YEAST
VOL.
8 669-672 (1992)
-V
VI
Yeast Mapping Reports
Mapping of Two New Codon-specific Suppressors in Saccharomyces cerevisiae BUN-ICHIRO ONO*, YUJIRO ARAOt AND KAYO MORIYOSHI Laboratory of Environmental Hygiene Chemistry, Faculty of Pharmaceutical Sciences, Okayama Unviversity, Tsushima Naka 1-1-1,Okayama 700, Japan
Received 15 November 1991; accepted 26 February 1992
KEY WORDS - Saccharomyces cerevisiae; nonsense suppressors; tRNA genes; yeast chromosome V; yeast chromosome VI.
A large number of nonsense suppressors of Saccharomyces cerevisiae have been compiled (see Sherman, 1982). They are divided into two categories, codon-specific and codon-non specific (omnipotent). The codon-specific suppressors are classified by codons on which they act, UAA, UAG and UGA. Eight tyrosine-inserting (Gilmore et al., 1971),four serine-inserting (On0 et al., 1979a, 1981) and six leucine-inserting (On0 et al., 1979b) UAAspecific suppressors have been identified and mapped. In addition, eight tyrosine-inserting (allelic to tyrosine-inserting UAA suppressors) (Liebman et al., 1976), six leucine-inserting (allelic to leucineinserting UAA suppressors) (Liebman et al., 1977; Reed and Liebman, 1979) and one serine-inserting (Brandriss et al., 1976) UAG-specific suppressors have been identified and mapped. Although a total of 17 UGA-specific suppressors have been mapped (Hawthorne, 1976, 1981; Ono et al., 1988), their amino acid insertions are not known yet. The codon-specific suppressors are known or presumed to represent tRNA genes. They are useful not only for studying tRNA genes but also for alignment of the physical map against the genetic map because of their dispersed distribution over the entire genome. However, it should be noted that no UAA/G-specific suppressor causing insertion of *Corresponding author. ?Present address: Department of Pathology, National Institute of Health in Japan, Tokyo,Japan. 0749-503X/92/080669-04 $07.00 0 1992 by John Wiley & Sons Ltd
glutamine, lysine or glutamic acid has been obtained so far, although tRNAG'"CAA/G, tRNALysAAA/G and tRNAG1"GAA/Gshould give rise to UAA/Gdecoding tRNA species by single base substitutions. In this respect, two suppressors we uncovered in strains of our stock collection are of interest. One of them, designated SUPx, acted only on the leu2-1 (UAA) mutation as far as we examined; it did not suppress met8-1 (UAG), aro7-1 (UAG) nor adel-14 (UGA). While SUPx was present in a Y' strain, its action spectrum was not changed by treatment with guanidine-HC1, which is known to eliminate effectively the Y cytoplasmic determinant (Tuite et al., 1981). It suppressed cycl-72 (UAA) very weakly, if at all, in both the Y' and Y- cytoplasms. Although SUPx was a highly inefficient suppressor as judged by its action spectrum, its action on leu2-1 was dominant in both the Y' and Y- cytoplasms. SUPx showed linkages with the centromere (3 1 cM) and with hisl (18 cM) (Table 1). We therefore placed it proximal to hisl on the right arm of chromosome V (Figure 1). Its distances to SUP19 (24 cM) and i l v l ( 3 6 cM) are in accord with the previous linkage data (Mortimer and Schild, 1980). The other, uncovered in a Y-strain and designated SUPy, acted on the met8-1 (UAG) mutation but not on nonsense mutations such as aro7-1 (UAG), trpl-1 (UAG), leu2-I (UAA), ilvl-2 (UAA) and adel-I4 (UGA). The action spectrum of SUPy was not affected by the Y' cytoplasm. Moreover, +
cen5-SUPx
11
49
Total
9
66
29
17 40
37
23
31
48
48
P
2
2
N
10
10
T
S UPx-his1
18
cM
16
16
P
0
0
N
15
15
T
24
18
cM
SUPX-SUP19
42
1
24
P
Cross Segregation*
3
20
2
N
34
34
T
SUPx-ilvl
36
cM
50
50
P
0
0
N
10
10
T
his1 -argS
8
cM
39
39
P
2
2
N
18
18
T
arg5-ilvl
25
cM
'Centromere was defined by segregation of trpl and m e t l l ; FDS and SDS represent first-division segregation and second-division segregation, respectively. Distances (cM) from the centromere were estimated by the formula 50 x DS/(FDS+SDS). P, N and T represent parental ditype, non-parental ditype and tetratype tetrads, respectively; SUPx and SUP19 were detected by their suppression of the leu2-1 (UAA) mutations.
11
49
KM13 YA277 YA292
FDSSDS cM FDSSDS cM
ura3-cenS
Table 1. Linkage analysis of SUPx
"0
2
F
wI
67 1
TWO NEW CODON-SPECIFIC SUPPRESSORS IN S. CEREVISIAE
Table 2. Linkage analysis of SUPy
Cross Segregation* SUP1 I-SUPy
P YA167 KM40 KM30
11 3
Total
14
N
T
1 0
1
c
SUP6-SUPy
M
P
N
T
c
M
17 6 23
38
71
0
21
71
0
21
11
*SUP11 and SUP4 were scored by suppression of the leu2-1 (UAA) mutation, while SUPy was by suppression of the metd-1 (UAG) mutation. P, Nand T represent parental ditype, non-parentalditype and tetratype tetrads, respectively. Genetic distances (cM) were estimated by the formula 50 x (T + 6N)/(P + N + T).
--
ura3
cen5
SUP1 9 his1 arg5
SUPx
ilvl
A
*
--
24 ~~
36 Figure I .
Genetic map of chromosome V with markers relevant to the present work.
m-
c e n 6 SUP11 - ,I
I 1
a
b C
38
3
SUPy
SUP6
0 0
11
30
Figure 2. Genetic map of chromosome VI with markers relevant to the present work. (a) and (b), this study; (c) Mortimer and Schild (1 990).
SUPy was dominant in action both in the Yand Y' cytoplasms. SUPy showed linkage to SUP6 (I 1 cM) and to SUPZZ (38 cM) (Table 2). We therefore place SUPy distal to SUP6 on the right arm of chromosome VI (Figure 2), where no nonsense suppressor has been mapped previously (Mortimer et al., 1990).
Omnipotent suppressors generally have narrow action spectra, in particular, some alleles of recessive omnipotent suppressors are scored as codon-specific or allosuppressors due to their limited actions (On0 et al., 1986, 1991). Although SUPx and SUPy have extremely narrow action spectra, they are unlikely to be alleles of omnipotent
672 suppresors because they are dominant in action. We contend that they represent new classes of codon-specific suppressors (and thus new classes of tRNA suppressors), presumably mutations in genes for tRNA of glutamic acid, lysine or glutamine.
B . 4 . ONO, Y. ARAO AND K. MORIYOSHI
Lower Eucaryotes. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 3 W 9 . Ono, B., Stewart, J. W. and Sherman, F. (1979a). Yeast UAA suppressors effective in Y t strains. Serineinserting suppressors. J . Mol. Biol. 128,81-100. Ono, B., Stewart, J. W. and Sherman, F. (1979b). Yeast UAA suppressors effective in Y strains: leucineinserting suppressors. J . Mol. Biol. 132,507-520. Ono, B., Wills, N., Stewart, J. W., Gestland, R. F. and Sherman, F. (1981). Serine-inserting UAA suppression mediated by yeast tRNA&'. J . Mol. Biol. 150,361-373. Ono, B., Ishino-Arao, Y., Tanaka, M., Awano, I. and Shinoda, S. (1986). Recessive nonsense suppressors in Saccharomyces cerevisiae: action spectra, complementation groups and map positions. Genetics 114, 363-374. Ono, B., Fujimoto, R., Ohno, Y., Maeda, N., Tsuchiya, Y., Usui, T. and Ishino-Arao, Y. (1988). UGA suppressors in Saccharomyces cerevisiae: allelism, action spectra and map positions. Genetics 118,4147. Ono, B., Chernoff,Y. O.,Ishino-Arao, Y. Yamagishi, N., Shinoda, S. and Inge-Vechtomov, S. G. (1991). Interactions between chromosomal omnipotent suppressors and extrachromosomal effectors in Saccharomyces cerevisiae. Curr. Genet. 19,243-248. Reed, C. R. and Liebman, S. W. (1979). New amber suppressors in Saccharomyces cerevisiae. Genetics 91, s102. Sherman, F. (1982). Suppression in the yeast Saccharomyces cerevisiae. In Strathern, J. N., Jones, E. W. and Broach, J. R. (Eds), The Molecular Biology of Yeast Saccharomyces. Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 463486. Tuite, M. F., Mundy, C. R. and Cox, B. S. (1981). Agents that cause a high frequency of genetic change from [psi'] to [psi-] in Saccharomyces cerevisiae. Genetics 98,69 1-7 1 1 . +
ACKNOWLEDGEMENTS We thank Yumiko Ishnino-Arao for her technical assistance during the study. REFERENCES Brandriss, M. C., Stewart, J. W., Sherman, F. and Botstein, D. (1976). Substitution of serine caused by a recessive lethal suppressor in yeast. J. Mol. Biol. 102, 467476. Gilmore, R. A., Stewart, J. W. and Sherman, F. (1971). Amino acid replacement resulting from supersuppression of nonsense mutations of iso-lcytochrome c from yeast. J. Mol. Biol. 61,157-173. Hawthorne, D. C. (1976). UGA mutations and UGA suppressors in yeast. Biochimie 58, 179-182. Hawthorne, D. C. (1981). UGA suppressors in yeast. In Von Wettstein, D., Friis, J., Kielland-Brandt, M. and Stenderup, A. (Eds), Molecular Genetics in Yeast. Munksgaard, Copenhagen, pp, 291-301, Liebman, S. W., Sherman, F. and Stewart, J. W. (1976). Isolation and characterization of amber suppressors in yeast. Genetics 82,251-272. Liebman, S . W., Stewart, J. W., Parker, J. H. and Sherman, F. (1977). Leucine insertion caused by a yeast amber suppressor. J. Mol. Biol. 109,13-22. Mortimer, R. and Schild, D. (1980). Genetic mapping of Saccharomyces cerevisiae. Microbiol. Rev. 44,s 19-571. Mortimer, R., Schild, D., Contopoulou, C. R. and Kans, J. A. (1990). Saccharomyces cerevisiae nuclear genes. In O'Brien, S. J. (Ed.), Genetic Maps, 5th edn, Book 3,
VOL.
8 669-672 (1992)
-V
VI
Yeast Mapping Reports
Mapping of Two New Codon-specific Suppressors in Saccharomyces cerevisiae BUN-ICHIRO ONO*, YUJIRO ARAOt AND KAYO MORIYOSHI Laboratory of Environmental Hygiene Chemistry, Faculty of Pharmaceutical Sciences, Okayama Unviversity, Tsushima Naka 1-1-1,Okayama 700, Japan
Received 15 November 1991; accepted 26 February 1992
KEY WORDS - Saccharomyces cerevisiae; nonsense suppressors; tRNA genes; yeast chromosome V; yeast chromosome VI.
A large number of nonsense suppressors of Saccharomyces cerevisiae have been compiled (see Sherman, 1982). They are divided into two categories, codon-specific and codon-non specific (omnipotent). The codon-specific suppressors are classified by codons on which they act, UAA, UAG and UGA. Eight tyrosine-inserting (Gilmore et al., 1971),four serine-inserting (On0 et al., 1979a, 1981) and six leucine-inserting (On0 et al., 1979b) UAAspecific suppressors have been identified and mapped. In addition, eight tyrosine-inserting (allelic to tyrosine-inserting UAA suppressors) (Liebman et al., 1976), six leucine-inserting (allelic to leucineinserting UAA suppressors) (Liebman et al., 1977; Reed and Liebman, 1979) and one serine-inserting (Brandriss et al., 1976) UAG-specific suppressors have been identified and mapped. Although a total of 17 UGA-specific suppressors have been mapped (Hawthorne, 1976, 1981; Ono et al., 1988), their amino acid insertions are not known yet. The codon-specific suppressors are known or presumed to represent tRNA genes. They are useful not only for studying tRNA genes but also for alignment of the physical map against the genetic map because of their dispersed distribution over the entire genome. However, it should be noted that no UAA/G-specific suppressor causing insertion of *Corresponding author. ?Present address: Department of Pathology, National Institute of Health in Japan, Tokyo,Japan. 0749-503X/92/080669-04 $07.00 0 1992 by John Wiley & Sons Ltd
glutamine, lysine or glutamic acid has been obtained so far, although tRNAG'"CAA/G, tRNALysAAA/G and tRNAG1"GAA/Gshould give rise to UAA/Gdecoding tRNA species by single base substitutions. In this respect, two suppressors we uncovered in strains of our stock collection are of interest. One of them, designated SUPx, acted only on the leu2-1 (UAA) mutation as far as we examined; it did not suppress met8-1 (UAG), aro7-1 (UAG) nor adel-14 (UGA). While SUPx was present in a Y' strain, its action spectrum was not changed by treatment with guanidine-HC1, which is known to eliminate effectively the Y cytoplasmic determinant (Tuite et al., 1981). It suppressed cycl-72 (UAA) very weakly, if at all, in both the Y' and Y- cytoplasms. Although SUPx was a highly inefficient suppressor as judged by its action spectrum, its action on leu2-1 was dominant in both the Y' and Y- cytoplasms. SUPx showed linkages with the centromere (3 1 cM) and with hisl (18 cM) (Table 1). We therefore placed it proximal to hisl on the right arm of chromosome V (Figure 1). Its distances to SUP19 (24 cM) and i l v l ( 3 6 cM) are in accord with the previous linkage data (Mortimer and Schild, 1980). The other, uncovered in a Y-strain and designated SUPy, acted on the met8-1 (UAG) mutation but not on nonsense mutations such as aro7-1 (UAG), trpl-1 (UAG), leu2-I (UAA), ilvl-2 (UAA) and adel-I4 (UGA). The action spectrum of SUPy was not affected by the Y' cytoplasm. Moreover, +
cen5-SUPx
11
49
Total
9
66
29
17 40
37
23
31
48
48
P
2
2
N
10
10
T
S UPx-his1
18
cM
16
16
P
0
0
N
15
15
T
24
18
cM
SUPX-SUP19
42
1
24
P
Cross Segregation*
3
20
2
N
34
34
T
SUPx-ilvl
36
cM
50
50
P
0
0
N
10
10
T
his1 -argS
8
cM
39
39
P
2
2
N
18
18
T
arg5-ilvl
25
cM
'Centromere was defined by segregation of trpl and m e t l l ; FDS and SDS represent first-division segregation and second-division segregation, respectively. Distances (cM) from the centromere were estimated by the formula 50 x DS/(FDS+SDS). P, N and T represent parental ditype, non-parental ditype and tetratype tetrads, respectively; SUPx and SUP19 were detected by their suppression of the leu2-1 (UAA) mutations.
11
49
KM13 YA277 YA292
FDSSDS cM FDSSDS cM
ura3-cenS
Table 1. Linkage analysis of SUPx
"0
2
F
wI
67 1
TWO NEW CODON-SPECIFIC SUPPRESSORS IN S. CEREVISIAE
Table 2. Linkage analysis of SUPy
Cross Segregation* SUP1 I-SUPy
P YA167 KM40 KM30
11 3
Total
14
N
T
1 0
1
c
SUP6-SUPy
M
P
N
T
c
M
17 6 23
38
71
0
21
71
0
21
11
*SUP11 and SUP4 were scored by suppression of the leu2-1 (UAA) mutation, while SUPy was by suppression of the metd-1 (UAG) mutation. P, Nand T represent parental ditype, non-parentalditype and tetratype tetrads, respectively. Genetic distances (cM) were estimated by the formula 50 x (T + 6N)/(P + N + T).
--
ura3
cen5
SUP1 9 his1 arg5
SUPx
ilvl
A
*
--
24 ~~
36 Figure I .
Genetic map of chromosome V with markers relevant to the present work.
m-
c e n 6 SUP11 - ,I
I 1
a
b C
38
3
SUPy
SUP6
0 0
11
30
Figure 2. Genetic map of chromosome VI with markers relevant to the present work. (a) and (b), this study; (c) Mortimer and Schild (1 990).
SUPy was dominant in action both in the Yand Y' cytoplasms. SUPy showed linkage to SUP6 (I 1 cM) and to SUPZZ (38 cM) (Table 2). We therefore place SUPy distal to SUP6 on the right arm of chromosome VI (Figure 2), where no nonsense suppressor has been mapped previously (Mortimer et al., 1990).
Omnipotent suppressors generally have narrow action spectra, in particular, some alleles of recessive omnipotent suppressors are scored as codon-specific or allosuppressors due to their limited actions (On0 et al., 1986, 1991). Although SUPx and SUPy have extremely narrow action spectra, they are unlikely to be alleles of omnipotent
672 suppresors because they are dominant in action. We contend that they represent new classes of codon-specific suppressors (and thus new classes of tRNA suppressors), presumably mutations in genes for tRNA of glutamic acid, lysine or glutamine.
B . 4 . ONO, Y. ARAO AND K. MORIYOSHI
Lower Eucaryotes. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 3 W 9 . Ono, B., Stewart, J. W. and Sherman, F. (1979a). Yeast UAA suppressors effective in Y t strains. Serineinserting suppressors. J . Mol. Biol. 128,81-100. Ono, B., Stewart, J. W. and Sherman, F. (1979b). Yeast UAA suppressors effective in Y strains: leucineinserting suppressors. J . Mol. Biol. 132,507-520. Ono, B., Wills, N., Stewart, J. W., Gestland, R. F. and Sherman, F. (1981). Serine-inserting UAA suppression mediated by yeast tRNA&'. J . Mol. Biol. 150,361-373. Ono, B., Ishino-Arao, Y., Tanaka, M., Awano, I. and Shinoda, S. (1986). Recessive nonsense suppressors in Saccharomyces cerevisiae: action spectra, complementation groups and map positions. Genetics 114, 363-374. Ono, B., Fujimoto, R., Ohno, Y., Maeda, N., Tsuchiya, Y., Usui, T. and Ishino-Arao, Y. (1988). UGA suppressors in Saccharomyces cerevisiae: allelism, action spectra and map positions. Genetics 118,4147. Ono, B., Chernoff,Y. O.,Ishino-Arao, Y. Yamagishi, N., Shinoda, S. and Inge-Vechtomov, S. G. (1991). Interactions between chromosomal omnipotent suppressors and extrachromosomal effectors in Saccharomyces cerevisiae. Curr. Genet. 19,243-248. Reed, C. R. and Liebman, S. W. (1979). New amber suppressors in Saccharomyces cerevisiae. Genetics 91, s102. Sherman, F. (1982). Suppression in the yeast Saccharomyces cerevisiae. In Strathern, J. N., Jones, E. W. and Broach, J. R. (Eds), The Molecular Biology of Yeast Saccharomyces. Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 463486. Tuite, M. F., Mundy, C. R. and Cox, B. S. (1981). Agents that cause a high frequency of genetic change from [psi'] to [psi-] in Saccharomyces cerevisiae. Genetics 98,69 1-7 1 1 . +
ACKNOWLEDGEMENTS We thank Yumiko Ishnino-Arao for her technical assistance during the study. REFERENCES Brandriss, M. C., Stewart, J. W., Sherman, F. and Botstein, D. (1976). Substitution of serine caused by a recessive lethal suppressor in yeast. J. Mol. Biol. 102, 467476. Gilmore, R. A., Stewart, J. W. and Sherman, F. (1971). Amino acid replacement resulting from supersuppression of nonsense mutations of iso-lcytochrome c from yeast. J. Mol. Biol. 61,157-173. Hawthorne, D. C. (1976). UGA mutations and UGA suppressors in yeast. Biochimie 58, 179-182. Hawthorne, D. C. (1981). UGA suppressors in yeast. In Von Wettstein, D., Friis, J., Kielland-Brandt, M. and Stenderup, A. (Eds), Molecular Genetics in Yeast. Munksgaard, Copenhagen, pp, 291-301, Liebman, S. W., Sherman, F. and Stewart, J. W. (1976). Isolation and characterization of amber suppressors in yeast. Genetics 82,251-272. Liebman, S . W., Stewart, J. W., Parker, J. H. and Sherman, F. (1977). Leucine insertion caused by a yeast amber suppressor. J. Mol. Biol. 109,13-22. Mortimer, R. and Schild, D. (1980). Genetic mapping of Saccharomyces cerevisiae. Microbiol. Rev. 44,s 19-571. Mortimer, R., Schild, D., Contopoulou, C. R. and Kans, J. A. (1990). Saccharomyces cerevisiae nuclear genes. In O'Brien, S. J. (Ed.), Genetic Maps, 5th edn, Book 3,
