MAPPING OF TRICHODERMIN RESISTANCE I N SACCHAROMYCES CEREVZSZAE: A GENETIC LOCUS FOR A COMPONENT OF THE 60s RIBSOMAL SUBUNIT PAUL G. GRANT, DANIEL SCHINDLER
AND
JULIAN E. DAVIES
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706 Manuscript received December 29, 1975 Revised copy received April 6, 1976 ABSTRACT
Resistance to the protein synthesis inhibitor trichodermin in Saccharomyces cereuisiae has been studied. A single recessive nuclear gene was responsible for resistance. The resistance locus, tcml was found to be closely linked (1 centimorgan) to the locus pet 17 on the right arm of chromosome XV. The mutation to trichodermin resistance conferred resistance to other 12,1S-epoxytrichothecenes and to the structurally unrelated antibiotic anisomycin.
HE availability of antibioltic resistant mutants in bacteria with altered riboTsomes has proven extremely useful in our understanding o€ the function and and DAVIES1968; GALEet al. 1972; genetics of the bacterial ribosome (WEISBLUM DAVIESand NOMURA 1972; JASKUNAS, NOMURA and DAVIES1974). By comparison, similar biochemical and genetic studies of antibiotic resistance mutants in eukaryotic organisms have been somewhat limited (MCLAUGHLIN 1974). The yeast Saccharomyces cereuisiae is well suited to studies of eukaryotic ribosomal genetics, structure, and function. S. cerevisiae has a well characterized genetic system (MORTIMER and HAWTHORNE 1973), a stable haploid phase that facilitates mutant selection and characterization. and is amenable to most biochemical techniques. Mutations to antibiotic resistance in S. cerevisiae which involve ribosomal alterations have been reported for the glutarimide antibiotics (COOPER,BANTHORPE and WILKIE 1967; JIMENEZ,LITTLEWOOD and DAVIES1972), the phenMCI,AUGHI,IN and WAKATAMA 1973; GRANT, anthrene alkaloids (SKOGERSON, SANCHEZand JIMENEZ1974) , and some of the 12,13-epoxytrichothecenes (SCHINDLER, GRANT and DAVIES1974). We have mapped the genetic locus for resistance to trichodermin tcml, and have found it to be located on the right arm of chromosome X V near the locus petl7. Surprisingly, mutations at this locus tcml also determined resistance to the unrelated antibiotic anisomycin.. MATERIALS A N D METHODS
Strains: The strains used in these studies are described in Table 1. The isolation and characterization of S. cereuisiae strains SRI, (JIMENEZ,LITTLEWOOD and DAVIES1972) and CLP-1 Genetics 83: 667-673 August, 1976
668
P. G. GRANT, D. S C H I N D L E R A N D J. E . DAVIES
TABLE I
Strains used in these studies Strain
CLP-1 CLP-8
SRI, E27c 2181-1A X3382-3A 115 SH361 IC 421-5A
Source
Genotype
a leuztcml a leu2 tcml LY his4 trp5 cyh2 ~i leu2 tcml a trpl adel his2 gall a arg4 asp5 pet17 tyr7 cdcl4 adel gall trpl his2 his6 a ade2 his4 cyh2 a sup3 can1100 leul-12 metl-I ade2-I lysl-I his5-2 CY ade2-I his5-2 tcml a his5-2 lysl-I arg4-17 trp5-48 ade2-I
D. SCHINDLER D. SCHINDLER B. LITTLEWOOD Cross of CLP-1 and SRI, B. LITTLEWOOD Yeast Genetics Stock Center B. LITTLEWOOD J. GORMAN Cross of CLP-1 and 421-5a
J. GORMAN
( SCHINDLER, GRANTand DAVIES1974) have been described elsewhere. Strain E27c was derived from a cross of strains CLPl and SR,,. Media: YED medium contained 1% yeast extract (Difco), 2% peptone (Difco), and 2% glucose. YNB medium contained 0.7% yeast nitrogen base without amino acids (Difco), 2% glucose, plus appropriate supplements. Presporulation medium contained 10% glucose, 0.8% yeast extract, and 0.3% peptone. Sporulation medium contained 1% potassium acetate, 0.1 % yeast extract, and 0.05% glucose. All solid media were prepared by adding 2% agar (Difco) to the above media. Genetic analyses: Matings were performed by mixing haploid strains of opposite mating type on YED plates and incubating overnight at 30". Diploids were isolated by prototrophic selection on YNB plates containing the appropriate supplements. Single colonies were used to inoculate 10 ml of presporulation medium which was incubated at 30" for 16 to 24 hrs without shaking. When fermentation appeared Iigorous, as evidenced by the rapid evolution of CO,, the cells were washed once with 5 ml of sporulation medium and resuspended in 5 ml of fresh sporulation medium. The cultures were then incubated 2 to 4 days at 30" with shaking. In crosses with strains possessing the temperature-sensitive allele cdcl4, incubation in sporulation medium was carried out at 23 to 25". Sporulation cultures were stored at 4" in 1 ml of fresh sporulation medium. Tetrads were dissected and analyzed using standard techniques (HAWTHORNE and MORTIMER 1968). The percentage of tetrads which germinated completely varied from about 60 to 90% depending on the cross. Only complete tetrads were analyzed. Poly-U-dependent phenylalanine incorporation: This was carried out as described previously (SCHINDLER, GRANTand DAVIES1974), but with the following modification: reactions were terminated by the addition of 2 drops of I N KOH followed by incubation at 30" for 20 minutes before addition of 10% trichloroacetic acid. RESULTS
Trichodermin resistance in S. cereuisiae strain CLP-1 was due to a single, recessive nuclear gene and we have named this gene tcml. Eight trichodermin resistant mutants isolated previously were allelic with tcml. The trichodermh resistant mutants have been shown to have a n altered 60s ribosomal subunit (SCHINDLER, GRANTand DAVIES1974). Since mutations in the gene cyh2 have been shown to produce cycloheximide-resistant 60s ribosomal subunits
TRICHODERMIN RESISTANCE OF
669
60s RIBSOMES
TABLE 2 Ascus-type ratios of tcml with cyh2 and mating-type locus Gene pair
tcml -cyh2 tcml-mating type
PD*
NPD'
T'
8 23
8 13
32 80
* PD,NPD,T refer to parental ditype, nonparental ditype and tetratype asci, respectively.
(MCLAUGHLIN 1974), strain CLP-1 was crossed with strain SR17, a mutant at the cyh2 locus (JIMENEZ,LITTLEWOOD and DAVIES 1973), to determine if tcml was linked to cyh2. The results of the cross, shown in Table 2, indicated that there was no linkage between these two loci. The results in Table 2 also show that there is no linkage between tcml and the mating-type locus. Since c r y l , a gene producing 40s ribosomal subunits resistant to phenanthrene alkaloids, is closely linked to the mating type locus, tcml is not linked to cry1 (SKOGERSON, MCLAUGHLIN and WAKATAMA 1973; GRANT,SANCHEZand JIMENEZ 1974). Neither cyh2 nor tcml appeared to have any effect on the expression of the other resistance allele as it was possible to score all tetrads for resistance to cycloheximide and trichodermin, without ambiguity. The possibility of centromere linkage of tcml was checked in crosses involving tcml and trpl as a marker for centromere segregation. The results show a second division segregation frequency of 39% placing tcml 19-20 centimorgans (cm) from its centromere. tcml containing strains were crossed with a series of strains containing established centromere-linked markers to determine the centromere to which tcml was linked. In the course of screening these crosses tcml was found to be tightly linked to petl7, a gene marking the centromere of chromosome X V . The ascus type ratios are shown in Table 3 ; these results placed tcml 1 cm from petl7. The three tetratype asci all showed a first division segregation pattern from tcml and a second division segregation pattern for petl7, indicating the tcml was closer to the centromere than petl7. This suggests the order; centromere, tcml, pet17 on the right arm of chromosome X V . To confirm the location of icml on chromosome XV, crosses were performed with strains containing ade2 and SUP3. The data from these crosses, presented in Table 3, confirmed that tcml was located on chromosome X V . The above results placed tcml on the right arm of chromosome X V , 19-20 cm from the centromere, 1 cm nearer the centromere than petl7. TABLE 3
Linkage data for tcml with loci on chromosome X V Gene pair
tcml-pet17 tcml-ade2 tcml -SUP3
PD
130 15 53
NPD
7
0 3 0
3 91 34
670
P. G . G R A N T , D. S C H I N D L E R A N D J. E. DAVIES
Resistance to anisomycin: The trichodermin-resistant mutants that we have isolated were resistant to the antibiotic anisomycin as well as to other 12,13epoxytrichothecenes such as nivalenol (SCHINDLER, GRANTand DAVIES 1974; SCHINDLER, 1974). Resistance to anisomycin appeared to be the result of the same mutation in tcml that conferred resistance to trichodermin, as no recombinant spores were found in 190 tetrads analyzed for both trichodermin and anisomycin resistance. Ribosomes from strains CLP-1 and CLP-8 were shown to be resistant to several of the 12,13-epoxytrichothecenes in vitro ( SCHINDLER,GRANTand
I
0.I
I .o Drug
10.0
IOO.0
Concentration, pM
FIGURE 1.-Inhibition of poly (U)-dependent phenylalanine incorporation on A224A and CLP-8 ribosomes by various drugs. Results are given as '% control activity measured by acid-precipitable radioactive polyphenylalanine formed after a 30-minute incubation at 30" as described in MATERIALS AND METHODS. Control activity (no drug) for A224 ribosomes (0) is 62.0 pmoles of phenylalanine and for CLP-8 ribosomes (x), 42.2 pmoles. Solid lines-nivalenol. Dashed lines-anisomycin.
TRICHODERMIN RESISTANCE OF
60s RIBSOMES
671
DAVIES1974; SCHINDLER, 1974). However, ribosomes prepared from strain CLP-8 appear to be only weakly resistant to anisomycin in the same in vitro assay (Figure 1). These ribosomes are as sensitive as the ribosomes from the parental strain to inhibition by cycloheximide, blasticidin S, and sparsomycin (data not shown). JIMENEZ, SANCHEZ and VASQUEZ (1975) have made similar observations on mutants derived from different strains of S. cerevisiae. Resistance to trichothecin: Mutants selected for resistance to trichothecin, another of the 12,13-epoxytrichothecenes,were allelic with tcml and were crossresistant to trichodermin and anisomycin. DISCUSSION
Trichodermin is a peptidyl transferase inhibitor ( CARRASCO, BARBACID and VAZQUEZ 1973). The isolation of mutants of S. cereuisiae resistant to trichodermin and other 12,13-epoxytrichothecenesprovides a gentic marker for a 60s ribosomal function and perhaps for a component of the peptidyl transferase centre. The gene determining this resistance, tcml, is a single, recessive nuclear gene located on the right arm of chromosome X V , 1 cm nearer the centromere than petl7. At first sight, simultaneous resistance to 12,13-epoxytrichothecenesand anisomycin, two structurally unrelated drugs, was surprising. However it has been shown that these drugs bind at mutually exclusive sites (BARBACID and VAZQUEZ 1974a, b; WEIet al. 1974). The genetic evidence in this paper strongly indicates that the simultaneous resistance to anisomycin and 12,13-epoxytrichothecenesis the result of a single mutation, although biochemically the resistance to each drug in vitro differs greatly. A possible explanation for the weak expression of resistance to anisomycin may be that a factor is missing from our in vitro assay system which is necessary for the full expression of anisomycin resistance, or that the ribosomes we use are structurally or functionally defective without affecting poly-U-dependent incorporation. We cannot eliminate the possibility that tcml may be closely linked to a genetic locus altering drug permeability and that we are dealing with two very closely linked mutations. The fact that ribosomes from the tcml strain show some degree of resistance to anisomycin would tend to argue against this. It is interesting to note that the loci known to affect ribosomal structure in S. cerevisiae tcml, cyh2, cry1 and the genes for ribosomal RNA (rRNA) are not clustered within the same genetic region although some clustering of redundant genes for rRNA does occur (CRAMER, BHARGAVA and HALVORSON 1972; FINKELSTEIN, BLAMIRE and MARMUR 1972; KABACK, BHARGAVA and HALVORSON 1973; @YEN, 1973). It has recently been found that the ribosomal structural genes in E. coli are also scattered throughout the chromosome and often occur in clusters of several loci (JASKUNAS, LINDAHL and NOMURA 1975; LINDAHL et al. 1975; WATSON et al. 1975). While no evidence is available to demonstrate that the loci tcml, cyh2, and cry1 are ribosomal proteins and not modifying enzymes, the organization of ribosomal structural genes in S. cerevisiae may prove different from that in E . coli, since there is presently very little evidence of clustering of
6 72
P. G. GRANT, D. S C H I N D L E R A N D J. E. DAVIES
genes of biochemically related functions in yeast (MORTIMER and HAWTHORNE 1973). It would appear that in S. cerezlisiae mutations affecting ribosomes and protein synthesis are often closely linked to mutations involving mitochondrial function. This linkage occurs between prt2 and pet2, cyh2 and tsm437 (MORTIMER and HAWTHORNE 1973), cryl and pet18 ( SKOGERSON, MCLAUGHLIN and WAKATAMA 1973), and between tcml and petl7. One may speculate that these petite mutations lead to the production OP altered, nonfunctional mitochondrial ribosomal proteins and that mitochrondrial ribosomal protein genes may be linked to cytoplasmic ribosomal protein genes. It is known that most, if not all, mitochondrial ribosomal proteins are made on cytoplasmic ribosomes and presumably coded by nuclear genes (GROOT1974) and also that mitochondrial protein synthesis is et al. 1966). necessary for mitochondrial fimction (HUANG We wish to thank B. LITTLEWOOD and J. GORMAN for strains and advice on genetic mapping and other yeast lore. Trichodermin was provided by DR. W. 0. GODTFREDSEN, nivalenol by DR. F. STRONG, blasticidin S by DR. R. METZENBERG and cycloheximide by DR. G. B. WHITFIELD.This work was supported by NIH grant 1-R01-12448-01and by an Upjohn Graduate Fellowship. L I T E R A T U R E CITED
BARBACID, M. and D. VAZQUEZ, 1974a [3H]-anisomycin binding to eukaryotic ribosomes. J. Mol. 1974b Binding of [acetyl-14-C] trichodemin to the peptidyl Biol. 84.: 603-623. -, transferase centre of eukaryotic ribosomes. Eur. J. Biochem. 4: 437-444. CARRASCO, L., M. BARBACID and D. VAZQUEZ, 1973 The trichodermin group of antibiotics, inhibitors of peptide bond formation by eukaryotic ribosomes. Biochim. Biophys. Acta 312: 368376. COOPER, D., D. V. BANTHORPE and D. WILKIE, 1967 Modified ribosomes conferring resistance to cycloheximide in mutants of Saccharomyces cereuisiae. J. Mol. Biol. 25: 347-350. CRAMER, J. H., M. M. BHARGAVA and H. 0. HALVORSON, 1972 Isolation and characterization of ./-DNA of Saccharomyces cereuisiae. J. Mol. Biol. 71: 11-20. 1972 The genetics of bacterial ribosomes. Ann. Rev. Genet. 6: 203DAVIES,J. and M. NOMURA, 234. FINKELSTEIN, D. B., J. BLAMIRE and J. MARMUR, 1972 Location of ribosomal RNA cistrons in yeast. Nature New Biol. 240: 279-281. P. E. REYNOLDS, M. H. RICHMONDand M. J. WARING,1972 The GALE,E. F., E. CUNDLIFFE, Molecular Basis of Antibiotic Action. John Wiley and Sons, London. GRANT,P., L. SANCHEZ and A. JIMENEZ,1974 Cryptopleurine resistance: A genetic locus for a 40s ribosomal component in Saccharomyces cereuisiae. J. Bacteriol. 120: 1308-1314. GROOT,G. S. P., 1974 The biosynthesis of mitochondrial ribosomes in Saccharomyces cereuisine. pp. 443-452. In: The Biogenesis of Mitochondria. Edited by A. M. KROONand C. SACCONE. Academic Press, New York. HAWTHORNE, D. C. and R. K. MORTIMER, 1968 Genetic mapping of nonsense suppressors in yeast. Genetics 60 : 735-742. HUANG, M., D. R. BIGGS,G. D. CLARK-WALKER and A. W. LINNANE,1966 Chloramphenicol inhibition of the formation of particulate mitochondrial enzymes of S. cereuisiae. Biochim. Biophys. Acta 114: 434-436. JASKUNAS, S. R., L. LINDAHLand M. NOMURA,1975 Specialized transducing phages for ribosomal protein genes of Escherichia coli. Proc. Natl. Acad. Sci. U.S. 72: 6-10.
T R I C H O D E R M l N RESISTANCE O F
60s RIBSOMES
6 73
JASKUNAS,S. R., M. NOMURA and J. DAVIES,1974 Genetics of bacterial ribosomes. pp. 333-368. In: Ribosomes. Edited by M. NOMURA, A. TISSIERES and P. LENGYEL.Cold Spring Harbor Laboratory, New York. JIMENEZ, A., B. LITTLEWOOD and J. E. DAVIES,1972 Inhibition of protein synthesis in yeast. pp. 292-306. In: Molecular Mechanisms of Antibiotic Action on Protein Biosynthesis and Membranes. Edited by E. MUNOZ,F. GARCIA-FERRANDEZ and D. VAZQUEZ. Elsevier, Amsterdam. JIMENEZ,A., L. SANCHEZ and D. VAZQUEZ, 1975 Simultaneous ribosomal resistance to trichodermin and anisomycin in Sacchnromyces cereuisicre mutants. Biochim. Biophys. Acta 383 : 427-434. KABACK, D. B., M. M. BHARGAVA and H. 0. HALVORSON, 1973 Location and arrangement of genes coding for ribosomal RNA in Saccharomyces cereuisiae. J. Mol. Biol. 79: 735-739. hvDAHL, L., s. R. JASKUNAS, P. P. DENNISand M. NOMURA,1975 Cluster of genes in Escherichia coli for ribosomal proteins, ribosomal RNA, and RNA polymerase subunits. Proc. Natl. Acad. Sci. US. 72: 2743-2747. MCL~UGHLIN,C. S., 1974 Yeast ribosomes: Genetics. pp. 815-827. In: Ribosomes. Edited by M. NOMURA, A. TISSIERES, and P. LENGYEL. Cold Spring Harbor Laboratory, New York. MORTIMER, R. K. and D. C. HAWTHORNE, 1973 Genetic mapping in Saccharomyces. IV. Mapping of temperature-sensitive genes and use of disomic strains in localizing genes. Genetics 74: 33-54. OYEN,T. B., 1973 Chromosome I as a possible site for some rRNA cistrons in Saccharomyces cereuisiae. FEBS Letters 30: 53-56. SCHINDLER, D., 1974 Two classes of inhibitors of peptidyl transferase activity on eukaryotes. Nature 249: 3841. SCHINDLER, D., P. GRANTand J. DAVIES,1974 Trichodermin resistance-mutation affecting eukaryotic ribosomes. Nature 248: 535-536. SKOGERSON, L., C. MCLAUGHLIN and E. WAKATAMA, 1973 Modification of ribosomes in cryptopleurine-resistant mutants of yeast. J. Bacteriol. 116: 818-822. WATSON, R. J., J. PARKER, N. P. FIILS,J. G. FLAKS and FRIESEN, J. D.. 1975 New chromosomal location for structural genes of ribosomal proteins. Proc. Natl. Acad. Sci. U.S. 72: 27652769. WEI,C.-M., I. M. CAMPBELL, C. S. MCLAUGHLINand M.H. VAUGHAN, 1974 Binding of trichodermin to mammalian ribosomes and its inhibition by other 12,13-epoxytrichothecenes.Mol. Cell. Biochem. 3: 215-219. WEISBLUM, B. and J. DAVIES,1968 Antibiotic inhibitors of the bacterial ribosome. Bacteriol. Rev. 32: 493-528. Corresponding Editor: D. SCHLESSINGER
AND
JULIAN E. DAVIES
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706 Manuscript received December 29, 1975 Revised copy received April 6, 1976 ABSTRACT
Resistance to the protein synthesis inhibitor trichodermin in Saccharomyces cereuisiae has been studied. A single recessive nuclear gene was responsible for resistance. The resistance locus, tcml was found to be closely linked (1 centimorgan) to the locus pet 17 on the right arm of chromosome XV. The mutation to trichodermin resistance conferred resistance to other 12,1S-epoxytrichothecenes and to the structurally unrelated antibiotic anisomycin.
HE availability of antibioltic resistant mutants in bacteria with altered riboTsomes has proven extremely useful in our understanding o€ the function and and DAVIES1968; GALEet al. 1972; genetics of the bacterial ribosome (WEISBLUM DAVIESand NOMURA 1972; JASKUNAS, NOMURA and DAVIES1974). By comparison, similar biochemical and genetic studies of antibiotic resistance mutants in eukaryotic organisms have been somewhat limited (MCLAUGHLIN 1974). The yeast Saccharomyces cereuisiae is well suited to studies of eukaryotic ribosomal genetics, structure, and function. S. cerevisiae has a well characterized genetic system (MORTIMER and HAWTHORNE 1973), a stable haploid phase that facilitates mutant selection and characterization. and is amenable to most biochemical techniques. Mutations to antibiotic resistance in S. cerevisiae which involve ribosomal alterations have been reported for the glutarimide antibiotics (COOPER,BANTHORPE and WILKIE 1967; JIMENEZ,LITTLEWOOD and DAVIES1972), the phenMCI,AUGHI,IN and WAKATAMA 1973; GRANT, anthrene alkaloids (SKOGERSON, SANCHEZand JIMENEZ1974) , and some of the 12,13-epoxytrichothecenes (SCHINDLER, GRANT and DAVIES1974). We have mapped the genetic locus for resistance to trichodermin tcml, and have found it to be located on the right arm of chromosome X V near the locus petl7. Surprisingly, mutations at this locus tcml also determined resistance to the unrelated antibiotic anisomycin.. MATERIALS A N D METHODS
Strains: The strains used in these studies are described in Table 1. The isolation and characterization of S. cereuisiae strains SRI, (JIMENEZ,LITTLEWOOD and DAVIES1972) and CLP-1 Genetics 83: 667-673 August, 1976
668
P. G. GRANT, D. S C H I N D L E R A N D J. E . DAVIES
TABLE I
Strains used in these studies Strain
CLP-1 CLP-8
SRI, E27c 2181-1A X3382-3A 115 SH361 IC 421-5A
Source
Genotype
a leuztcml a leu2 tcml LY his4 trp5 cyh2 ~i leu2 tcml a trpl adel his2 gall a arg4 asp5 pet17 tyr7 cdcl4 adel gall trpl his2 his6 a ade2 his4 cyh2 a sup3 can1100 leul-12 metl-I ade2-I lysl-I his5-2 CY ade2-I his5-2 tcml a his5-2 lysl-I arg4-17 trp5-48 ade2-I
D. SCHINDLER D. SCHINDLER B. LITTLEWOOD Cross of CLP-1 and SRI, B. LITTLEWOOD Yeast Genetics Stock Center B. LITTLEWOOD J. GORMAN Cross of CLP-1 and 421-5a
J. GORMAN
( SCHINDLER, GRANTand DAVIES1974) have been described elsewhere. Strain E27c was derived from a cross of strains CLPl and SR,,. Media: YED medium contained 1% yeast extract (Difco), 2% peptone (Difco), and 2% glucose. YNB medium contained 0.7% yeast nitrogen base without amino acids (Difco), 2% glucose, plus appropriate supplements. Presporulation medium contained 10% glucose, 0.8% yeast extract, and 0.3% peptone. Sporulation medium contained 1% potassium acetate, 0.1 % yeast extract, and 0.05% glucose. All solid media were prepared by adding 2% agar (Difco) to the above media. Genetic analyses: Matings were performed by mixing haploid strains of opposite mating type on YED plates and incubating overnight at 30". Diploids were isolated by prototrophic selection on YNB plates containing the appropriate supplements. Single colonies were used to inoculate 10 ml of presporulation medium which was incubated at 30" for 16 to 24 hrs without shaking. When fermentation appeared Iigorous, as evidenced by the rapid evolution of CO,, the cells were washed once with 5 ml of sporulation medium and resuspended in 5 ml of fresh sporulation medium. The cultures were then incubated 2 to 4 days at 30" with shaking. In crosses with strains possessing the temperature-sensitive allele cdcl4, incubation in sporulation medium was carried out at 23 to 25". Sporulation cultures were stored at 4" in 1 ml of fresh sporulation medium. Tetrads were dissected and analyzed using standard techniques (HAWTHORNE and MORTIMER 1968). The percentage of tetrads which germinated completely varied from about 60 to 90% depending on the cross. Only complete tetrads were analyzed. Poly-U-dependent phenylalanine incorporation: This was carried out as described previously (SCHINDLER, GRANTand DAVIES1974), but with the following modification: reactions were terminated by the addition of 2 drops of I N KOH followed by incubation at 30" for 20 minutes before addition of 10% trichloroacetic acid. RESULTS
Trichodermin resistance in S. cereuisiae strain CLP-1 was due to a single, recessive nuclear gene and we have named this gene tcml. Eight trichodermin resistant mutants isolated previously were allelic with tcml. The trichodermh resistant mutants have been shown to have a n altered 60s ribosomal subunit (SCHINDLER, GRANTand DAVIES1974). Since mutations in the gene cyh2 have been shown to produce cycloheximide-resistant 60s ribosomal subunits
TRICHODERMIN RESISTANCE OF
669
60s RIBSOMES
TABLE 2 Ascus-type ratios of tcml with cyh2 and mating-type locus Gene pair
tcml -cyh2 tcml-mating type
PD*
NPD'
T'
8 23
8 13
32 80
* PD,NPD,T refer to parental ditype, nonparental ditype and tetratype asci, respectively.
(MCLAUGHLIN 1974), strain CLP-1 was crossed with strain SR17, a mutant at the cyh2 locus (JIMENEZ,LITTLEWOOD and DAVIES 1973), to determine if tcml was linked to cyh2. The results of the cross, shown in Table 2, indicated that there was no linkage between these two loci. The results in Table 2 also show that there is no linkage between tcml and the mating-type locus. Since c r y l , a gene producing 40s ribosomal subunits resistant to phenanthrene alkaloids, is closely linked to the mating type locus, tcml is not linked to cry1 (SKOGERSON, MCLAUGHLIN and WAKATAMA 1973; GRANT,SANCHEZand JIMENEZ 1974). Neither cyh2 nor tcml appeared to have any effect on the expression of the other resistance allele as it was possible to score all tetrads for resistance to cycloheximide and trichodermin, without ambiguity. The possibility of centromere linkage of tcml was checked in crosses involving tcml and trpl as a marker for centromere segregation. The results show a second division segregation frequency of 39% placing tcml 19-20 centimorgans (cm) from its centromere. tcml containing strains were crossed with a series of strains containing established centromere-linked markers to determine the centromere to which tcml was linked. In the course of screening these crosses tcml was found to be tightly linked to petl7, a gene marking the centromere of chromosome X V . The ascus type ratios are shown in Table 3 ; these results placed tcml 1 cm from petl7. The three tetratype asci all showed a first division segregation pattern from tcml and a second division segregation pattern for petl7, indicating the tcml was closer to the centromere than petl7. This suggests the order; centromere, tcml, pet17 on the right arm of chromosome X V . To confirm the location of icml on chromosome XV, crosses were performed with strains containing ade2 and SUP3. The data from these crosses, presented in Table 3, confirmed that tcml was located on chromosome X V . The above results placed tcml on the right arm of chromosome X V , 19-20 cm from the centromere, 1 cm nearer the centromere than petl7. TABLE 3
Linkage data for tcml with loci on chromosome X V Gene pair
tcml-pet17 tcml-ade2 tcml -SUP3
PD
130 15 53
NPD
7
0 3 0
3 91 34
670
P. G . G R A N T , D. S C H I N D L E R A N D J. E. DAVIES
Resistance to anisomycin: The trichodermin-resistant mutants that we have isolated were resistant to the antibiotic anisomycin as well as to other 12,13epoxytrichothecenes such as nivalenol (SCHINDLER, GRANTand DAVIES 1974; SCHINDLER, 1974). Resistance to anisomycin appeared to be the result of the same mutation in tcml that conferred resistance to trichodermin, as no recombinant spores were found in 190 tetrads analyzed for both trichodermin and anisomycin resistance. Ribosomes from strains CLP-1 and CLP-8 were shown to be resistant to several of the 12,13-epoxytrichothecenes in vitro ( SCHINDLER,GRANTand
I
0.I
I .o Drug
10.0
IOO.0
Concentration, pM
FIGURE 1.-Inhibition of poly (U)-dependent phenylalanine incorporation on A224A and CLP-8 ribosomes by various drugs. Results are given as '% control activity measured by acid-precipitable radioactive polyphenylalanine formed after a 30-minute incubation at 30" as described in MATERIALS AND METHODS. Control activity (no drug) for A224 ribosomes (0) is 62.0 pmoles of phenylalanine and for CLP-8 ribosomes (x), 42.2 pmoles. Solid lines-nivalenol. Dashed lines-anisomycin.
TRICHODERMIN RESISTANCE OF
60s RIBSOMES
671
DAVIES1974; SCHINDLER, 1974). However, ribosomes prepared from strain CLP-8 appear to be only weakly resistant to anisomycin in the same in vitro assay (Figure 1). These ribosomes are as sensitive as the ribosomes from the parental strain to inhibition by cycloheximide, blasticidin S, and sparsomycin (data not shown). JIMENEZ, SANCHEZ and VASQUEZ (1975) have made similar observations on mutants derived from different strains of S. cerevisiae. Resistance to trichothecin: Mutants selected for resistance to trichothecin, another of the 12,13-epoxytrichothecenes,were allelic with tcml and were crossresistant to trichodermin and anisomycin. DISCUSSION
Trichodermin is a peptidyl transferase inhibitor ( CARRASCO, BARBACID and VAZQUEZ 1973). The isolation of mutants of S. cereuisiae resistant to trichodermin and other 12,13-epoxytrichothecenesprovides a gentic marker for a 60s ribosomal function and perhaps for a component of the peptidyl transferase centre. The gene determining this resistance, tcml, is a single, recessive nuclear gene located on the right arm of chromosome X V , 1 cm nearer the centromere than petl7. At first sight, simultaneous resistance to 12,13-epoxytrichothecenesand anisomycin, two structurally unrelated drugs, was surprising. However it has been shown that these drugs bind at mutually exclusive sites (BARBACID and VAZQUEZ 1974a, b; WEIet al. 1974). The genetic evidence in this paper strongly indicates that the simultaneous resistance to anisomycin and 12,13-epoxytrichothecenesis the result of a single mutation, although biochemically the resistance to each drug in vitro differs greatly. A possible explanation for the weak expression of resistance to anisomycin may be that a factor is missing from our in vitro assay system which is necessary for the full expression of anisomycin resistance, or that the ribosomes we use are structurally or functionally defective without affecting poly-U-dependent incorporation. We cannot eliminate the possibility that tcml may be closely linked to a genetic locus altering drug permeability and that we are dealing with two very closely linked mutations. The fact that ribosomes from the tcml strain show some degree of resistance to anisomycin would tend to argue against this. It is interesting to note that the loci known to affect ribosomal structure in S. cerevisiae tcml, cyh2, cry1 and the genes for ribosomal RNA (rRNA) are not clustered within the same genetic region although some clustering of redundant genes for rRNA does occur (CRAMER, BHARGAVA and HALVORSON 1972; FINKELSTEIN, BLAMIRE and MARMUR 1972; KABACK, BHARGAVA and HALVORSON 1973; @YEN, 1973). It has recently been found that the ribosomal structural genes in E. coli are also scattered throughout the chromosome and often occur in clusters of several loci (JASKUNAS, LINDAHL and NOMURA 1975; LINDAHL et al. 1975; WATSON et al. 1975). While no evidence is available to demonstrate that the loci tcml, cyh2, and cry1 are ribosomal proteins and not modifying enzymes, the organization of ribosomal structural genes in S. cerevisiae may prove different from that in E . coli, since there is presently very little evidence of clustering of
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P. G. GRANT, D. S C H I N D L E R A N D J. E. DAVIES
genes of biochemically related functions in yeast (MORTIMER and HAWTHORNE 1973). It would appear that in S. cerezlisiae mutations affecting ribosomes and protein synthesis are often closely linked to mutations involving mitochondrial function. This linkage occurs between prt2 and pet2, cyh2 and tsm437 (MORTIMER and HAWTHORNE 1973), cryl and pet18 ( SKOGERSON, MCLAUGHLIN and WAKATAMA 1973), and between tcml and petl7. One may speculate that these petite mutations lead to the production OP altered, nonfunctional mitochondrial ribosomal proteins and that mitochrondrial ribosomal protein genes may be linked to cytoplasmic ribosomal protein genes. It is known that most, if not all, mitochondrial ribosomal proteins are made on cytoplasmic ribosomes and presumably coded by nuclear genes (GROOT1974) and also that mitochondrial protein synthesis is et al. 1966). necessary for mitochondrial fimction (HUANG We wish to thank B. LITTLEWOOD and J. GORMAN for strains and advice on genetic mapping and other yeast lore. Trichodermin was provided by DR. W. 0. GODTFREDSEN, nivalenol by DR. F. STRONG, blasticidin S by DR. R. METZENBERG and cycloheximide by DR. G. B. WHITFIELD.This work was supported by NIH grant 1-R01-12448-01and by an Upjohn Graduate Fellowship. L I T E R A T U R E CITED
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