mutagenicity testing with yeast have been described by Zimmermann (1975). Cultures were started with an inoculum of about 200 cells per 5 ml yeast-extractpeptone-glucose medium in test tubes and grown to about 5 x 107 - 1 x 10a cells/ml. At this point, 0.1-ml samples were removed and plated onto a medium without tryptophan for D7 or on a yeastextract-peptone-glucose medium with 1.5 ppm cycloheximide for D61.M. The plates were incubated until they could be scored for prototrophic or resistant ceils, respectively. During this time, the test tubes were stored in a refrigerator and were us-
TABLE 1 THE EFFECTS OF 5-AZACYTIDINE ON THE INDUCTION OF MITOTIC SEGREGATION AND CHROMOSOMAL MALSEGREGATIONIN YEAST STRAIN D61.M mg/ml
Titer ( x 106)
Resistant colonies Direct
1:5
Resistants (x l0 -5)
Monosomics (x l0 -6)
0.0 0.4 0.8
18.9 29.5 30.0
1381 (1) 1399 (0) 1445 (2)
289 (0) 334 (0)
24.31 16.35 18.57
0.18 0.22
1.5 2.5 5.0
28.6 15.2 20.4
1939 (2) (6) (2)
411 (0) 695 (2) 1244 (0)
25.67 76.38 101.80
0.23 1.31 0.33
4.8
328 (15)
29.52
10.34
Bavistan 20 ppm Cells were plated, without a washing, directly or after a 1: 5 dilution, onto the selective medium with cycloheximide.The actual colony counts from three petri dishes are given under column heading 'Resistant colonies'. White and leucine-requiring colonies are listed in parentheses. Cell titers were computed from colony counts on a non-selective medium seeded with appropriate dilutions. Total exposure time: 17 h.
23
ed within 2 weeks. In this way it was possible always to use cultures with low spontaneous backgrounds.
TABLE 2
Mutagenic treatments. 5-Azacytidine (Serva, Heidelberg) was dissolved in the synthetic growth medium. The initial cell titers at the start of the treatment were about 1 x 106 ceils per ml. After an overnight incubation of 16-17 h on a reciprocal shaker at 28°C, the cultures were placed in ice and plated without washing, directly or after appropriate dilution, on the selective media and a non-selective medium to determine viable titers.
mg/ml
Results and discussion
Cells were plated, without washing or dilution~ onto the synthetic media lacking tryptophan to select for convertants or isoleucine to select for reverse mutants. Cell titers were computed from colony counts on a non-selective medium seeded with appropriate dilution. The colony counts ate from 3 petri dishes in each category. Total exposure time: 17 h.
The effects of 5-azacytidine on strain D61 .M are shown in Table 1. There was a distinct increase in the frequencies of red resistant colonies in cultures with higher concentrations o f the analog. This is indicative o f the induction o f mitotic recombination or mutation. However, there was no increase in white or leucine-requiring resistant colonies which would have signaled induction of mitotic c h r o m o s o m e loss. Bavistan is known to induce mitotic c h r o m o s o m e loss in yeast (Wood, 1982) and was used as a positive control agent. It induced monosomics in the experiment reported showing that the test was functioning properly. The genetic effects induced by 5-azacytidine in
I N D U C T I O N OF MITOTIC GENE CONVERSION AND REVERSE M U T A T I O N W I T H 5-AZACYTIDiNE IN YEAST STRAIN D7 Titer
Convertants
Revertants
( × 106) Colonies
( × 10 -~)
Colonies
( × 10 -6 )
0.0 1.0 2.0
27.5 25.7 27.3
15 218 273
0.18 1.69 2.00
• 48 130
1.27 3.73 9.52
3.0 4.0 5.0
25.2 24.6 25.2
395 525 710
3.13 4.27 5.64
200 292 439
15.86 23.76 34.87
7.5 10.0
28.6 29.2
1061 9009
7.41 6.23
657 774
45.88 53.09
strain D7 are shown in Table 2. There was a strong induction of both mitotic gene conversion and reverse mutation. It is important to note that the spontaneous frequency of mitotic convertants in the control was about one-fifth of the normal level. Th~s suggested that the culture used contained a high percentage of cells that were homoallelic for one of the trp5 alleles. 29 colonies from the control plating on the
TABLE 3 I N D U C T I O N OF M I T O T I C S E G R E G A T I O N W I T H 5-AZACYTIDINE IN YEAST STRAIN D7 mg/ml
Unselected colonies Total
Aberrant
Percent
Aberrant convertant colonies
Percent
Aberrant revertant colonies
Percent
0.0 1.0 2.0
826 1287 1365
0 0 4
0,29
0 2 1
0.92 0.37
0 0 0
-
3.0 4.0 5.0
1261 1229 1259
5 6 5
0.40 0.49 0.40
1 2 5
0.25 0.38 0,70
1 0 4
0.50 0.91
7.5 10.0
1432 1458
10 7
0.70 0.48
8 9
0,75 0.99
6 10
0.91 1.29
The data shown here are from the experiment described in Table 2. Mitotic segregation was followed among unselected (~olumns 2-4), convertant and reverse mutant colonies. The total numbers of convertant and reverse mutant colonies are listed in Table 2 (columns 3 and 5) and are not repeated here.
24 non-selective medium were tested for heteroallelic instability. Only 7 o f those single-colony clones showed the typical heteroallelic instability. This clearly indicated that continued selection for cultures with a low spontaneous background in convertants had resulted in cultures that consisted mostly of homoallelic and stable cells which were unable to signal mitotic gene conversion at the trp5 locus. Mitotic segregation was observed among colonies on all media: the non-selective medium where all viable cells could grow as well as on the media selective for mitotic convertants and reverse mutants (Table 3). This unequivocally showed that 5-azacytidine did not select cycloheximide-resistant cells or cells prototrophic for tryptophan or isoleucine but induced genetic effects. 5-Azacytidine has to be considered as a strong mutagen in yeast. It induces genetic effects at the D N A level, but it does not induce chromosomal malsegregation. Apparently, its primary effects are directed at D N A integrity itself possibly through incorporation into D N A or interference with D N A metabolism. As reviewed by Hori (1983), it has various effects on chromosome condensation, ac-
tivation of genes in heterochromatic regions and cell differentiation. Yeast has become a prime organism in the study of eukaryotic molecular genetics, and 5-azacytidine may become a powerful research tool in this organism.
References Hori, T.-A. (1983) Induction of chromosomedecondensation, sister-chromatid exchanges and endoreduplication by 5-azacytidine, an inhibitor of DNA-methylation, Mutation Res., 121, 47-52. Parry, J.M., and F.K. Zimmermann (1976) The detection of monosomic colonies by mitotic non-disjunction in the yeast Saccharomyces cerevisiae, Mutation Res., 36, 49-66. Podger, D.M. (1983) Mutagenicity of 5-azacytidine in Salmonella typhimurium, Mutation Res., 121, 1-6. Wood, J.S. (1982) Genetic effects of methylbenzimidazole-2-ylcarbamate on Saccharomyces cerevisiae, Mol. Cell Biol., 2, 1069-1074. Zimmermann, F.K. (1975) Procedures used in the induction of mitotic recombination and mutation in the yeast Saccharomyces cerevisiae, Mutation Res., 31, 71-86. Zimmermann, F.K., R. Kern and H. Rasenberger (1975) A yeast strain for the simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381-388.