Current Genetics 1, 177-183 (1980)

CurrentGens ©

by Springer-Verlag 1980

Altered Ribosomal Protein L29 in a Cycloheximide-Resistant Strain of

Saccharomyces cerevisiae* Walter StScklein and Wolfgang Piepersberg

Lehrstuhl ftir Mikrobiologieder Universit~t,Universit~tsstrasse31, D-8400 Regensburg,Federal Republic of Germany

Summary. A spontaneous high-level cycloheximideresistant mutant of the yeast Saccharomyces cerevisiae (strain cy32) is found to have an altered protein of the large subunit (60S) of cytoplasmic ribosomes, namely protein L29. The resistance character segregates together with this biochemical defect and is semidominant in heterozygous diploids. Judged from in vitro susceptibility to inhibition by cycloheximide there are at least 50% resistant ribosomes present in such diploid strains. From these results it is concluded that cycloheximide resistance of mutant cy32 is caused by mutation of a single gene and that it is the structural gene for L29 which is affected. Preliminary genetic mapping data are also reported. They indicate a location of cyhx-32 marker on chromosome 7 near met13. Key words: Yeast Cyclohexirnide.

Ribosomal protein alteration -

Introduction

Cycloheximide is an itthibitor of eucaryotic cytoplasmic protein synthesis and seems to act on nearly all ribosome promoted stages of this process (for ref. see Pestka, 1977; Vazquez, 1978). Recent in vivo studies revealed initiation of translation to be the most sensitive step, though also elongation and possibly termination are

Offprint requests to: W. Piepersberg * Some of the data delt with in this paper were reported on the IX. Steenbock Symposium on Ribosomes, July 5th to 8th, 1979, Madison(Wisc.),U.S.A.

affected at higher drag concentrations (Cooper and Bossinger, 1976; Oleinick, 1977). These differential concentration dependent effects may indicate multiple interactions of cycloheximide with 80S ribosomes or other components of the translational apparatus. Recently, a cellular fraction (called "factor P") of yeast Saccharomyces cerevisiae could be isolated from a high salt ribosome-free extract, which was necessary for sensitivity to cycloheximide but not for activity in a cell-free protein synthesizing system (Somasundaran and Skogerson, 1976). Mutations leading to cycloheximide resistance have been shown to be 80S ribosome associated in Saccharomyces cerevisiae (Cooper et al., 1967; Jim6nez et al., 1972), Schizosaccharomyces pombe (Coddington and Fluff, 1977), Neurospora crassa (Pongartz and Klin~gniiller, 1973), Podospora anserina (Begueret et al., 1977), Physarurn polycephalum (Haugli et al., 1972), Tetrahymena pyriforrnis (Sutton et al., 1978) and a Chinese hamster ovary cell line (P6che et al., 1975). In addition naturally occuring resistance to cycloheximide in Saccharomyces fragilis turned out to be a property of the 60S ribosomal subunit (Rao and Grollman, 1967). In mutants of Saccharomyces cerevisiae, Podospora anserina, Schizosaccharomyces pombe and Tetrahymena pyriformis resistance could be attributed to the large subunit of the organelle, but it has been reported that also 40S alterations could bring about this effect (Jim6nez et al., 1972; Coddington and Fluff, 1977; B~gueret et al., 1977; Sutton et al., 1978). Altered ribosomal proteins in such mutants were demonstrated until today only for Schizosaccharomyces pombe and Podospora anserina (Coddington and Fluff, 1977; B~gueret et al., 1977). Many different genes can contribute to cycloheximide resistance in Saccharomyces cerevisiae, five of which have been mapped at different locations on the yeast 0172-8083/80/0001/0177/$ 01.40

178

W. St6cklein and W. Piepersberg: Cycloheximide-Resistant Yeast

Table 1. Strains of Saccharomyces cerevisiae used Strain

Genotype

A364A Y166 XS144-$19 XS144-S22

a ~ a ~

X3144-11A

~

cy32 3d

a ~

XSY-6

~

a

adel+2 urai his4 trp5 met13 leul met13 leul gall gal2 leu2 trpl pet19 adel+2 ural ade(1,2?) ural cyhx-32 met13 leul

Origin

gall MALl trp5 trp5

tyrl

his7

lys2

cyh2 cyh2

aro2 aro2

lys5 lys5

ade5 ade5

Hartwell A. Jim6nez YGSC a YGSC a

arg9

his6

ilv3

metl4

pet8

YGSC a

gall tyrl his(4,7?)

his7 tyrl

lys2 trp5

cyhx-32 gall

this paper this paper

trp5

lys5

ade5

aro2

this paper

YGSC = yeast genetic stock center, Berkeley (California, USA)

c h r o m o s o m e s ( f o r ref. see H a w t h o r n e a n d M o r t i m e r , 1976). S o m e o f these m a r k e r s s h o w t h e p h e n o m e n o n o f s e m i d o m i n a n c e , b o t h in vivo a n d v i t r o ( C o o p e r et al., 1 9 6 7 ; J i m 6 n e z et. al., 1 9 7 2 ; M c L a u g h l i n , 1974). I n this p a p e r we s h o w t h a t high-level c y c l o h e x i m i d e resistance in a r e c e n t l y isolated m u t a n t o f Saccharo-

myces

cerevisiae is associated w i t h an a l t e r e d 60S s u b u n i t p r o t e i n L 2 9 a n d t h a t t h e m u t a t i o n seems t o be

in t h e L 2 9 s t r u c t u r a l gene.

Materials a n d M e t h o d s

Organisms, Media and Growth Conditions. All strains of Saccharomyces cerevisiae used in this study are listed in Table 1. The complete and minimal media employed were YPAD and SD media, respectively, as described by Sherman et al. (1977). Cells were grown at 30 °C.

Genetic Procedures. All genetic manipulations were carried out as given by Sherman et al. (1979).

(15 min at 15.000 g). Ribosomes were pelleted by centrifugation at 48.000 rpm for 2 h (50 Ti, Beckman), resuspended in buffer 2 (20 mM Tris/HC1, pH 7.4, 100 mM Mg-acetate, 500 mM NH4C1, 5 mM mercaptoethanol) and centrifuged for 15 h at 48.000 rpm (60 Ti) through a layer of 8 ml 20% sucrose and 12 ml 40% sucrose in buffer 2. They were resuspended in a small volume of buffer la (like buffer 1 with Tris at 20 mM), freed from aggregates (15.000 rpm for 10 min) and stored frozen at - 7 0 °C. The S100 fraction used was a 30-70% (NH4)2SO 4 cut of the supernatant of the first ultracentrifugation step dissolved in a small volume of and dialyzed against buffer 3 (20 mM Tris/HCl, pH 7.4, 0.5 mM dithiothreitol). 0.1 ml portions were kept frozen at 70 °C. Ribosomal subunits were isolated from 30.000 g supernatants prepared in buffer 1 (see above) in the following manner: For dissociation of 80S ribosomes the extract was diluted with buffer 1 to an A260nm of 150 per ml, brought to 0.5 M with solid KCI and incubated for 10 min at 30 °C. Subunit separation was performed at 10 °C by centrifugation in 10 to 30% sucrose gradients in the same buffer for 1 h at 41.000 rpm (VTi 50). Subunits were sedimented by centrifugation for 14 h at 50.000 rpm (60 Ti), resuspended in the same buffer, and purified by a second density gradient centrifugation. Particles were resuspended in buffer 1, clarified by a 10 min centrifugation at 15.000 g and stored at - 7 0 °C until used.

Determination of Minimal Inhibitory Concentration (MIC). MIC values were obtained by a plate dilution method using YPAD medium with the following concentrations of cycloheximide: 0.1, 0.2, 0.5, 1, 2, 5, 7.5, 10, 20, 50, 75, 100, 150, 200 ~g/ml. Streaks were made from a suspension of cells with an A436ran of about 1.

Preparation of Ribosomes, Ribosomal Subunits and Supernatant Fraction. With some modification the methods de_scribed by Grant et al. (1974) were used. Ceils were grown in 500 ml volumes of YPAD at 30 °C to an A56onm of about 2. Before harvesting, cultures were treated with 1 mM NaN 3 for 15 rain to get run-off of polysomes. Cultures were then chilled by the addition of crashed ice; the cells were collected by centrifugation, washed twice with H20 and stored frozen at - 2 0 °C or used immediately. Extracts were made in 2 volumes of buffer 1 (100 mM Tris/HC1, pH 7.4, 80 mM KCI, 12.5 mM Mg-acetate, 1 mM dithiothreitol) per g wet weight of cells. Cells were broken either by grinding with acid-washed sea sand in a mortar or by passing through a French pressure cell press (Aminco). Cell-free supernatants were obtained after two consecutive centrifugations

PolyU-directed Polyphenylalanine Synthesis. A test system similar to that of Grant et al. (1974) was employed. Reaction mixtures contained in a final volume of 50 ~1 50 mM Tris/HC1, pH 7.4, 80 mM KC1, 12.5 mM Mg-acetate, 1 mM dithiothreitol0 1 mM ATP, 0.05 mM GTP, 4 mM creatine phosphate, 2 #g of creatine phosphate kinase, 25 ~zg of crude yeast tRNA, 15 #g of polyU, 10 ~1 of S100 fraction, 0.8 A26o rma units of ribosomes or 0.5 A260 nm units each of ribosomal subunits and 0.012 mM 14C-phenylalanine (50 ~Ci/~zmole). Tests were incubated for 20 rain at 30 °C; after addition of 1 ml of 10% trichloroacetic acid containing 1% of casamino acids and head treatment (15 rain at 90 °C) the precipitate was collected on glassfibre filters, dried and counted in a toluene based szintillation fluid. 100 cpm correspond to 1 pmole of phenylalanine incorporated.

Two-dimensional Polyacrylamide Gel Electrophoresis of Ribosomal Proteins. Ribosomal proteins were extracted from whole ribosomes or ribosomal subunits by acetic acid (Hardy et al., 1969). Three different systems of two-dimensional gel electrophoresis were used: (I) The method described my Mets and

W. St6cklein and W. Piepersberg: Cycloheximide-ResistantYeast

179

Fig. 1. Two-dimensional gel electropherograms of 80S ribosomal proteins according to method I (see Materials and Methods) from (A) wild-type strain A364A, (B) mutant cy32. The arrow indicates the altered ribosomal protein

Bogorad (1974) with the modification that the second dimension SDS gel was 15% in polyacrylamide and was prepared and run in the buffer system of Laemmli (1970). First dimension gels (0.5 x 9 cm) were run at 4 mA per gel for 3.5 h, equilibrated for 45 min by shaking in 0.5 M Tris/HCl, pH 6.8, 1% SDS, fixed to the running gel by a stacking gel of 4.5% polyacrylamide and run at 40 mA per slab for about 4 h. (II) The second procedure was that given by Kaltschmidt and Wittmann (1970). (III) As a third system a combination of the first dimension of method I with the second dimension of method II was also used. All second dimension gels were 0.3 x 9 x 9 em and five gel slabs each were made up and run in a special apparatus similar to that originally designed by Kaltschmidt and Wittmann (1970).

One-dimensionalPolyaerylamide Gel Eleetrophoresis. Ribosomes were precipitated from buffer 1 by adding an at least 10-fold excess of acetone, collected by centrifugation (5 min at 10,000 rpm) dried in vacuum, and dissolved in lysis buffer (60 mM Tris/ HC1, pH 7.5, 10% glycerol, 5% mercaptoethanol, 2% SDS). Samples were applied to 10 to 25% gradient slab gels with the dimensions 0.15 x 13 x 10 cm. The buffer system was essentially that of Laemmli (1970). Gels were run at 30 mA until the bromphenol blue marker left the slab.

Chemicals. Cycloheximide was from Serva, Heidelberg, FRG, [14C]labelled phenylalanine from Amersham Buchler, Braunschweig, FRG, ATP, GTP, creatine phosphate, creatine phosphate kinase and yeast tRNA were from Boehringer-Mannheim, FRG. Sucrose and (NH4)2SO4 were ultrapure grade (Schwarz and Mann, Orangeburg, USA); all other chemicals were analytical grade.

Results

Isolation of Mutants and Screening for Altered Ribosomal Proteins. A set o f spontaneous cycloheximideresistant strains of Saecharomyees eerevisiae were independently isolated from stein A364A by selection on complete medium plates containing 1 /ag/ml of the drug. Out o f 40 clones originally obtained 15 showed a comparatively high-level resistance with MIC values higher than 10/~g cycloheximide per ml and were chosen for further studies.

Ribosomal proteins were extracted from 80S ribosomes o f these strains and separated by two-dimensional polyacrylamide gel electrophoresis according to m e t h o d II (see Materials and Methods). No differences could be detected in the patterns obtained. Since there are many overlapping or not clearly separated spots we introduced another system with better resolution (method I) using SDS in the second dimension. Employing this procedure strain cy32 could be shown to have a change in migration of a certain ribosomal protein (Fig. 1). Its position on the electropherogram indicates that it has a more basic isoelectric point and possibly a higher molecular weight than the wild-type form.

Subunit Localization and Identity of the Altered Ribosomal Protein in cy32. To investigate whether the altered protein was a constituent of the large (60S) or the small (40S) ribosomal subunit, proteins from both separated subparticles were run on two-dimensional gels. Figure 2 (A, B and C) shows the result. The defect is d e a f l y in one of the 60S ribosomal proteins, whereas the 40S pattern is unaffected. F o r identification o f the altered component in the widely used Kaltschmidt and Wittmann (1970) procedure 60S ribosomal proteins were also separated by use of methods II and III (see Materials and Methods). Here it could be shown that it is protein L29 (nomenclature of Kmiswijk and Planta, 1974) (Fig. 2D and E), which is affected. In gels prepared according to methods II and III the respective spot also shows some differences in migration in comparison to the wild-type protein, but since there is considerable overlapping with a 40S ribosomal protein in m e t h o d II it was not detected during the first screen (see above). A very similar electrophoretic procedure to our m e t h o d I was used by Otaka and Kobata (1978). Within their numbering system the protein altered in strain cy32 should be identical to protein 41, which is a large

180

W. St6cklein and W. Piepersberg: Cycloheximide-Resistant Yeast

Fig. 2. Two-dimensional gel electropherograms of 60S (A, B, D and E) and 40S ribosomal proteins (C). Separation was according to method I (A-C), method III (D) or method II (E). Ribosomal protein was from A364A (A, C) or from cy32 (B, D, E). The arrows indicate the altered ribosomal protein L29.

.g lO0-

"~

tx tx

50-

10-

-'

011

i

lb

10'0

s60

cyc[oheximide [~Jg/mt]

Fig. 3. Effect of different cycloheximide concentrations on polyU-directed polyphenylalanine synthesis on ribosomes from A364A (e) and cy32 (o) or on mixed subunits: 40S from A364A with 60S from cy32 (A) and 408 from cy32 with 60S from A364A(A)

subunit protein; its molecular weight has been determined to be 16,500 daltons.

Cellular Localization of Cycloheximide Resistance in ey32. To confirm that it is the 80S cytoplasmic ribosome which bears cycloheximide resistance in cy32 the sensitivity of polyU-directed polyphenylalanine synthesis

for this antibiotic was tested for both wild type (A364A) and cy32 ribosomes (Fig. 3). 50% inhibition occurs at about 0.3 /~g/ml with ribosomes from strain A364A and at 30 /~g/ml with those from cy32. Since supernatant factors were the same (from strain Y166) in each case this result proves that resistance resides in cy32 80S ribosomes. In a subunit exchange experiment employing subparticles from A364A and cy32 it could be shown that the 60S subunit provides resistance (Fig. 3): Cycloheximide resistant polyphenylalanine synthesis is only observed when 60S subunits from mutant cy32 are present.

cyhx-32 and the Biochemical Defect Segregate Together. To confirm that the observed alteration of protein L29 is causally connected with cycloheximide-resistance, tetrad analysis from a cross between cy32 and Y166 was performed: a two to two segregation for high-level cyclohexirnide resistance was revealed. This proves that there is a single nuclear gene involved; the respective marker was named cyhx-32 because of its still unclear relation to other cycloheximide resistance mutations described (Hawthorne and Mortimer, 1976). In order to identify the ribosomal protein change in the progeny of the spores ribosomal protein was extracted by acetone and

w. St6cklein and W. Piepersberg: Cycloheximide-ResistantYeast

181

separated on one-dimensional SDS slab gels. Since there is a slight difference in migration of the mutant and wild-type L29 forms on those gels (see above) the defect could be clearly demonstrated by use of this system (Fig. 4). A total of 15 tetrads were investigated in this way and in each case only in the cycloheximide-resistant derivatives an altered position for protein L29 could be discovered. A direct correlation of the observed biochemical defect with the resistance character of strain cy32 is indicated by these experiments.

Semidominance of cyhx-32 Marker. Most of the cycloheximide resistance markers investigated so far in Saccharomyces cerevisiae are recessive, but not completely: Heterozygous diploids show intermediate resistance phenotypes (Wilkie and Lee, 1967; McLanghlin, 1974). This phenomenon, called semidominance, can also be seen in diploids resulting from crosses between strains bearing the cyhx-32 marker and those which are wildtype. The MIC values for cycloheximide of several haploid and diploid strains are given in Table 2. Heterodiploids are about tenfold more resistant than wild-type whereas diploid clones homozygous for eyhx32 show about hundred fold higher resistance. This behaviour can only be explained on the basis of the existence of mixed populations of resistant and sensitive ribosomes within the same strain. For further analysis ribosomes were isolated from three diploid strains and tested for sensitivity to the antibiotic in polyU-directed polyphenylalanine synthesis. From the data presented in Table 3 a content of about 50% or even more of resistant ribosomes present in the heterozygous diploids can be calculated, which is in agreement with earlier findings (Cooper et al., 1967; McLaughlin, 1974). Even more convincing would be the direct estimation of the amount of wild-type and mutant proteins present in these strains. But unfortunately due to the very small proteinchemical difference no conditions were found, under which clear separation of both proteins could be achieved by electrophoretic techniques. Nevertheless a broad double band in SDS slab gels and an extended spot in method I two-dimensional gels (not shown) indicated that both proteins are present. In any case the results allow the following conclusions: First, semidominance shows that it is the structural gene for protein L29 which is mutated in strain cy32 and secondly, as one should expect, ribosomal genes in yeast show a gene dosage effect and both genomes are obviously expressed equally well in diploid strains. Gene Location of the cyhx-32 Marker. It was tested whether cyhx-32 exhibits any linkage to rnet13, a marker present on chromosome 7 and linked to cyh2 (Hawthorne and Mortimer, 1976). For this purpose, a strain (XSY-6) was constructed by mating strain

Fig. 4. Ten to 25% exponential gradient slab gels run in the presence of SDS. Ribosomal protein was extracted by acetone as described under Materials and Methods. The arrowsindicate the positions of L29

Table 2. Minimal inhibitory concentrations for cycloheximide in various haploid and diploid yeast strains Strains

A haploids A364A Y166 cy32 3d XS144-S 19 XS144-$22

cyh alleles

MIC (ug/ml)

wt. a wt.

0.25 0.25-0.5 100- 200 100-150 2.5

eyhx.32 eyhx-32 cyh2 cyh2

5

B homozygous diploids A364A x Y166 wt./wt. 0.25-0.5 cy32 x 3d eyhx-32/cyhx-32 100-150 XS144-$19 x XS144-$22 eyh2/cyh2 5 C heterozygous diploids A364A x 3d cy32 x Yl66 A364A x XSl44-$22 XS144-$19 x Y166 cy32 x XS144-$22 XS144-$19 x 3d a wild-type

wt./cyhx.32 eyhx-32/wt. wt./cyh2 cyh2/wt. cyhx-32/cyh2 cyh2/cyhx-32

1-2.5 2.5-5 0.5-1

0.25 7.5-10 25-50

182

W. StScklein and W. Piepersberg: Cycloheximide-Resistant Yeast

Table 3. Effect of cycloheximide on in vitro polypeptide synthesis With ribosomes from different diploid strains

(i) Heterozygous cells possess a mixture of cycloheximide resistant and sensitive ribosomes, which says that the altered L29 is assembled into 60S particles as well as the wild-type L29 form. From in vitro experiments about equal proportions of each class seem to be present. This indicates that there is a gene dosage effect for ribosomal protein genes in diploid yeast cells and that both alleles are expressed equally well. In vitro in the polyU system, the resistant ribosomes apparently function as well as sensitive ones. (ii) The rather low in vivo resistance of heterodiploid strains reveals a strong interdependence of sensitive and resistant ribosomes in the cell. Apparently, interaction of cycloheximide with sensitive ribosomes blocks resistant organdies in their movement and thereby "freezes" polysomes. At low drug concentrations, however, resistant ribosomes are able to reinitiate since under this condition the drug preferentially acts at the initiation process (Cooper and Bossinger, 1976; Oleinick, 1977) and not on elongation. Therefore, in heterozygous cells the minimal inhibitory concentration directly reflects the antibiotic's action on elongation. (iii) Semidominance strongly indicates that the cyhx-32 mutation is in the structural gene for protein L29. All other possible mechanisms, such as deficiency in posttranslational maturation or modification should be enzyme promoted steps and deliver recessive phenotypes. The genetic studies reported indicate a position of cyhx-32 close to met13 on chromosome 7, a location similar to that reported for cyh2. Detailed genetic and biochemical analysis will reveal whether both genes are allelic or not.

Cycloheximide (~g/ml)

Activity in polyU system (% of controla)Percent age of reA B C sistant riA364A x cy32 × 3d cy32 x bosomes Y166 Y166 in Cb

0.1

70.4

1

32.3

10 100

20.1 14.5

92.0 85.0 58.7 33.4

82.8 62.4 47.3 24.9

57.5 57.0 70.5 55.0

a 100% values (without cycloheximide) were in the range of 245-260 pmoles phenylalanine incorporated per 50 pl test volume (24.500-26.000 cpna); each value is the average of two determinations b calculated assuming that both resistant and sensitive ribosomes are equally and independently active in the polyU system; values from lanes A and B are taken as reference

XS144-S19

with Y166 (see Table 1) which lacks

cyh2 but has retained all other markers of this chromosome. Spores from a cross between cy32 and XSY-6 showed normal viability and complete tetrads could be obtained. A linkage between cyhx-32 and met13 was found with both markers, 13 centi-Morgans (cM) apart from each other. For comparison, distances of 23 and 9 cM were measured for cyhx-32/aro2 and met13/aro2, respectively. These preliminary data may indicate that cyhx-32 is an allele of the cyh2 gene, but further and more detailed studies are necessary to confirm this result.

Discussion

cy32 is the first mutant ofSaccharomyces cerevisiae for which a defined phenotype could be attributed to an altered component of cytoplasmic ribosomes, namely protein L29. Since ribosomal protein alterations were recently also detected in mutants of yeast resistant to narciclasine or cycloheximide (Gonzales et al., personal communication; Adoutte and Davies, personal communication) there is hope that with the development of more sensitive electrophoretic screening methods and with the use of mutagens causing more drastic structural alterations (Piccard-Bonnoun, personal communication) the previous difficulty in the identification of altered gene products (Waldron and Cox, 1978) in ribosomal mutants will be overcome. As demonstrated for other, biochemically not identified, cycloheximide resistance mutations (Cooper et al., 1967; Jirn6nez et al., 1972; McLaughlin, 1974) cyhx-32 is semidominant. The following conclusions can be drawn from this phenomenon:

Acknowledgments. We thank A. BSck for reading the manuscript and for many helpful discussions. We are grateful to A. Jhn6nez for advice in in vitro procedures with yeast and to R. Contopoulou from the yeast genetic stock center, Berkeley, for delivering strains. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to W. P.

References

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W. Strcklein and W. Piepersberg: Cycloheximide-Resistant Yeast Hawthorne, D. C., Mortimer, R. K.: In: Handbook of biochemistry and molecular biology (G. D. Fasman, ed.), 3rd edition, nucleic acids - vol. 2, pp. 765-832. Cleveland: CRC Press 1976 Jimrnez, A., Littlewood, B., Davies,J.: In: Molecularmechanisms of antibiotic action on protein synthesis and membranes (E. Munoz, F. Garaia-Ferrandiz, D. Vazquez, eds.), pp. 292-306. New York: Elsevier 1972 Kaltschmidt, E., Wittmann, H. G.: Anal. Biochem. 36,401-412 (1970) Kruiswijk, T., Planta, R. J.: Mol. Biol. Rep. 1,409-415 (1974) Laemmli, U. K.: Nature 227,680-685 (1970) McLaughlin, C. S.: In: Ribosomes (M. Nomura, A. Tissi~res, P. Lengyel, eds.). Cold Spring Harbor Monograph Series, pp. 815-827. New York: Cold Spring Harbor Laboratory 1974

Mets, L. J., Bogorad, L.: Anal. Biochem. 57, 200-210 (1974) Oleinick, N. L.: Arch. Biochem. Biophys. 182, 171-180 (1977) Otaka, E., Kobata, K.: Mol. Gen. Genet. 162, 259-268 (1978) Pestka, S.: In: Molecular mechanisms of protein biosynthesis (H. Weissbach, S. Pestka, eds.), pp. 467-553. New York, San Francisco, London: Academic Press 1977 Prche, H., Junghahn, J., Geissler,E., Bielka, H,: Mol. Gen. Genet. 138, 173-177 (1975)

183 Pongratz, M., Klingmiiller;W.: Mol. Gen. Genet. 124, 359-363 (1973) Rao, S. S., Grollmann, A. P.: Biochem. Biophys. Res. Commun. 29, 696-704 (1967) Sherman, F., Fink, G. R., Lawrence, C. W.: Methods in yeast genetics (Laboratory manual for a course). New York: Cold Spring Harbor Laboratory 1977 (revised version) Somasundaran, U., Skogerson, L.: Biochemistry 15, 4760-4764 (1976) Sutton, C. A., Ares, M., Hallberg, R. L.: Proc. Nat. Acad. Sci. USA 75, 3158-3162 (1978) Vazquez, D.: In: Molecular biology, biochemistry and biophysics (A. Kleinzeller, G. F. Springer, H. G. Wittmann, eds.), Vol. 30. Berlin, Heidelberg, New York: Springer 1979 Waldron, C., Box, B. S.: Mol. Gen. Genet. 159, 223-225 (1978) Wilkie, D., Lee, B. K.: Genet. Res. Cam. 6, 130-138 (1965)

Communicated by R. J. Schweyen Received August 30/November 19, 1979