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DNA-length polymorphisms of chromosome III in the yeast Saccharomyces cerevisiae
JOUgNALor FERMENTATIONANDBIOENGINEERING VO1. 75, NO. 6, 395-398. 1993
DNA-Length Polymorphisms of Chromosome III in the Yeast Saccharomyces cerevisiae AKIRA YODA, TATSUYA KONTANI, SEIKI OHSHIRO, NOBUHARU YAGISHITA, TAISUKE HISATOMI, AND M I C H I O TSUBOI* Department o f Biotechnology, Faculty o f Engineering, F u k u y a m a University, Gakuen-cho, F u k u y a m a City, Hiroshima 729-02, Japan
Received 2 March 1992/Accepted 18 March 1993 The chromosome-sized DNAs of sporulation-deficient mutants, which had been isolated by mutagenizing spores of a homothallic diploid strain (MT9$a-3D) of Saccharomyces cerevisiae, were analyzed by pulsed-field gel electrophoresis. While the size of chromosome HI D N A of the parent strain was 450 kb, some mutants had one or more chromosome IH DNAs of 350 kb, 450 kb, 530 kb and 630 kb. No size variation was observed for other chromosomes. Chromosome IH D N A s of laboratory-stock strains, except MT9ga-3D, were in the neighborhood of 350 kb. Size variation of chromosome IIl was observed at a high frequency in spore clones derived from MT9ga-3D strain. The results suggest that DNA-length polymorphisms of chromosome III are generated by the loss or addition of a specific D N A unit of approximately 100 kb.
In a previous study, we isolated sporulation-deficient mutants by treating spores of a homothallic diploid strain with UV light, and identified 23 sporulation specific genes ( s p o T l - s p o T 2 3 ) by genetic analyses in the yeast Saccharom y c e s cerevisiae (1). These mutants cease meiosis and sporulation at a specific stage when they are cultured in a sporulation medium. In order to determine whether these mutations affect the organization of chromosomal DNAs, we examined chromosome-sized DNAs of the parent and mutant strains using pulsed-field gel electrophoresis. All sporulation mutants had the same pattern of chromosomesized DNAs before and after cultivation in the sporulation medium. However, we noticed that the length of chromosome III DNA of the parent strain, MT98a-3D, was 450 kb, a value which was remarkably different from the reported value of 350 kb (2--4), and that the length of chromosome III DNA of some mutants varied from that of the parent strain. Electrophoretic karyotypes of several yeasts, including S a c c h a r o m y c e s , Candida, and K i u y v e r o m y c e s species, have been studied, and it was shown that chromosome-length polymorphisms are not rare events in these yeasts (5-8). In the present communication, we have analyzed the characteristics of MT98a-3D strain from the point of view of generation of chromosome-length polymorphisms. We found that DNA-length polymorphisms of chromosome III are generated during meiosis, sporulation or the spore germination process of a strain which carries the 450 kb DNA of chromosome III.
Pulsed-field gel electrophoresis Agarose plugs containing intact chromosomal DNAs were prepared from cells grown to an early stationary phase in YPD medium according to the method of Carle and Olson (5). Chromosome-sized DNAs were separated by gel electrophoresis with a contour-clamped homogeneous electric field (CHEF-DRII system, Bio-Rad Laboratories, Richmond, CA, USA). The gel used was 1.0% agarose (Ultra Pure, Bethesda Research Laboratories, MD, USA) in a buffer composed of 25 mM Tris, 25 mM boric acid and 0.5 mM EDTA. Switch time was 60 s for 15 h at 200 V followed by 90 s for 8 h at 200 V. The running temperature was 15°C. The gel was then stained with 0.5/zg/ml ethidium bromide for 30 min and photographed. The chromosomal DNAs of YNN295, commercially available in the state of agarose plugs from Bio-Rad Laboratories, were used as size standards. TABLE 1. List of strains used Strain MT98a-3D MTM3-1228 MTM3-1466 MTM4-185 MTM4-263 MTM4-868 MTM4-1084 MTM4-1982 MTM2-786 MTM4-714 MTM3-937 MTM3-1342 MTM3-1422 MTM4-255 MTM4-1858 MTM4-1431 MT607a-7D MT13 C5672-5D
MATERIALS AND METHODS Yeast strains and culture conditions
The strains of
S a c c h a r o m y c e s cerevisiae used in this study are listed in
Table 1. YPD medium contained 1% Difco yeast extract, 2% Difco Bacto-peptone and 2% glucose. KAc medium containing 1% potassium acetate was used as the sporulation medium. For solid media, agar was added to the medium at 2%. The incubation temperature was 26°C.
Genotype HO a adel trpl ural HO spoT1 adel trpl ural HO spoT2 adel trpl ural HO spoT3 adel trpl ural HO spoT4 adel trpl ural 1t0 spo T7 adel trpl ural HO spoT9 adel trpl ural HO spoTll adel trpl ural 1t0 spoTl2 adel trpl ural HO spoTl5 adel trpl ural 1-10 spoTl7 adel trpl ural HO spoT20 adel trpl ural HO spoT21 adel trpl ural HO spoT22 adel trpl ural HO spoT23-1 adel trpl ural HO spoT23-3 adel trpl ural HO leu2 his4 HO his4 lysl ura3 ho MA Ta his4 leu2 thr4 ura3
Reference 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
present study 11, 12 presentstudy
a Strains carrying the HO gene are diploid homozygousfor all the genes, except MA Ta and M A T a, for which they are heterozygous.
* Corresponding author. 395
396
YODA ET AL.
J. FERMENT. BIOElqG.,
Southern blot hybridization Chromosomal DNAs in the gel were transferred to a membrane (GeneScreen, New England Nuclear, Boston, MA, USA) by the method of Southern (9). A radioactive method using 32p-labeled probes (10, 11) and a non-radioactive DNA labeling and a detection kit (Boehringer Mannheim, G m b H , Germany) were used for the hybridization analysis. The procedure recommended by the supplier was employed. Plasmid DNAs used as probes were p2.5, pDX, pKI101, YIp5 and YRp7 containing M A Ta, M A Ta, LEU2, URA3 and TRP1 genes of S. cerevisiae, respectively.
1 2 3 4 5 6
1 2 3 4 5 6
(530kb
~450kb ~350kb
A RESULTS AND DISCUSSION
FIG. 1. Gel separation of chromosome-sized DNAs from a parent and sporulation deficient mutant strains. (A) The ethidium-bromide staining pattern of the gel. (B) An autoradiogram probed with the MA Ta gene after blotting to a membrane from the gel. Lane 1, a size standard YNN295; lane 2, a parent strain MT98a-3D; lane 3, MTM4-868 (spoT7); lane 4, MTM2-786 (spoTl2); lane 5, MTM3-937 (spoT17); and lane 6, MTM4-1858 (spoT23).
Chromosome-sized DNAs of sporulation-deficient mutants Strains MTM4--868, MTM2-786, MTM3-937 and MTM4-1858, which carried spoT7, spoT12, spoT17 and spoT23, respectively, were sporulation-deficient mutants isolated from a parent strain (MT98a-3D) by UVlight irradiation, and have been maintained in our laboratory (1, 11, 12). Chromosome-sized DNAs from these mutants and the parent strain were separated by pulsed-field gel electrophoresis and stained with ethidium bromide (Fig. 1A). A 350 kb band was observed in MTM2-786 and MTM4-1858, but not in MT98a-3D, MTM4-868 and MTM3-937. This band corresponded to chromosome III of the size-standard strain, YNN295. These separated DNAs were blotted to a membrane and Southern-hybridized with a probe of plasmid p2.5 containing the M A Ta gene located on the chromosome III (Fig. 1B). The M A Ta gene hybridized to 350 kb DNA of the standard strain YNN295. On the contrary, this gene hybridized to 450 kb DNA of MT98a-3D, to 530 kb DNA of MTM4-868, to both 450 kb and 350 kb DNAs of MTM2-786, to 450 kb DNA of MTM3-937, and to 350 kb DNA of MTM4-1858. Next, the plasmid pK1101 which contains the LEU2 gene, also located on chromosome III, was used as a probe. The hybridization pattern with the LEU2 gene was the same as with the M A T gene (Fig. 2A). Hence, the possibility that the mating type locus might transpose to other chromosomes in sporulation mutants was ruled out. In order
1
2
3
A
4
5
1
2
B
to determine whether the DNA lengths of other chromosomes had varied in these sporulation mutants, chromosome-sized DNAs were analyzed by Southern hybridization using probes, specifically YIp5 and YRp7 containing the URA3 gene on chromosome V and the TRP1 gene on chromosome IV, respectively. Each gene hybridized to DNAs at the same position among the tested strains (Fig. 2B and 2C). Thus, the DNA lengths of the two chromosomes, IV and V, did not vary. In addition, no significant variation was observed in band patterns stained with ethidium bromide, except for chromosome III (Fig. 1A). These results indicate that variations in DNA length occurrs specifically in chromosome III. Chromosome-sized DNAs of other sporulation-deficient mutants derived from MT98a-3D strain were analyzed using the M A Ta gene as a probe. The lengths of DNAs hybridized to the M A Ta gene are summarized in Table 2. DNAs of 350 kb, 450 kb, 530 kb and 630 kb were detected as hybridization bands. Nine of the 15 strains examined had any one of these four bands, while the others
3
B
4
5
1
2
3
4
5
C
FIG. 2. Southern hybridization of chromosome-sized DNAs from a parent and sporulation-deficient mutant strains probed with (A) LEU2, (B) URA3, and (C) TRPI genes. Lane 1, a parent strain MT98a-3D; lane 2, MTM4-868 (spoT7); lane 3, MTM2-786 (spoT12); lane 4, MTM3-937 (spoT17); and lane 5, MTM4-1858 (spoT23).
VoL 75, 1993
POLYMORPHISMS OF CHROMOSOME III IN YEAST
TABLE 2. DNA lengths hybridized to the M A T a g e n e in 14 sporulation-deficientmutants derived fromMT98a-3D Strain
spo mutation
MT98a-3D MTM3-1228 MTM3-1466 MTM4-185 MTM4-263 MTM4-868 MTM4-1084 MTM4-1982 MTM2-786 MTM4-714 MTM3-937 MTM3-1342 MTM3-1422 MTM4-255 MTM4-1858 MTM4-1431
Wild
DNA length (kb) 350
450
530
630
+a
spoT1 spoT2 spoT3 spoT4 spoT7 spoT9 spoTll spoT12 spoT15 spoT17 spoT20 spoT21 spoT22 spoT23-1 spo T23-3
+ + + +
+ + +
+ + +
+ +
+ + + +
+
+ + +
+
" + indicates hybridization occurred.
had two or three of the four bands. These strains have been maintained in our laboratory after several singlecell colony isolation procedures. Hence, strains showing multiple bands are thought to be trisomic or polysomic for chromosome III. It is interesting that the DNA lengths of chromosome III were 350 kb, 450 kb, 530 kb or 630 kb, and that no other lengths were observed. D N A lengths of chromosome III in our laboratory stock strains The DNA length of chromosome III of MT98a3D strain was 450 kb in contrast with the value of 350 kb reported (2-4). We examined the DNA lengths of chromosome III in ten strains of our laboratory stock. The representative patterns of hybridization with the M A T a gene are shown in Fig. 3. All strains, except MT98a-3D, possessed about 350 kb DNA of chromosome III. Hence, it is concluded that the DNA length of 450 kb of chromosome III in the MT98a-3D strain was uncommon. D N A lengths of chromosome III of spore clones from MT98a-3D strain Our sporulation-deficient mutants 1
2
3
4
5
397
had been isolated by treating spores of the MT98a-3D strain with UV light. We examined DNA lengths of chromosome III of spore clones derived from the MT98a-3D strain. MT98a-3D was cultured on KAc medium and the spores produced were dissected with a micromanipulator. DNA lengths of chromosome III of 16 spore clones derived from 4 asci were examined by hybridization using the M A T a gene. As shown in Fig. 4, 13 of 16 spore clones possessed 350 kb and/or 530 kb DNA of chromosome III. All of these spore clones had sporulation ability. Hence, it is concluded that these polymorphisms of chromosome III do not affect sporulation. The frequency of spore clones indicated the DNA-length variation of chromosome III was about 80%. On the contrary, when spore clones from the MT13 strain which possessed 350 kb DNA of chromosome III were examined, no variation in the DNA length of chromosome III was observed (data not shown). Spore clones 1A, 1C, 2A and 4D from the MT98a-3D strain showed three hybridization bands with the probe M A T gene, and there was a difference in intensity among hybridization bands in some spore clones (Fig. 4). When several single-cell colonies were isolated from the spore clone 2A and their chromosome III examined, this spore clone was shown to be a mixture of cells containing various lengths of chromosome III DNA. Furthermore, some cells still exhibited three hybridization bands (data not shown). The sporulation deficient mutant MTM4-1982, which has been maintained after several single-cell colony isolations, also possessed three chromosome III DNA (Table 2). It is not easy to explain why three kinds of chromosome III DNA exist in a single cell. The above results indicate that variation in DNA lengths of chromosome III occur during meiosis, sporulation or the spore germination process in MT98a-3D strain, resulting in chromosome III DNA-length polymorphisms in spore clones at a high frequency. The length of chromosome III DNA found in the present experiments were 350 kb, 450 kb, 530 kb and 630 kb. No intermediate lengths were observed. This phenomenon indicates that the chromosome III DNA-length polymorphisms are generated by the loss or addition of a specific DNA unit at approximately 100 kb. 1 ABC
D
2 ABCD
3 ABCD
4 AB CD
~530kb ,~
450kb
,~
350kb
~,450kb ~350kb
i,il FIG. 3. The DNA lengths of chromosome III in our laboratory stock strains. Chromosome-sized DNAs of our laboratory stock strains were separated and Southern hybridized with the M A Ta gene. Lane 1, C5672-5D; lane 2, MTI3; lane 3, MT607a-7D; lane 4, MT98a3D; and lane 5, a size standard YNN295.
FIG. 4. DNA lengths of chromosome III of spore clones derived from MT98a-3D strain. Four asci 0--4) from MT98a-3D strain were dissected into four spores (A-D) each. Chromosome-sized DNAs of spore clones were separated and Southern hybridized with the M A Ta gene.
398
J. FERMENT.BIOENO.,
YODA ET AL.
In homothaUic strains o f S. cerevisiae, one mating type gene on c h r o m o s o m e III frequently converts to another mating type through the action o f H O - c o d e d en d o n u clease (13). Since the MT98a-3D strain is a homothallic strain, we are currently studying whether the product o f the H O gene affects the generation o f D N A - l e n g t h polymorphisms in c h r o m o s o m e III. Recently, a physical m a p o f the g e n o m e and the entire D N A sequence o f c h r o m o s o m e III o f S. cerevisiae have been determined (3, 4). H o w e v e r , the structure o f c h r o m o some III carrying 450 kb D N A remains to be elucidated. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research (No. 01540570) to M. Tsuboi from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Tsuboi, M.: The isolation and genetic analysis of sporulationdeficient mutants in Saccharomyces cerevisiae. Mol. Gen. Genet., 191, 17-21 (1983). 2. Mortimer, R. K., Contopoulou, C. R., and King, J. S.: Genetic map of Saccharomyces cerevisiae, edition 11. Yeast, 8, 817-902 (1992). 3. Link, A.J. and Olson, M.V.: Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution. Genetics,
127, 681-698 (1991). 4. Oliver, S. G. ctal.: The complete DNA sequence of yeast chromosome III. Nature, 357, 38-46 (1992). 5. Carle, G. F. and OIson, M. V.: An electrophoretic karyotype for yeast. Proc. Natl. Acad. Sci. USA, 82, 3756-3760 (1985). 6. Ono, B. and Ishlno-Arao, Y.: Inheritance of chromosome length polymorphisms in Saccharomyces cerevisiae. Curr. Genet., 14, 413-418 (1988). 7. Iwaguchi, S., Homma, M., and Tanaka, K.: Variation in the electrophoretic karyotype analysed by the assignment of DNA probes in Candida albicans. J. Gen. Microbiol., 136, 2433-2442 (1990). 8. Sor, F. and Fukuhara, H.: Analysis of chromosomal DNA patterns of the genus Kluyveromyees. Yeast, 5, 1-10 (1989). 9. Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98, 503-517 (1975). 10. Sambrook, J., Fritsch, E. F., and Maniatis, T.: Molecular Cloning. A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989). 11. Tsuboi, M. and Takahashi, T.: Genetic analysis of the nonsporulating phenotype of brewer's yeasts. J. Ferment. Technol., 66, 605-613 (1988). 12. Tanaka, H. and Tsuboi, M.: Cloning and mapping of the sporulation gene, 8poTT, in Saeeharomyees cerevisiae. Mol. Gen. Genet., 199, 21-25 (1985). 13. Kostriken, R., Strathern, J. N., Klar, A. J. S., Hicks, J. B., and Hetfron, F.: A site-specific endonuclease essential for matingtype switching in Saeeharomyee8 eerevisiae. Cell, 35, 167-174 (1983).
DNA-Length Polymorphisms of Chromosome III in the Yeast Saccharomyces cerevisiae AKIRA YODA, TATSUYA KONTANI, SEIKI OHSHIRO, NOBUHARU YAGISHITA, TAISUKE HISATOMI, AND M I C H I O TSUBOI* Department o f Biotechnology, Faculty o f Engineering, F u k u y a m a University, Gakuen-cho, F u k u y a m a City, Hiroshima 729-02, Japan
Received 2 March 1992/Accepted 18 March 1993 The chromosome-sized DNAs of sporulation-deficient mutants, which had been isolated by mutagenizing spores of a homothallic diploid strain (MT9$a-3D) of Saccharomyces cerevisiae, were analyzed by pulsed-field gel electrophoresis. While the size of chromosome HI D N A of the parent strain was 450 kb, some mutants had one or more chromosome IH DNAs of 350 kb, 450 kb, 530 kb and 630 kb. No size variation was observed for other chromosomes. Chromosome IH D N A s of laboratory-stock strains, except MT9ga-3D, were in the neighborhood of 350 kb. Size variation of chromosome IIl was observed at a high frequency in spore clones derived from MT9ga-3D strain. The results suggest that DNA-length polymorphisms of chromosome III are generated by the loss or addition of a specific D N A unit of approximately 100 kb.
In a previous study, we isolated sporulation-deficient mutants by treating spores of a homothallic diploid strain with UV light, and identified 23 sporulation specific genes ( s p o T l - s p o T 2 3 ) by genetic analyses in the yeast Saccharom y c e s cerevisiae (1). These mutants cease meiosis and sporulation at a specific stage when they are cultured in a sporulation medium. In order to determine whether these mutations affect the organization of chromosomal DNAs, we examined chromosome-sized DNAs of the parent and mutant strains using pulsed-field gel electrophoresis. All sporulation mutants had the same pattern of chromosomesized DNAs before and after cultivation in the sporulation medium. However, we noticed that the length of chromosome III DNA of the parent strain, MT98a-3D, was 450 kb, a value which was remarkably different from the reported value of 350 kb (2--4), and that the length of chromosome III DNA of some mutants varied from that of the parent strain. Electrophoretic karyotypes of several yeasts, including S a c c h a r o m y c e s , Candida, and K i u y v e r o m y c e s species, have been studied, and it was shown that chromosome-length polymorphisms are not rare events in these yeasts (5-8). In the present communication, we have analyzed the characteristics of MT98a-3D strain from the point of view of generation of chromosome-length polymorphisms. We found that DNA-length polymorphisms of chromosome III are generated during meiosis, sporulation or the spore germination process of a strain which carries the 450 kb DNA of chromosome III.
Pulsed-field gel electrophoresis Agarose plugs containing intact chromosomal DNAs were prepared from cells grown to an early stationary phase in YPD medium according to the method of Carle and Olson (5). Chromosome-sized DNAs were separated by gel electrophoresis with a contour-clamped homogeneous electric field (CHEF-DRII system, Bio-Rad Laboratories, Richmond, CA, USA). The gel used was 1.0% agarose (Ultra Pure, Bethesda Research Laboratories, MD, USA) in a buffer composed of 25 mM Tris, 25 mM boric acid and 0.5 mM EDTA. Switch time was 60 s for 15 h at 200 V followed by 90 s for 8 h at 200 V. The running temperature was 15°C. The gel was then stained with 0.5/zg/ml ethidium bromide for 30 min and photographed. The chromosomal DNAs of YNN295, commercially available in the state of agarose plugs from Bio-Rad Laboratories, were used as size standards. TABLE 1. List of strains used Strain MT98a-3D MTM3-1228 MTM3-1466 MTM4-185 MTM4-263 MTM4-868 MTM4-1084 MTM4-1982 MTM2-786 MTM4-714 MTM3-937 MTM3-1342 MTM3-1422 MTM4-255 MTM4-1858 MTM4-1431 MT607a-7D MT13 C5672-5D
MATERIALS AND METHODS Yeast strains and culture conditions
The strains of
S a c c h a r o m y c e s cerevisiae used in this study are listed in
Table 1. YPD medium contained 1% Difco yeast extract, 2% Difco Bacto-peptone and 2% glucose. KAc medium containing 1% potassium acetate was used as the sporulation medium. For solid media, agar was added to the medium at 2%. The incubation temperature was 26°C.
Genotype HO a adel trpl ural HO spoT1 adel trpl ural HO spoT2 adel trpl ural HO spoT3 adel trpl ural HO spoT4 adel trpl ural 1t0 spo T7 adel trpl ural HO spoT9 adel trpl ural HO spoTll adel trpl ural 1t0 spoTl2 adel trpl ural HO spoTl5 adel trpl ural 1-10 spoTl7 adel trpl ural HO spoT20 adel trpl ural HO spoT21 adel trpl ural HO spoT22 adel trpl ural HO spoT23-1 adel trpl ural HO spoT23-3 adel trpl ural HO leu2 his4 HO his4 lysl ura3 ho MA Ta his4 leu2 thr4 ura3
Reference 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
present study 11, 12 presentstudy
a Strains carrying the HO gene are diploid homozygousfor all the genes, except MA Ta and M A T a, for which they are heterozygous.
* Corresponding author. 395
396
YODA ET AL.
J. FERMENT. BIOElqG.,
Southern blot hybridization Chromosomal DNAs in the gel were transferred to a membrane (GeneScreen, New England Nuclear, Boston, MA, USA) by the method of Southern (9). A radioactive method using 32p-labeled probes (10, 11) and a non-radioactive DNA labeling and a detection kit (Boehringer Mannheim, G m b H , Germany) were used for the hybridization analysis. The procedure recommended by the supplier was employed. Plasmid DNAs used as probes were p2.5, pDX, pKI101, YIp5 and YRp7 containing M A Ta, M A Ta, LEU2, URA3 and TRP1 genes of S. cerevisiae, respectively.
1 2 3 4 5 6
1 2 3 4 5 6
(530kb
~450kb ~350kb
A RESULTS AND DISCUSSION
FIG. 1. Gel separation of chromosome-sized DNAs from a parent and sporulation deficient mutant strains. (A) The ethidium-bromide staining pattern of the gel. (B) An autoradiogram probed with the MA Ta gene after blotting to a membrane from the gel. Lane 1, a size standard YNN295; lane 2, a parent strain MT98a-3D; lane 3, MTM4-868 (spoT7); lane 4, MTM2-786 (spoTl2); lane 5, MTM3-937 (spoT17); and lane 6, MTM4-1858 (spoT23).
Chromosome-sized DNAs of sporulation-deficient mutants Strains MTM4--868, MTM2-786, MTM3-937 and MTM4-1858, which carried spoT7, spoT12, spoT17 and spoT23, respectively, were sporulation-deficient mutants isolated from a parent strain (MT98a-3D) by UVlight irradiation, and have been maintained in our laboratory (1, 11, 12). Chromosome-sized DNAs from these mutants and the parent strain were separated by pulsed-field gel electrophoresis and stained with ethidium bromide (Fig. 1A). A 350 kb band was observed in MTM2-786 and MTM4-1858, but not in MT98a-3D, MTM4-868 and MTM3-937. This band corresponded to chromosome III of the size-standard strain, YNN295. These separated DNAs were blotted to a membrane and Southern-hybridized with a probe of plasmid p2.5 containing the M A Ta gene located on the chromosome III (Fig. 1B). The M A Ta gene hybridized to 350 kb DNA of the standard strain YNN295. On the contrary, this gene hybridized to 450 kb DNA of MT98a-3D, to 530 kb DNA of MTM4-868, to both 450 kb and 350 kb DNAs of MTM2-786, to 450 kb DNA of MTM3-937, and to 350 kb DNA of MTM4-1858. Next, the plasmid pK1101 which contains the LEU2 gene, also located on chromosome III, was used as a probe. The hybridization pattern with the LEU2 gene was the same as with the M A T gene (Fig. 2A). Hence, the possibility that the mating type locus might transpose to other chromosomes in sporulation mutants was ruled out. In order
1
2
3
A
4
5
1
2
B
to determine whether the DNA lengths of other chromosomes had varied in these sporulation mutants, chromosome-sized DNAs were analyzed by Southern hybridization using probes, specifically YIp5 and YRp7 containing the URA3 gene on chromosome V and the TRP1 gene on chromosome IV, respectively. Each gene hybridized to DNAs at the same position among the tested strains (Fig. 2B and 2C). Thus, the DNA lengths of the two chromosomes, IV and V, did not vary. In addition, no significant variation was observed in band patterns stained with ethidium bromide, except for chromosome III (Fig. 1A). These results indicate that variations in DNA length occurrs specifically in chromosome III. Chromosome-sized DNAs of other sporulation-deficient mutants derived from MT98a-3D strain were analyzed using the M A Ta gene as a probe. The lengths of DNAs hybridized to the M A Ta gene are summarized in Table 2. DNAs of 350 kb, 450 kb, 530 kb and 630 kb were detected as hybridization bands. Nine of the 15 strains examined had any one of these four bands, while the others
3
B
4
5
1
2
3
4
5
C
FIG. 2. Southern hybridization of chromosome-sized DNAs from a parent and sporulation-deficient mutant strains probed with (A) LEU2, (B) URA3, and (C) TRPI genes. Lane 1, a parent strain MT98a-3D; lane 2, MTM4-868 (spoT7); lane 3, MTM2-786 (spoT12); lane 4, MTM3-937 (spoT17); and lane 5, MTM4-1858 (spoT23).
VoL 75, 1993
POLYMORPHISMS OF CHROMOSOME III IN YEAST
TABLE 2. DNA lengths hybridized to the M A T a g e n e in 14 sporulation-deficientmutants derived fromMT98a-3D Strain
spo mutation
MT98a-3D MTM3-1228 MTM3-1466 MTM4-185 MTM4-263 MTM4-868 MTM4-1084 MTM4-1982 MTM2-786 MTM4-714 MTM3-937 MTM3-1342 MTM3-1422 MTM4-255 MTM4-1858 MTM4-1431
Wild
DNA length (kb) 350
450
530
630
+a
spoT1 spoT2 spoT3 spoT4 spoT7 spoT9 spoTll spoT12 spoT15 spoT17 spoT20 spoT21 spoT22 spoT23-1 spo T23-3
+ + + +
+ + +
+ + +
+ +
+ + + +
+
+ + +
+
" + indicates hybridization occurred.
had two or three of the four bands. These strains have been maintained in our laboratory after several singlecell colony isolation procedures. Hence, strains showing multiple bands are thought to be trisomic or polysomic for chromosome III. It is interesting that the DNA lengths of chromosome III were 350 kb, 450 kb, 530 kb or 630 kb, and that no other lengths were observed. D N A lengths of chromosome III in our laboratory stock strains The DNA length of chromosome III of MT98a3D strain was 450 kb in contrast with the value of 350 kb reported (2-4). We examined the DNA lengths of chromosome III in ten strains of our laboratory stock. The representative patterns of hybridization with the M A T a gene are shown in Fig. 3. All strains, except MT98a-3D, possessed about 350 kb DNA of chromosome III. Hence, it is concluded that the DNA length of 450 kb of chromosome III in the MT98a-3D strain was uncommon. D N A lengths of chromosome III of spore clones from MT98a-3D strain Our sporulation-deficient mutants 1
2
3
4
5
397
had been isolated by treating spores of the MT98a-3D strain with UV light. We examined DNA lengths of chromosome III of spore clones derived from the MT98a-3D strain. MT98a-3D was cultured on KAc medium and the spores produced were dissected with a micromanipulator. DNA lengths of chromosome III of 16 spore clones derived from 4 asci were examined by hybridization using the M A T a gene. As shown in Fig. 4, 13 of 16 spore clones possessed 350 kb and/or 530 kb DNA of chromosome III. All of these spore clones had sporulation ability. Hence, it is concluded that these polymorphisms of chromosome III do not affect sporulation. The frequency of spore clones indicated the DNA-length variation of chromosome III was about 80%. On the contrary, when spore clones from the MT13 strain which possessed 350 kb DNA of chromosome III were examined, no variation in the DNA length of chromosome III was observed (data not shown). Spore clones 1A, 1C, 2A and 4D from the MT98a-3D strain showed three hybridization bands with the probe M A T gene, and there was a difference in intensity among hybridization bands in some spore clones (Fig. 4). When several single-cell colonies were isolated from the spore clone 2A and their chromosome III examined, this spore clone was shown to be a mixture of cells containing various lengths of chromosome III DNA. Furthermore, some cells still exhibited three hybridization bands (data not shown). The sporulation deficient mutant MTM4-1982, which has been maintained after several single-cell colony isolations, also possessed three chromosome III DNA (Table 2). It is not easy to explain why three kinds of chromosome III DNA exist in a single cell. The above results indicate that variation in DNA lengths of chromosome III occur during meiosis, sporulation or the spore germination process in MT98a-3D strain, resulting in chromosome III DNA-length polymorphisms in spore clones at a high frequency. The length of chromosome III DNA found in the present experiments were 350 kb, 450 kb, 530 kb and 630 kb. No intermediate lengths were observed. This phenomenon indicates that the chromosome III DNA-length polymorphisms are generated by the loss or addition of a specific DNA unit at approximately 100 kb. 1 ABC
D
2 ABCD
3 ABCD
4 AB CD
~530kb ,~
450kb
,~
350kb
~,450kb ~350kb
i,il FIG. 3. The DNA lengths of chromosome III in our laboratory stock strains. Chromosome-sized DNAs of our laboratory stock strains were separated and Southern hybridized with the M A Ta gene. Lane 1, C5672-5D; lane 2, MTI3; lane 3, MT607a-7D; lane 4, MT98a3D; and lane 5, a size standard YNN295.
FIG. 4. DNA lengths of chromosome III of spore clones derived from MT98a-3D strain. Four asci 0--4) from MT98a-3D strain were dissected into four spores (A-D) each. Chromosome-sized DNAs of spore clones were separated and Southern hybridized with the M A Ta gene.
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In homothaUic strains o f S. cerevisiae, one mating type gene on c h r o m o s o m e III frequently converts to another mating type through the action o f H O - c o d e d en d o n u clease (13). Since the MT98a-3D strain is a homothallic strain, we are currently studying whether the product o f the H O gene affects the generation o f D N A - l e n g t h polymorphisms in c h r o m o s o m e III. Recently, a physical m a p o f the g e n o m e and the entire D N A sequence o f c h r o m o s o m e III o f S. cerevisiae have been determined (3, 4). H o w e v e r , the structure o f c h r o m o some III carrying 450 kb D N A remains to be elucidated. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research (No. 01540570) to M. Tsuboi from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Tsuboi, M.: The isolation and genetic analysis of sporulationdeficient mutants in Saccharomyces cerevisiae. Mol. Gen. Genet., 191, 17-21 (1983). 2. Mortimer, R. K., Contopoulou, C. R., and King, J. S.: Genetic map of Saccharomyces cerevisiae, edition 11. Yeast, 8, 817-902 (1992). 3. Link, A.J. and Olson, M.V.: Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution. Genetics,
127, 681-698 (1991). 4. Oliver, S. G. ctal.: The complete DNA sequence of yeast chromosome III. Nature, 357, 38-46 (1992). 5. Carle, G. F. and OIson, M. V.: An electrophoretic karyotype for yeast. Proc. Natl. Acad. Sci. USA, 82, 3756-3760 (1985). 6. Ono, B. and Ishlno-Arao, Y.: Inheritance of chromosome length polymorphisms in Saccharomyces cerevisiae. Curr. Genet., 14, 413-418 (1988). 7. Iwaguchi, S., Homma, M., and Tanaka, K.: Variation in the electrophoretic karyotype analysed by the assignment of DNA probes in Candida albicans. J. Gen. Microbiol., 136, 2433-2442 (1990). 8. Sor, F. and Fukuhara, H.: Analysis of chromosomal DNA patterns of the genus Kluyveromyees. Yeast, 5, 1-10 (1989). 9. Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98, 503-517 (1975). 10. Sambrook, J., Fritsch, E. F., and Maniatis, T.: Molecular Cloning. A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989). 11. Tsuboi, M. and Takahashi, T.: Genetic analysis of the nonsporulating phenotype of brewer's yeasts. J. Ferment. Technol., 66, 605-613 (1988). 12. Tanaka, H. and Tsuboi, M.: Cloning and mapping of the sporulation gene, 8poTT, in Saeeharomyees cerevisiae. Mol. Gen. Genet., 199, 21-25 (1985). 13. Kostriken, R., Strathern, J. N., Klar, A. J. S., Hicks, J. B., and Hetfron, F.: A site-specific endonuclease essential for matingtype switching in Saeeharomyee8 eerevisiae. Cell, 35, 167-174 (1983).