Current Genetics
Current Genetics (1983) 7:357 362
~) Springer-Verlag 1983
Drug-Dependent Mutants in Yeast Saccharomyces cerevisiae M. D. Ter-Avanesyan 1, L. N. Mironova 1, S. G. Inge-Vechtomov 1 , I. V. Zlatkin 2, V. N. Smirnov 2, and A. P. Surguchov 2 1 Department of Genetics, Leningrad State University, Leningrad 199164, USSR 2 USSR Research Center of Cardiology, Petroverigsky Lane 10, Moscow 101837, USSR
Summary. Mutations in s u p l and sup2 genes may cause cycloheximide-dependent growth in yeast Saccharornyces cerevisiae. Two classes of such mutants are described in the paper: 1) high temperature sensitive mutants, which do not express their sensitivity to nonpermissive temperature in the presence of cycloheximide (conditionally dependent) and 2) mutants unable to grow in the absence o f the drug (true dependent). Some o f the mutants of both classes express dependence toward another antibiotic - trichodermine. The binding o f H 3labelled cycloheximide studied by equilibrium dialysis has demonstrated that both 80S ribosomes and 60S subunits isolated from conditionally dependent mutant showed a higher affinity for the drug compared to that o f a parent strain. The number of binding sites per ribosome or per 60S subunit in the cycloheximide dependent mutant remains unchanged. Circular dichroism spectra o f a mutant ribosomes in the presence as well as in the absence o f antibiotic revealed that s u p l and sup2 mutations alter conformation of the yeast cytoplasmic ribosomes. The binding o f cycloheximide to mutant ribosomes induces a conformational shift, which presumably compensates for their functional defect. Key words: Cycloheximide Ribosomal suppressors
dependent
mutants
Introduction A significant body o f knowledge in ribosomal genetics has been obtained due to the study of drug-dependent mutants of E. coli (Dabbs 1978; Dabbs 1979; Dabbs and Offprint requests to." A. P. Surguchov
Looman 1981; Dabbs and Wittman 1976). However, a similar class of mutants has not been available for eukaryotic organisms. Recently in the course of the study o f s u p l and sup2 ribosomal suppressors in yeast Sacchar o m y c e s cerevisiae we have described a temperature sensitive sup2 mutant which did not express sensitivity to restrictive temperature in the presence of cycloheximide, a glutarimide antibiotic (Ter-Avanesyan et al. 1982b). In the present paper a more detailed characterisation o f the cycloheximide-dependent mutants o f yeast is presented.
Materials and Methods Routine methods of yeast genetics were used (Inge-Vechtomov 1971; Ter-Avanesyan et al. 1982a). Hypertonic media contained glycerol of KC1 as osmotic agents (Ter-Avanesyan et al. 1982a). If necessary, media were supplemented with cycloheximide (Calbiochem), and trichodermine (kindly supplied by Doctor Godtfredsen, Leo Pharmaceutical Products Ltd., Denmark). The following strains of yeast S. cerevisiae from Peterhoff genetic collection were used as originals for selection of cycloheximide-dependent mutants: 8A-P3532 29V-P2156
~ adel-14 his7-1 m e t a l
aadel-14 his7-1 m e t a l
The inheritance of drug-dependence was analyzed in meiotic progeny of hybrids derived from the crosses of mutants with the strain 2A-P3663 (a his7-1 lys2-A12 thr4-B15 leu2-2 canl-88). supl and sup2 mutants derived previously from strains 29V-P2156 and 125A-P2156 were used as testers for allelism of the suppressors (Ter-Avanesyan et al. 1982a). The designations of markers have been described previously (Inge-Vechtomov 1971). The following designations of phenotype were used: H t s - and Hts + - high temperature sensitivity and resistance, respectively; Cyh d - cycloheximide dependence; Cyh cd - conditional cycloheximide dependence (inability to grow at 30 °C and/or 36 °C without cycloheximide); Cyh td true cycloheximide dependence (inability to grow without cycloheximide not only at 30 °C and 36 °C, but also at 20 °C);
358
M.D. Ter-Avanesyan et al.: Drug-Dependent Mutants in Yeast
Table 1. Growth pattern of the Hts- mutants in the presence of cycloheximide at restrictive temperatures Phenotype of the mutant a
Number of mutants, carrying mutations in gene
Number of mutants able to grow at 30° or 36 oC in the presence of cycloheximide in concentration (~g/ml) 0.1
Hts- (30 °) Hts- (36 °)
0.2
0.5
Total number of Cyh cd mutants
supl
72
54
1
0
55
sup2
3
2
1
0
3
supl sup2
8 3
5 1
0 0
0 0
5 1
a The restrictive temperatures are indicated in the brackets
Cyhnd - nondependence - ability to grow without cycloheximide at all temperature mentioned above, Sup + and Sup suppressor activity and its absence, respectively. Equilibrium dialysis has been performed as described in (Stocklein and Piepersberg 1980a) in the range of antibiotic concentrations 0.22-1.79 ~M. The specific activity of cycloheximide was 1.2 Ci/mmole. Post-ribosomal supernatant was present in the final concentration 0.8 mg/ml in all the samples for equilibrium dialysis. Ribosomes and ribosomal subunits were isolated as described in (Surguchov et al. 1980). Ribosomes prepared from freshly-grown yeast cells were suspended in buffer I (10 mM MgC12, 25 mM KC1, 35 mM Tris-HC1, 1 mM DTT, pH 7.6) up to 0.4-0.9 A260/ml. Circular dichroic (CD) spectra were registered in 1 cm quartz cuvette on Dichrographe III (Roussel-Jouan) (sensitivity 2 x 10-6/mm). The molar elfipticity of the samples was calculated according to AA(X) the formula [0] = 3300 x - ,where AA(X)is the measured C CD-absorption at certain X and C is ribosomes concentration in moles of nucleotides. The latter value was determined on the basis of absorbancy of the solution after the fight scattering substraction using the molar extinction of ribosomes e260 = 6.1 x 103 (van der Zeijst 1973). After the titration of the ribosome sample by cycloheximide the increase of the volume did not exceed 1/25 of the original value. The correction for dilution was made by calculation of [01.
Results The Effect o f Cycloheximide on the Growth Patterns o f Hts- supl and sup2 Mutants o f Yeast Earlier we have described the Hts- sup2 mutant, which was able to grow at non-permissive temperature in the presence of cycloheximide (Ter-Avanesyan et al. 1982b). In the present paper the data on the expression o f high temperature sensitivity of a large number of supl and sup2 mutants in the presence of cycloheximide are presented. 86 Hts- suppressor mutants selected earlier using the strain 8A-P3532 as original were studied (Mironova et al. 1982). The data presented in Table 1 demonstrate that
temperature sensitivity of the majority of such mutants is not expressed in the presence of 0.1 pg/ml cycloheximide. The "cycloheximide-repairable" phenotype was attributed both for supl and sup2 mutants. From these data it is evident that the Cyh cd phenotype described earlier is not unique for one strain, but is a comm o n feature for supl and sup2 mutations.
Selection and Analysis o f Cyclohexirnide-Dependent Mutants Since a high proportion o f Hts- sup1 and sup2 mutants was able to grow at restrictive temperature in the presence o f cycloheximide we had proposed that it was possible to select mutants unable to grow without drug at ahy temperature tested. As demonstrated previously, the revertants to prototrophy for both adenine and histidine in strains, carrying adel-14 and his7-1 mutations arise only due to mutations in supl or sup2 suppressor genes (Inge-Vechtomov and Andrianova 1972). In this study we have carried out selection of such mutants on selective media supplemented with cycloheximide (0.1 /~g/ml). All the subsequent procedures with these mutants were carried out using the drug-containing media. F r o m 567 selected mutants, 167 express altered growth patterns in YEPD medium. Some of them were temperature sensitive, whereas others were unable to grow on this medium at all temperatures tested. The data presented in Table 2 demonstrate that cycloheximide stimulates growth of a large number of these mutants derived both from the strains 8A-P3532 and 29V-P2156. These mutants were distributed into two classes according to the effect of the drug upon their growth: 1) previously described class of high temperature sensitive mutants, which grow at the restrictive temperature in the presence of the drug (CyhCd); 2) a new class, which is comprised by mutants able to grow
359
M. D. Ter-Avanesyan et al.: Drug-Dependent Mutants in Yeast
Table 2. Cycloheximide-dependence of selected mutants Original strain
Total number of mutants analyzed
Absence of growth on the YEPD media at (t°)
Number of mutants of this phenotype
Number of Cyhd mutants
8A-P3532
95
36° 30°,36 ° 20°,30°,36 °
30 45 20
7 21 20
29V-P2156
72
36° 30°,36 ° 20°,30°,36 °
10 39 23
3 17 23
The dependence of growth was tested at the minimal temperature for given class of mutants
Table 3. Meiotic segregation for suppressor activity (Sup+) and cycloheximide dependence (Cyhd) in hybrids, heterozygous for sup1 and sup2 mutations Parental strainsa
2A-p3663 (Cyhnd)
a b c d
Hybrids
Number of tetrades analyzedb
Total number of segregants analyzedc
Number of segregants with phenotypes Cyhd Sup+
Cyhnd Sup-
5-8A-P3532 (sup2, Cyhtd)
P2995
12
233
76
157 d
2-8A-P3532 (sup1, Cyhcd)
P2991
17
92
45
47
44-8A-P3532 (sup2, Cyhtd)
P2997
11
63
30
33
49-8A-P3532 (supl, Cyhtd)
P2996
9
138
60
78
Loci of suppressor mutations and character of c'ycloheximide dependence of corresponding mutant are indicated in brackets In all tetrades the segregation 2CyhdSup+: 2CyhndSup - was observed The summarized data on complete and noncomplete tetrades The deviation from the 1 CyhdSup+:icyhndSup- ratio for hybrid P2995 is probably due to a selective advantage of segregar~ts without suppressor mutation
only on the medium supplemented with cycloheximide (Cyh td) at all temperatures tested. For further characteristic of the cycloheximide dependence phenotype 25 Cyh cd and Cyh td mutants were crossed with testers for supl and sup2 mutations. All selected mutations were found to be recessive and allelic for either supl or sup2. Therefore, the modification of the selection procedure for revertants did not influence the spectrum of the suppressor mutations. To study the relationship between the cycloheximide dependence of the mutants and the suppressor mutations the meiotic segregation of the heterozygous hybrids was analyzed. No recombination of cycloheximide dependence and suppressor phenotype for all hybrids analyzed has been observed (Table 3).
Growth of Cycloheximide Dependent Mutants on the Hyperto nic Media
The Cyh cd sup2 mutant described in our previous publication (Ter-Avanesyan et al. 1982b) was able to grow at restrictive temperature not only in the presence of the drug but in addition on hypertonic media. In the present study the osmotic remediality of some suppressor mutants was also observed. All of the Cyh cd and Cyh td mutants tested were able to grow on high osmolarity media (0.1 M glycerol; 0.2 M KC1 or 0.5 M KC1). All suppressor carrying segregants of hybrids heterozygous for supl and sup2 mutations (Table 3) were also osmotic remedial.
360
M.D. Ter-Avanesyan et al.: Drug-Dependent Mutants in Yeast i
A
16
14
12
10 ¢o I
O ,-x
8
| I I
\
-2
250
270
290
,310 "'250
270
290
310
/~ (rim) Fig. 2 a and b. Circular dichroic (CD) spectra of ribosomes for parent (29V-P2156) a and Cyh cd mutant (63-29V-P2156) b yeast cells without cycloheximide ( ) and after the addition of 36 nM cycloheximide ( - - - ) . The concentration of ribosomes was 11-12 nM
Fig. la and b. Test for antibiotic dependence of the suppressor mutants. The white rings around the crystalls of antibiotics in the center of the Petri dishes indicate a drug dependent growth: a cycloheximide dependent growth, b trichodermine dependent growth. Other details are described in the text
Trichodermine Dependence of Mutants For better understanding of the nature of cycloheximide dependence the growth of mutants in the presence of another antibiotic - trichodermine was analyzed. The cells o f mutants (growth o f two Cyh ca and two Cyh td mutants has been studied in these experiments)
were spead on YEPD plates containing several crystalls o f trichodermine in the center of each plate. Growth o f mutants at nonpermissive conditions was followed at 30 °C or 36 °C for Cyh cd mutants and at 20 ° for Cyh td mutants. Figure 1 presents a typical example of the drug dependence phenomenon. In addition to these mutants, four suppressor carrying segregants from the meiotic progeny of hybrid P2991 (corresponding mutant was conditionally dependent) and six segregants of hybrid P2997 (corresponding mutant was true dependent) were also analysed. All of these mutants expressed a trichodermine dependence phenotype.
Circular Dichroism Study of Mutant Ribosomes As can be seen from Fig. 2, the CD-spectmm o f ribosomes from Cyh cd mutant strain differs from that o f the parental strain. The main difference between the CD-spectra of mutant and parent ribosomes was found at 265 nm. The addition of cycloheximide to ribosome preparation is accompanied by the change in the spectrum of the mutant but not of the parent strain ribosomes. As a result the CD-spectra for mutant and parent ribosomes become practically identical.
M. D. Ter-Avanesyanet al.: Drug-DependentMutants in Yeast
e~
0 x
8os
3
6 )S
2
"I(,.)
\ 0,2
o,3
0,4
0,5
0,2
0,4
-7-
Fig. 3. Scatchard plots for cycloheximide binding to 80S ribosomes and 60S subunits isolated from parent strain ( - - o - - ) and Cyhcd mutant 63-29V-P2156 ( - - e - - )
Binding of Ha-Cycloheximide to Ribosomes and Ribosomal Subparticles In equilibrium dialysis experiments with [H3]cycloheximide the Ka values of 0.35 -+ 0.12 x 107 M-1 and 1.26 -+ 0.14 x 10 v M-1 were estimated for parent and mutant 80S ribosomes, respectively (Fig. 3a). The corresponding values for 60S subparticles were found to be 0 . 4 6 + 0 . 1 0 x l 0 V M -1 and 2 . 5 x 1 0 7 M -1 for parent and mutant subunits, respectively (Fig. 3b). Within the range of drug concentration used no indication of binding of cycloheximide either to 40S subunits or to post-ribosomal supernatant was found. The number of binding sites was found to be equal to 0.4-0.5 both for parent and mutant 80S and 60S particles.
Discussion Earlier we have described a yeast mutant sensitive to a high temperature which did not express its sensitivity to restrictive temperature in the presence of cycloheximide (Ter-Avanesyan et al. 1982). In the present study we have found that this property is a common feature of high temperature sensitive supl and sup2 mutants. This observations allows us to suggest a possibility of selection of supl and sup2 mutants unable to grow in the absence of cycloheximide irrespectively of incubation temperature. This type of suppressor mutants was indeed selected on medium containing a low concentration of the drug. We have also shown that some of such conditionally and true dependent mutants express their dependence towards trichodermine, another protein synthesis inhibitor. The observation that supl and sup2 mutations may cause a simultaneous dependence for cycloheximide and trichodermine, both of which are known to act upon the 60S subunit of the yeast cytoplasmic ribosomes (Stock-
361 lein and Piepersberg 1980b; Fried and Warner 1981) supports our suggestion that supl and sup2 genes code for proteins of the large ribosomal subunit (Surguchov et al. 1981a, 1981b; Ter-Avanesyan et al. 1982). In the course of the characterization of the drug dependent mutants it has also been found that some of the mutants can grow at nonpermissive conditions on hypertonic media in the absence of antibiotics. It is usually accepted that osmotic remediality is an indication in favour of the conformational lability of mutant proteins (Fincham and Baron 1977). If this is indeed the case, the common effect of antibiotics and hypertonic conditions on growth pattern of the mutant strains allows us to suggest that the phenomenon of the drug dependence is explained by the conformational changes of the mutant ribosomes in the presence of antibiotic. Moreover, one can propose that this conformational shift of mutant ribosomes compensates for their functional defects induced by suppressor mutation. This suggestion was confirmed by the comparison of CD-spectra of mutant and parent ribosomes. The main differences between these spectra are found at 265 nm. The magnitude of the peak height in this range is known to be proportional to the base-stacking in rRNA (Allen and Wong 1979). Since the CD-absorbance at 265 nm of mutant ribosomes is higher than that of parent strain one can assume that ribosomes from Cyhcd mutant strain have a more ordered rRNA structure in comparison to that of parent ribosomes. After the addition ofcycloheximide to ribosomes preparations the CD-spectra for mutant and parent strain ribosomes become practically identical. Based on this fact one may suppose that the binding of cycloheximide to mutant ribosomes probably leads to the relaxation of the secondary structure of rRNA and the resulting ribosomes conformation becomes comparable to that of parent strain ribosomes. The data of equilibrium dialysis revealing the differences in K a values for 80S and 60S particles from parent and mutant strains demonstrate the alteration of ribosomal structure in the domain of 60S subunit responsible for cycloheximide binding. We conclude that the phenomenon of the drug dependence is related to the conformational state of the mutant ribosomes. The use of supl and sup2 drug dependent mutants allows us to show that the binding of cycloheximide and trichodermine to cytoplasmic eukaryotic ribosomes may induce a conformational shift, as it was earlier demonstrated for some antibiotics, acting on prokaryotic ribosomes (Noreau et al. 1980; Martinez et al. 1978).
Acknowledgement. The authors are grateful to Mrs. G. Zaretzki for assistance in some experiments.
362 References Allen SH, Wong KP(1979)Arch Biochem Biophys 195:112-120 Dabbs ER (1978) Mol Gen Genet 165:73-78 Dabbs ER (1979) J Bacteriol 140:734-734 Dabbs ER, Looman K (1981) Mol Gen Genet 184:224-229 Dabbs ER, Wittmann HG (1976) Mol Gen Genet 149:303-309 Fincham JRS, Baron YA (1977) J Mol Biol 11.0:627-634 Fried HM, Warner JR (1981) Proc Natl Acad Sci USA 78:238 242 Inge-Vechtomov SG (1971) Genetika 7:113-120 (in Russian) lnge-Vechtomov SG, Andrianova VM (1972) In: Dubinin NP, Goldfarb DM (eds) The molecular mechanisms of genetic processes. Nauka, Moscow, pp 189-195 (in Russian) Martinez O, Vazques D, Modolell J (1978) FEBS Letters 87: 21-24 Mironova LN, Provorov NA, Ter-Avanesyan MD, Inge-Vechtomov SG, Surguchov AP, Smirnov VN (1982) Curt Genet 5:149-152 Noreau J, Grise-Miron L, Brakier-Gingras L (1980) Biochem Biophys Acta 608:72-81 Stocklein W, Piepersberg W (1980a) Antimicrobiol Agents and Chemotherapy 18:863-867
M.D. Ter-Avanesyan et al.: Drug-Dependent Mutants in Yeast Stocklein W, Piepersberg W (1980b) Curt Genet 1:177-183 Surguchov AP, Berestetskaya YuV, Fominykch ES, Pospelova EM, Smimov VN, Ter-Avanesyan MD, Inge-Vechtomov SG (1980) FEBS Letters 111:175-178 Surguchov AP, Berestetskaya YuV, Smirnov VN, Ter-Avanesyan MD, Inge-Vechtomov SG (1981 a) FEMS Microbiology Letters 12:381-384 Surguchov AP, Fominykch ES, Smirnov VN, Ter-Avanesyan MD, Mironova LN, lnge-Veehtomov SG (1981b) Biochim Biophys Acta 654:149-155 Ter-Avanesyan MD, Schubochkina EA, Inge-Vechtomov SG (1982a) Genetika 18:215 222 (in Russian) Ter-Avanesyan MD, Schubochkina EA, lnge-Veehtomov SG (1982b) Genetika 18:223-226 (in Russian) van der Zeist BAM, Kees JM, Engel JM, Bloemers HPI (1973) Biochim Biophys Acta 294:517-526
Communicated by F. Kaudewitz Received April 6, 1983
Current Genetics (1983) 7:357 362
~) Springer-Verlag 1983
Drug-Dependent Mutants in Yeast Saccharomyces cerevisiae M. D. Ter-Avanesyan 1, L. N. Mironova 1, S. G. Inge-Vechtomov 1 , I. V. Zlatkin 2, V. N. Smirnov 2, and A. P. Surguchov 2 1 Department of Genetics, Leningrad State University, Leningrad 199164, USSR 2 USSR Research Center of Cardiology, Petroverigsky Lane 10, Moscow 101837, USSR
Summary. Mutations in s u p l and sup2 genes may cause cycloheximide-dependent growth in yeast Saccharornyces cerevisiae. Two classes of such mutants are described in the paper: 1) high temperature sensitive mutants, which do not express their sensitivity to nonpermissive temperature in the presence of cycloheximide (conditionally dependent) and 2) mutants unable to grow in the absence o f the drug (true dependent). Some o f the mutants of both classes express dependence toward another antibiotic - trichodermine. The binding o f H 3labelled cycloheximide studied by equilibrium dialysis has demonstrated that both 80S ribosomes and 60S subunits isolated from conditionally dependent mutant showed a higher affinity for the drug compared to that o f a parent strain. The number of binding sites per ribosome or per 60S subunit in the cycloheximide dependent mutant remains unchanged. Circular dichroism spectra o f a mutant ribosomes in the presence as well as in the absence o f antibiotic revealed that s u p l and sup2 mutations alter conformation of the yeast cytoplasmic ribosomes. The binding o f cycloheximide to mutant ribosomes induces a conformational shift, which presumably compensates for their functional defect. Key words: Cycloheximide Ribosomal suppressors
dependent
mutants
Introduction A significant body o f knowledge in ribosomal genetics has been obtained due to the study of drug-dependent mutants of E. coli (Dabbs 1978; Dabbs 1979; Dabbs and Offprint requests to." A. P. Surguchov
Looman 1981; Dabbs and Wittman 1976). However, a similar class of mutants has not been available for eukaryotic organisms. Recently in the course of the study o f s u p l and sup2 ribosomal suppressors in yeast Sacchar o m y c e s cerevisiae we have described a temperature sensitive sup2 mutant which did not express sensitivity to restrictive temperature in the presence of cycloheximide, a glutarimide antibiotic (Ter-Avanesyan et al. 1982b). In the present paper a more detailed characterisation o f the cycloheximide-dependent mutants o f yeast is presented.
Materials and Methods Routine methods of yeast genetics were used (Inge-Vechtomov 1971; Ter-Avanesyan et al. 1982a). Hypertonic media contained glycerol of KC1 as osmotic agents (Ter-Avanesyan et al. 1982a). If necessary, media were supplemented with cycloheximide (Calbiochem), and trichodermine (kindly supplied by Doctor Godtfredsen, Leo Pharmaceutical Products Ltd., Denmark). The following strains of yeast S. cerevisiae from Peterhoff genetic collection were used as originals for selection of cycloheximide-dependent mutants: 8A-P3532 29V-P2156
~ adel-14 his7-1 m e t a l
aadel-14 his7-1 m e t a l
The inheritance of drug-dependence was analyzed in meiotic progeny of hybrids derived from the crosses of mutants with the strain 2A-P3663 (a his7-1 lys2-A12 thr4-B15 leu2-2 canl-88). supl and sup2 mutants derived previously from strains 29V-P2156 and 125A-P2156 were used as testers for allelism of the suppressors (Ter-Avanesyan et al. 1982a). The designations of markers have been described previously (Inge-Vechtomov 1971). The following designations of phenotype were used: H t s - and Hts + - high temperature sensitivity and resistance, respectively; Cyh d - cycloheximide dependence; Cyh cd - conditional cycloheximide dependence (inability to grow at 30 °C and/or 36 °C without cycloheximide); Cyh td true cycloheximide dependence (inability to grow without cycloheximide not only at 30 °C and 36 °C, but also at 20 °C);
358
M.D. Ter-Avanesyan et al.: Drug-Dependent Mutants in Yeast
Table 1. Growth pattern of the Hts- mutants in the presence of cycloheximide at restrictive temperatures Phenotype of the mutant a
Number of mutants, carrying mutations in gene
Number of mutants able to grow at 30° or 36 oC in the presence of cycloheximide in concentration (~g/ml) 0.1
Hts- (30 °) Hts- (36 °)
0.2
0.5
Total number of Cyh cd mutants
supl
72
54
1
0
55
sup2
3
2
1
0
3
supl sup2
8 3
5 1
0 0
0 0
5 1
a The restrictive temperatures are indicated in the brackets
Cyhnd - nondependence - ability to grow without cycloheximide at all temperature mentioned above, Sup + and Sup suppressor activity and its absence, respectively. Equilibrium dialysis has been performed as described in (Stocklein and Piepersberg 1980a) in the range of antibiotic concentrations 0.22-1.79 ~M. The specific activity of cycloheximide was 1.2 Ci/mmole. Post-ribosomal supernatant was present in the final concentration 0.8 mg/ml in all the samples for equilibrium dialysis. Ribosomes and ribosomal subunits were isolated as described in (Surguchov et al. 1980). Ribosomes prepared from freshly-grown yeast cells were suspended in buffer I (10 mM MgC12, 25 mM KC1, 35 mM Tris-HC1, 1 mM DTT, pH 7.6) up to 0.4-0.9 A260/ml. Circular dichroic (CD) spectra were registered in 1 cm quartz cuvette on Dichrographe III (Roussel-Jouan) (sensitivity 2 x 10-6/mm). The molar elfipticity of the samples was calculated according to AA(X) the formula [0] = 3300 x - ,where AA(X)is the measured C CD-absorption at certain X and C is ribosomes concentration in moles of nucleotides. The latter value was determined on the basis of absorbancy of the solution after the fight scattering substraction using the molar extinction of ribosomes e260 = 6.1 x 103 (van der Zeijst 1973). After the titration of the ribosome sample by cycloheximide the increase of the volume did not exceed 1/25 of the original value. The correction for dilution was made by calculation of [01.
Results The Effect o f Cycloheximide on the Growth Patterns o f Hts- supl and sup2 Mutants o f Yeast Earlier we have described the Hts- sup2 mutant, which was able to grow at non-permissive temperature in the presence of cycloheximide (Ter-Avanesyan et al. 1982b). In the present paper the data on the expression o f high temperature sensitivity of a large number of supl and sup2 mutants in the presence of cycloheximide are presented. 86 Hts- suppressor mutants selected earlier using the strain 8A-P3532 as original were studied (Mironova et al. 1982). The data presented in Table 1 demonstrate that
temperature sensitivity of the majority of such mutants is not expressed in the presence of 0.1 pg/ml cycloheximide. The "cycloheximide-repairable" phenotype was attributed both for supl and sup2 mutants. From these data it is evident that the Cyh cd phenotype described earlier is not unique for one strain, but is a comm o n feature for supl and sup2 mutations.
Selection and Analysis o f Cyclohexirnide-Dependent Mutants Since a high proportion o f Hts- sup1 and sup2 mutants was able to grow at restrictive temperature in the presence o f cycloheximide we had proposed that it was possible to select mutants unable to grow without drug at ahy temperature tested. As demonstrated previously, the revertants to prototrophy for both adenine and histidine in strains, carrying adel-14 and his7-1 mutations arise only due to mutations in supl or sup2 suppressor genes (Inge-Vechtomov and Andrianova 1972). In this study we have carried out selection of such mutants on selective media supplemented with cycloheximide (0.1 /~g/ml). All the subsequent procedures with these mutants were carried out using the drug-containing media. F r o m 567 selected mutants, 167 express altered growth patterns in YEPD medium. Some of them were temperature sensitive, whereas others were unable to grow on this medium at all temperatures tested. The data presented in Table 2 demonstrate that cycloheximide stimulates growth of a large number of these mutants derived both from the strains 8A-P3532 and 29V-P2156. These mutants were distributed into two classes according to the effect of the drug upon their growth: 1) previously described class of high temperature sensitive mutants, which grow at the restrictive temperature in the presence of the drug (CyhCd); 2) a new class, which is comprised by mutants able to grow
359
M. D. Ter-Avanesyan et al.: Drug-Dependent Mutants in Yeast
Table 2. Cycloheximide-dependence of selected mutants Original strain
Total number of mutants analyzed
Absence of growth on the YEPD media at (t°)
Number of mutants of this phenotype
Number of Cyhd mutants
8A-P3532
95
36° 30°,36 ° 20°,30°,36 °
30 45 20
7 21 20
29V-P2156
72
36° 30°,36 ° 20°,30°,36 °
10 39 23
3 17 23
The dependence of growth was tested at the minimal temperature for given class of mutants
Table 3. Meiotic segregation for suppressor activity (Sup+) and cycloheximide dependence (Cyhd) in hybrids, heterozygous for sup1 and sup2 mutations Parental strainsa
2A-p3663 (Cyhnd)
a b c d
Hybrids
Number of tetrades analyzedb
Total number of segregants analyzedc
Number of segregants with phenotypes Cyhd Sup+
Cyhnd Sup-
5-8A-P3532 (sup2, Cyhtd)
P2995
12
233
76
157 d
2-8A-P3532 (sup1, Cyhcd)
P2991
17
92
45
47
44-8A-P3532 (sup2, Cyhtd)
P2997
11
63
30
33
49-8A-P3532 (supl, Cyhtd)
P2996
9
138
60
78
Loci of suppressor mutations and character of c'ycloheximide dependence of corresponding mutant are indicated in brackets In all tetrades the segregation 2CyhdSup+: 2CyhndSup - was observed The summarized data on complete and noncomplete tetrades The deviation from the 1 CyhdSup+:icyhndSup- ratio for hybrid P2995 is probably due to a selective advantage of segregar~ts without suppressor mutation
only on the medium supplemented with cycloheximide (Cyh td) at all temperatures tested. For further characteristic of the cycloheximide dependence phenotype 25 Cyh cd and Cyh td mutants were crossed with testers for supl and sup2 mutations. All selected mutations were found to be recessive and allelic for either supl or sup2. Therefore, the modification of the selection procedure for revertants did not influence the spectrum of the suppressor mutations. To study the relationship between the cycloheximide dependence of the mutants and the suppressor mutations the meiotic segregation of the heterozygous hybrids was analyzed. No recombination of cycloheximide dependence and suppressor phenotype for all hybrids analyzed has been observed (Table 3).
Growth of Cycloheximide Dependent Mutants on the Hyperto nic Media
The Cyh cd sup2 mutant described in our previous publication (Ter-Avanesyan et al. 1982b) was able to grow at restrictive temperature not only in the presence of the drug but in addition on hypertonic media. In the present study the osmotic remediality of some suppressor mutants was also observed. All of the Cyh cd and Cyh td mutants tested were able to grow on high osmolarity media (0.1 M glycerol; 0.2 M KC1 or 0.5 M KC1). All suppressor carrying segregants of hybrids heterozygous for supl and sup2 mutations (Table 3) were also osmotic remedial.
360
M.D. Ter-Avanesyan et al.: Drug-Dependent Mutants in Yeast i
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/~ (rim) Fig. 2 a and b. Circular dichroic (CD) spectra of ribosomes for parent (29V-P2156) a and Cyh cd mutant (63-29V-P2156) b yeast cells without cycloheximide ( ) and after the addition of 36 nM cycloheximide ( - - - ) . The concentration of ribosomes was 11-12 nM
Fig. la and b. Test for antibiotic dependence of the suppressor mutants. The white rings around the crystalls of antibiotics in the center of the Petri dishes indicate a drug dependent growth: a cycloheximide dependent growth, b trichodermine dependent growth. Other details are described in the text
Trichodermine Dependence of Mutants For better understanding of the nature of cycloheximide dependence the growth of mutants in the presence of another antibiotic - trichodermine was analyzed. The cells o f mutants (growth o f two Cyh ca and two Cyh td mutants has been studied in these experiments)
were spead on YEPD plates containing several crystalls o f trichodermine in the center of each plate. Growth o f mutants at nonpermissive conditions was followed at 30 °C or 36 °C for Cyh cd mutants and at 20 ° for Cyh td mutants. Figure 1 presents a typical example of the drug dependence phenomenon. In addition to these mutants, four suppressor carrying segregants from the meiotic progeny of hybrid P2991 (corresponding mutant was conditionally dependent) and six segregants of hybrid P2997 (corresponding mutant was true dependent) were also analysed. All of these mutants expressed a trichodermine dependence phenotype.
Circular Dichroism Study of Mutant Ribosomes As can be seen from Fig. 2, the CD-spectmm o f ribosomes from Cyh cd mutant strain differs from that o f the parental strain. The main difference between the CD-spectra of mutant and parent ribosomes was found at 265 nm. The addition of cycloheximide to ribosome preparation is accompanied by the change in the spectrum of the mutant but not of the parent strain ribosomes. As a result the CD-spectra for mutant and parent ribosomes become practically identical.
M. D. Ter-Avanesyanet al.: Drug-DependentMutants in Yeast
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Fig. 3. Scatchard plots for cycloheximide binding to 80S ribosomes and 60S subunits isolated from parent strain ( - - o - - ) and Cyhcd mutant 63-29V-P2156 ( - - e - - )
Binding of Ha-Cycloheximide to Ribosomes and Ribosomal Subparticles In equilibrium dialysis experiments with [H3]cycloheximide the Ka values of 0.35 -+ 0.12 x 107 M-1 and 1.26 -+ 0.14 x 10 v M-1 were estimated for parent and mutant 80S ribosomes, respectively (Fig. 3a). The corresponding values for 60S subparticles were found to be 0 . 4 6 + 0 . 1 0 x l 0 V M -1 and 2 . 5 x 1 0 7 M -1 for parent and mutant subunits, respectively (Fig. 3b). Within the range of drug concentration used no indication of binding of cycloheximide either to 40S subunits or to post-ribosomal supernatant was found. The number of binding sites was found to be equal to 0.4-0.5 both for parent and mutant 80S and 60S particles.
Discussion Earlier we have described a yeast mutant sensitive to a high temperature which did not express its sensitivity to restrictive temperature in the presence of cycloheximide (Ter-Avanesyan et al. 1982). In the present study we have found that this property is a common feature of high temperature sensitive supl and sup2 mutants. This observations allows us to suggest a possibility of selection of supl and sup2 mutants unable to grow in the absence of cycloheximide irrespectively of incubation temperature. This type of suppressor mutants was indeed selected on medium containing a low concentration of the drug. We have also shown that some of such conditionally and true dependent mutants express their dependence towards trichodermine, another protein synthesis inhibitor. The observation that supl and sup2 mutations may cause a simultaneous dependence for cycloheximide and trichodermine, both of which are known to act upon the 60S subunit of the yeast cytoplasmic ribosomes (Stock-
361 lein and Piepersberg 1980b; Fried and Warner 1981) supports our suggestion that supl and sup2 genes code for proteins of the large ribosomal subunit (Surguchov et al. 1981a, 1981b; Ter-Avanesyan et al. 1982). In the course of the characterization of the drug dependent mutants it has also been found that some of the mutants can grow at nonpermissive conditions on hypertonic media in the absence of antibiotics. It is usually accepted that osmotic remediality is an indication in favour of the conformational lability of mutant proteins (Fincham and Baron 1977). If this is indeed the case, the common effect of antibiotics and hypertonic conditions on growth pattern of the mutant strains allows us to suggest that the phenomenon of the drug dependence is explained by the conformational changes of the mutant ribosomes in the presence of antibiotic. Moreover, one can propose that this conformational shift of mutant ribosomes compensates for their functional defects induced by suppressor mutation. This suggestion was confirmed by the comparison of CD-spectra of mutant and parent ribosomes. The main differences between these spectra are found at 265 nm. The magnitude of the peak height in this range is known to be proportional to the base-stacking in rRNA (Allen and Wong 1979). Since the CD-absorbance at 265 nm of mutant ribosomes is higher than that of parent strain one can assume that ribosomes from Cyhcd mutant strain have a more ordered rRNA structure in comparison to that of parent ribosomes. After the addition ofcycloheximide to ribosomes preparations the CD-spectra for mutant and parent strain ribosomes become practically identical. Based on this fact one may suppose that the binding of cycloheximide to mutant ribosomes probably leads to the relaxation of the secondary structure of rRNA and the resulting ribosomes conformation becomes comparable to that of parent strain ribosomes. The data of equilibrium dialysis revealing the differences in K a values for 80S and 60S particles from parent and mutant strains demonstrate the alteration of ribosomal structure in the domain of 60S subunit responsible for cycloheximide binding. We conclude that the phenomenon of the drug dependence is related to the conformational state of the mutant ribosomes. The use of supl and sup2 drug dependent mutants allows us to show that the binding of cycloheximide and trichodermine to cytoplasmic eukaryotic ribosomes may induce a conformational shift, as it was earlier demonstrated for some antibiotics, acting on prokaryotic ribosomes (Noreau et al. 1980; Martinez et al. 1978).
Acknowledgement. The authors are grateful to Mrs. G. Zaretzki for assistance in some experiments.
362 References Allen SH, Wong KP(1979)Arch Biochem Biophys 195:112-120 Dabbs ER (1978) Mol Gen Genet 165:73-78 Dabbs ER (1979) J Bacteriol 140:734-734 Dabbs ER, Looman K (1981) Mol Gen Genet 184:224-229 Dabbs ER, Wittmann HG (1976) Mol Gen Genet 149:303-309 Fincham JRS, Baron YA (1977) J Mol Biol 11.0:627-634 Fried HM, Warner JR (1981) Proc Natl Acad Sci USA 78:238 242 Inge-Vechtomov SG (1971) Genetika 7:113-120 (in Russian) lnge-Vechtomov SG, Andrianova VM (1972) In: Dubinin NP, Goldfarb DM (eds) The molecular mechanisms of genetic processes. Nauka, Moscow, pp 189-195 (in Russian) Martinez O, Vazques D, Modolell J (1978) FEBS Letters 87: 21-24 Mironova LN, Provorov NA, Ter-Avanesyan MD, Inge-Vechtomov SG, Surguchov AP, Smirnov VN (1982) Curt Genet 5:149-152 Noreau J, Grise-Miron L, Brakier-Gingras L (1980) Biochem Biophys Acta 608:72-81 Stocklein W, Piepersberg W (1980a) Antimicrobiol Agents and Chemotherapy 18:863-867
M.D. Ter-Avanesyan et al.: Drug-Dependent Mutants in Yeast Stocklein W, Piepersberg W (1980b) Curt Genet 1:177-183 Surguchov AP, Berestetskaya YuV, Fominykch ES, Pospelova EM, Smimov VN, Ter-Avanesyan MD, Inge-Vechtomov SG (1980) FEBS Letters 111:175-178 Surguchov AP, Berestetskaya YuV, Smirnov VN, Ter-Avanesyan MD, Inge-Vechtomov SG (1981 a) FEMS Microbiology Letters 12:381-384 Surguchov AP, Fominykch ES, Smirnov VN, Ter-Avanesyan MD, Mironova LN, lnge-Veehtomov SG (1981b) Biochim Biophys Acta 654:149-155 Ter-Avanesyan MD, Schubochkina EA, Inge-Vechtomov SG (1982a) Genetika 18:215 222 (in Russian) Ter-Avanesyan MD, Schubochkina EA, lnge-Veehtomov SG (1982b) Genetika 18:223-226 (in Russian) van der Zeist BAM, Kees JM, Engel JM, Bloemers HPI (1973) Biochim Biophys Acta 294:517-526
Communicated by F. Kaudewitz Received April 6, 1983
