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Mitochondrial antibiotic resistance in yeast: Ribosomal mutants resistant to chloramphenicol, erythromycin and spiramycin
Biochimica et Biophysica Acta, 312 (1973) 358-367 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97711
M I T O C H O N D R I A L A N T I B I O T I C R E S I S T A N C E I N YEAST: RIBOSOMAl_, M U T A N T S R E S I S T A N T TO C H L O R A M P H E N I C O L , E R Y T H R O M Y C I N A N D SPIRAMYCIN
L. A. GRIVELL*, P. NETTER**, P. BORST* and P. P. SLONIMSKI**
Section for Medical Enzymolooy*, Laboratory of Biochemistry, University of Amsterdant, Eerste Constantijn Huygensstraat 20, Amsterdam (The Netherlands) and Centre de G~ndtique Mol~culaire du C.N.R.S. ~*, 91-Gif-sur-Yvette (France)
(Received January 25th, 1973)
SUMMARY 1. Mutants of Saccharomyces cerevisiae, resistant to chloramphenicol, erythromycin and/or spiramycin as a result of a mutation in m t D N A have been characterized biochemically. 2. Three erythromycin-resistant mutants, mapping at two distinct loci on a linkage map of mitochondrial genes, have been examined. Mutants 354 and 514, highly resistant in vivo, possess altered mitochondrial ribosomes. Besides being genetically distinct, these two mutants can be distinguished on the basis of their response to lincomycin and in their quantitative response to erythromycin, carbomycin and spiramycin. Weakly resistant mutant 553 is also a ribosomal one. 3. Chloramphenicol-resistant mutants 321 and 323 also possess altered mitochondrial ribosomes. 4. Acrylamide gel electrophoresis of proteins from the 50-S mitochondrial ribosomal subunit of the wild type and several of the mutants has failed to reveal differences in composition. The possibility that resistance is determined by ribosomal R N A is discussed.
INTRODUCTION A number of mutants of Saccharomyces cerevisiae, resistant to a series of antibacterial antibiotics as a result of a mutation in m t D N A have recently been isolated and characterized genetically 1-3 and physiologically4. We describe in this paper the biochemical characterization of these mutants and first attempts to identify the component responsible for resistance. A preliminary account of this work has appeared in a recent abstract 5. METHODS AND MATER[ALS lsochromosomal diploid strains were used for all experiments. Their construction, genetic and physiological characterization are described elsewhere 3'4. These
YEAST MITOCHONDRIAL RIBOSOMAL MUTANTS
359
strains are: PS30 carrying the mitochondrial mutation CAR21", PS40, C R323," PS136, ER553," IL249, E R354,"IL246, E R353,"IL257, SR352," ILl7, E~I 4. 1L46 is the standard reference strain sensitive to the antibiotics. They were grown on the appropriate antibiotic-containing glycerol medium 3, before being used to produce pre-inocula for large-scale preparations. Methods for further growth of cells, isolation of mitochondria and purification of mitochondrial ribosomes have been described previously 6, as have also procedures for assay of mitochondrial protein synthesis in vitro and ribosomal peptidyl transferase activity 7.
Ribosomal subunit preparation 50-S ribosomal subunits were obtained by centrifugation of mitochondrial 74-S monosomes through 15-30 % (w/w) isokinetic sucrose gradients containing 500 mM NH4CI, 10 mM Tris-HCl (pH 7.5), 10 mM magnesium acetate, 6 mM 2-mercaptoethanol (Spinco SW-41 rotor, 5 h at 41 000 rev./min and at 4 °C) and pooling of appropriate fractions. Subunits were conveniently recovered by polyethyleneglycol precipitation. This was carried out by addition of solid carbowax 6000 to a final concentration of 10 % (w/v) and allowing the suspension to stand at 0 °C overnight. The subunits were then recovered by centrifugation at 15 000 × 9 and 2 °C for 10 rain. Acrylamide gel electrophoresis Subunits were taken up in a buffer containing 0.03 M methylamine-acetate, 6 mM urea, 3 mM 2-mercaptoethanol and 0.25 mM EDTA (pH 5.6) at a concentration corresponding to 20 A260 nm units per ml. T 1 and pancreatic ribonucleases (previously heated at 70 °C for 10 rain) were then added to final concentrations of 50 units and 10 pg/ml, respectively, and RNA was digested by incubation of the mixture at 37 °C for 45 rain. 0.1-ml samples, containing protein derived from about 2 A26 on,, units of ribosomes and 0.0005 % basic fuchsin as tracking dye were applied to 10 % acrylamide gels containing 8 M urea (pH 4.5) 8. Gels were contained in perspex tubes of internal diameter 0.6 cm and length 13 era. Electrophoresis was at 2.5 mA per tube and 4 °C for about 5 h. After removal from the tubes, gels were fixed for 30 min in about 20 vols 12.5 % (w/v) trichloroacetic acid and stained overnight in a 0.05 % solution of Coomassie Brilliant Blue in the same solvent. Destaining was by several rinses in 12.5 % trichloroacetic acid. Gels containing radioactively labelled proteins were stained, frozen in solid CO2 and cut in 1-mm slices by means of a mechanical gel slicer (Mickle Engineering Co., Surrey). Slices were treated with 0.5 ml of a 9 : 1 (v/v) mixture of Nuclear Chicago Solubilizer and water at 50 °C for 16 h. Distribution of radioactivity was determined in a PPO-POPOP-toluene scintillator, with a Nuclear Chicago Unilux III liquid scintillation counter. Radioactive labellin 9 of mitoehondrial ribosomal proteins Cells were grown for approximately 8 generations in a minimal medium composed of 0.67 % (w/v) Difco yeast nitrogen base (without amino acids), 0.1% glucose and 2 % potassium lactate (pH 4.5). 4,5-[3H]Leucine (specific activity 36 Ci/mmole) or [U-14C]leucine (specific activity 331 Cifmole; Amersham) were present at con-
360
L.A. GRIVELL et al.
centrations o f 0.5 mCi and 0.05 mCi per 1, respectively. G r o w t h was stopped by the addition o f [12C]leucine (1 mg/ml) and rapid chilling to 0 °C. Equal quantities o f differentially labelled m u t a n t and wild-type strains were then mixed and harvested. All subsequent steps in the isolation o f mitochondrial ribosomal proteins were carried out on the mixture. Chloramphenicol was obtained f r o m Roussel or Brocades; erythromycin (free base) f r o m Roussel or Sigma; spiramycin (free base) f r o m Rh6ne-Poulenc, lincomycin f r o m U p j o h n and c a r b o m y c i n f r o m Pfizer. RESULTS Characterization o f antibiotic-resistant mutants in vivo
Table I summarizes relevant properties o f mutants selected for further biochemical study. Full physiological characterization will be described in a separate article 4. TABLE I PROPERTIES OF MITOCHONDR1AL ANTIBIOTIC-RESISTANT MUTANTS: EFFECTS OF CHLORAMPHENICOL, ERYTHROMYCIN AND SPIRAMYCIN ON THE RATE OF GROWTH DURING EXPONENTIAL PHASE Resistance is expressed in terms of the concentration of antibiotic (in mg/ml) required to inhibit by 25 and 50 %, respectively, the rate of growth during exponential phase of cells in liquid, glycerolcontaining media. All mutants grew with similar generation times in media lacking antibiotics 4. Data taken from ref. 4. Inhibition (%)
Wild type CRaZl
cR323 ERs14
ER354 ER553
Chloramphenicol
Erythromycin
Spiramycin
25
50
25
50
25
50
1.1 4.0 3.6 1.0 0.4 0.59
2.4 3> 4 :> 4 2.4 1.2 2.0
0.025 0.039 0.063 ~> 5 3> 5 0.58
0.046 0.078 0.14 ~ 5 3> 5 4.2
0.22 0.8 0.76 ~ 8 4.4 0.2
0.39 1.4 1.5 ~- 8 6.7 0.31
Mutants selected for resistance to erythromycin fall into two distinct classes. First, as exemplified by 514 and 354, a class highly resistant to both erythromycin and spiramycin. 514 is as sensitive as wild type to chloramphenicol while 354 is slightly more sensitive than wild type. Second, exemplified by 553, a class weakly erythromycin resistant and sensitive to chloramphenicol and spiramycin. Mutants 321 and 323, selected for resistance to chloramphenicol, are also slightly cross-resistant to erythromycin and spiramycin. The exact extent o f resistance o f these strains to chloramphenicol is largely masked in vivo, by insensitivity o f the wild type to this antibiotic. This is evidently a result o f impermeability o f the cell membrane, since protein synthesis by isolated mitochondria o f the wild type is very sensitive (see Table II).
YEAST MITOCHONDRIAL
RIBOSOMAL MUTANTS
361
Observed cross-resistances result from the mutation responsible for the primary resistance to the selecting antibiotic, since all characteristics are inseparable in genetic crosses 3. These mutants have been assigned by recombination to positions on a linkage map of mitochondrial D N A (Fig. 1). Chloramphenicol-resistant mutations map at a locus very close to the gene influencing the polar recombination of mitochondrial genes in o~+ ×co- crosses, while erythromycin-resistant mutations occur at two other distinct loci. Mutants of widely differing phenotypes map at each of these loci. Mutants mapping at the same locus are homo- or possibly hetero-alleles, while those mapping at distinct loci are clearly non-alleles.
(d cR21 ER54 cR23 EgRS3
5R52 ERE~
ER~4 Fig. 1. Linkage m a p o f mitochondrial antibiotic-resistance genes. ( F r o m rel: 3). T A B L E II I N H I B I T I O N BY A N T I B I O T I C S O F P R O T E I N S Y N T H E S I S BY M I T O C H O N D R I A LATED FROM WILD-TYPE AND ANTIBIOTIC-RESISTANT STRAINS
ISO-
M i t o c h o n d r i a were incubated at a concentration o f 1-2 m g p r o t e i n / m l u n d e r conditions described previously 5. 15-30 pmoles L-[14C]leucine (specific activity 62 Ci/mole) were incorporated per m g p r o t e i n in 30 m i n at 30 °C. A linear rate o f i n c o r p o r a t i o n is observed in control incubations u n d e r these conditions (Grivell, L. A., u n p u b l i s h e d observations). Figures are the m e a n o f two or in s o m e cases three determinations with separate m i t o c h o n d r i a l preparations.
% Activity remaining
C h l o r a m p h e n i c o l (/zg/ml) 10 25 50 100 E r y t h r o m y c i n Q~g/ml) 0.4 1
10 100 250 L i n c o m y c i n (/~g/ml) 2 20 200 400
ER354
ER553
22 13 10 9
18 11 5 3
16 4 5 2
94 89 102 103 98
93 89 87 89 65
75 -34 28
85 80 36 22
95 78 59 49
52 48 10 5
60 30 3 2
86 53 16
88 78 30
81 23 6
20 9 10
Wild type
cR321
cR323
22 11 6 5
69 54 38 35
94 90 80 74
19 16 I1 10
38 33 27 16
55 47 35 24
- -
- -
43 35 6 4
52 81 41 26
33 9 4
76 31 15
ERs 1,,
- -
Spiramycin (/tg/ml) 1
10 100
L . A . G R I V E L L et al.
362
Biochemical characterization Resistance of mitochondrial protein synthesis to antibiotics might result either from impermeability of the mitochondrial membrane or from a change in the mitochondrial ribosome itself. A form of detoxification mechanism is a third, but unlikely possibility. A comparison of the response to antibiotics of intact, isolated mitochondria with that of purified mitochondrial ribosomes should reveal which of the mutants possess altered mitochondrial ribosomes. Chloramphenicol, erythromycin, spiramycin and lincomycin were, therefore, tested for their effects on protein synthesis by isolated mitochondria (Table II) and on the peptidyl transferase activity of mitochondrial ribosomes (Table III). In the latter assay, response to carbomycin was also tested. Sensitivity to chloramphenicol,
T A B L E III EFFECTS OF ANTIBIOTICS ON THE SYNTHESIS OF ACETYL- [aH]LEUCYL-PU ROMYCIN BY M I T O C I - [ O N D R I A L R I B O S O M E S I S O L A T E D F R O M W I L D - T Y P E A N D A N T I B I O T I C RESISTANT STRAINS Ribosomal peptidyl transferase activity was assayed by a modified fragment reaction using acetyl[3H]leucyl-tRNA from E. coli (specific activity 10 Ci/mmole; 5000 cpm/assay). The reaction mixture (0.15 ml after addition o f ethanol) contained the equivalent of between 0.6 and 1.0 A26o nm units o f mitochondrial ribosomes. 100 % reaction corresponds to between 1500 and 4600 cpm. The results are the mean of up to four determinations with two separate ribosomal preparations. They have been corrected for blanks without ribosomes (approx. 250 cpm). The extent of the reaction in the absence o f puromycin was never greater than 2 % and usually zero.
% Activity remaininy
cR321
Wild type Chloramphenicol (pg/ml) 26 66 132 Erythromycin (ttg/ml) 6 13 20
cR323
ERs14
ERasa
--
22 12 10
65 48 34
107 102 106
--
I10 113
120 115
167 165
-
-
-
-
ERSS3
4
--
--
5 4 2
113 -115
113 -107
118 -113
7 7 10
11 14 21
57 67 85
6
Chloramphenicol (66/~g/ml) ÷ Erythromycin (/~g/ml) 6 13 20 Lincomycin (~g/ml) 5 50
25 5
88 35
94 33
84 60
3 1
5 2
Spiramycin (/~g/ml) 66 132 198
38 27 23
54 49 49
61 59 58
92 106 94
82 92 86
56 56 44
Carbomycin (ktg/ml) 66 132
16 13
13
84 78
68 55
25 23
88 85
90 92 -
157 174
-
-
-
23 -
-
-
-
YEAST M1TOCHONDRIAL RIBOSOMAL M U T A N T S
363
spiramycin, lincomycin and carbomycin was measured directly, while response to erythromycin was determined by the ability of this antibiotic to displace chloramphenicol from erythromycin-sensitive, but not -resistant ribosomes. Table IV summarizes data from the two assays. TABLE IV A COMPARISON OF THE RESPONSE OF INTACT MITOCHONDR1A A N D ISOLATED RIBOSOMES TO ANTIBIOTICS
Phenotype intact mitochondria/isolated ribosomes Chloramphenicol CSEs
Erythromycin
Lincomycin
Spiramycin S
S
S
S
g
g
~
CR321
2R 2R
R 9.
R ~
R
CR323
3R 3R
R ~
R R
R
ER51,,
S
3R 3R
2R 2R
2R 2R
ERas,,
S
3R 3R
S S
R 2R
ER553
S
R
S
S
For chloramphenicol-resistant mutants 321 and 323, activities of both intact mitochondria and purified ribosomes are highly resistant to chloramphenicol and moderately resistant to spiramycin and lincomycin. The two mutations were easily distinguishable by their quantitative response to the antibiotics. Since the test of ribosomal erythromycin sensitivity requires a chloramphenicol-sensitive ribosome, the slight erythromycin resistance of intact mitochondria has not yet been verified at the ribosomal level. Antibiotic resistance in these strains is, however, clearly a result of the possession of altered mitochondrial ribosomes. Similarly, erythromycin-resistant mutations 354, 514 and 553 also affect the mitochondrial ribosome. Mutants 354 and 514, besides being genetically distinct, differ in their response to lincomycin in that 514 is resistant and 354 highly sensitive to this antibiotic. Quantitative differences in the levels of resistance to erythromycin, spiramycin and carbomycin are also evident. Mutant 553, as might be predicted from the low levels of resistance exhibited in vivo, yields mitochondria and ribosomes with low resistance to erythromycin, although the anomalous dose response of this strain in vivo (see ref. 4) is not seen in vitro. The significance of the slight resistance of isolated ribosomes to spiramycin is not clear. By the criteria of Bunn et al. 9 this strain would qualify as a membrane mutant, since it yields "sensitive" mitochondria (i.e. > 60 o/inhibited at a concentration of < 0.1 mM antibiotic). /o
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L.A. GRIVELL et al
The component altered in antibiotic-resistant ribosomes Resistance to erythromycin in Escherichia coli or Bacillus subtilis is accompanied by a change in one protein of the large ribosomal subunit, detectable by its altered mobility during chromatography on carboxymethyl cellulose or gel electrophoresis in urea at pH 4.5 (refs. 10 and 11). Fig. 2 compares separations of proteins from the 50-S mitochondrial ribosomal subunits of wild-type and mutant strains 354 and 514, obtained by electrophoresis in urea-acrylamide gels at pH 4.5. Despite reproducible resolution of the proteins in up to 28 components, no difference in band pattern can be detected. Similar results were obtained for mutants 321 and 323 (profiles not shown). Separations run at pH 8.7 also failed to reveal any differences between these mutants and the wild type.
Fig. 2. 50-S ribosomal proteins from wild-type and erythromycin-resistant mutants. See Methods and Materials for details of sample preparation and electrophoresis.
In order to increase the sensitivity of the method, separations at pH 4.5 were repeated using differentially-labelled mixtures of wild-type and mutant 50-S ribosomal proteins. These were obtained by growth of the strains for several generations in the presence of [3H]- or [14C]leucine and mixing of mutant with wild-type cells directly before harvesting. The results of such an experiment with chloramphenicol-resistant mutant 323 are displayed in Fig. 3. For comparison, differentially-labelled mixtures of wild-type cells and of wild-type plus mutant cells with labels reversed were also analysed (results not shown). The 14C/3H ratio throughout the gel does not deviate significantly from the mean, implying that the 50-S ribosomal proteins of mutant 323 are not detectably different from those of the wild type. Similar results were obtained for erythromycin-resistant mutant 514 (experiment not shown).
YEAST M I T O C H O N D R I A L R I B O S O M A L M U T A N T S
365
b
46
64
~
2
8 .,-~4
C~23 (3HI + C~[ 14c1
,%.,i 0
20
4~
'a 40
60
80 ~ '120 Distence migreted (mm)
Fig. 3. Gel analysis of a differentially labelled mixture of 50-S ribosomal proteins from wild-type a n d chloramphenicol-resistant strain 323. See Methods and Materials. - - - - - - , 14C; - - - , 3H. DISCUSSION
We find that several genetically distinguishable mutants of m t D N A in yeast are resistant to chloramphenicol, erythromycin and/or spiramycin as the result of the possession of altered mitochondrial ribosomes. The mutants exhibit widely differing phenotypes and in several cases are cross-resistant to other antibiotics with related modes of action (e.g. lincomycin, carbomycin). It was initially surprising to find mutants with such widely different biochemical characteristics mapping at the same locus, e. g. 354 and 553. Such phenotypic variation, not separable by genetic recombination, may originate from various substitutions of a single nucleotide at the same position as observed for mutants of E. coli resistant to, or dependent on, streptomycin 12. It may also be that the genetic techniques used at present are incapable of the necessary resolution in the case of mutations resulting from substitutions at closely adjacent positions. In contrast to corresponding bacterial mutants 1°, none of the mitochondrial mutations seem to have deleterious effects on mitochondrial ribosomal function, detectable either by reduced growth rate in the absence of antibiotics 4, or function in vitro (Tables II and III). Furthermore, mutants 354 and 514, primarily resistant to erythromycin, are many times more resistant than corresponding bacterial ribosomal mutants 13. As such, they may be representatives of novel classes of ribosomal mutations. A cytoplasmic erythromycin-resistant mutant was previously shown by us to possess altered mitochondrial ribosomes 7. This mutant resembled 514 in its high resistance to erythromycin and cross-resistance to lincomycin. 514 is in fact the type of erythromycin-resistant mutant arising, most frequently, spontaneously 3. Use of the peptidyl transferase assay has permitted rapid routine screening of ribosomes from many mutants. The assay may be limited, however, in its ability to pick up all types of antibiotic-resistant mutants. Erythromycin-resistant mutant 353, and spiramycin-resistant mutant 352, both mapping at the same locus as 514, were also studied using this technique (results not shown). Although both mutants yielded
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L.A. GR1VELL et al.
antibiotic-resistant ribosomes, the levels of resistance observed were lower than predicted from in vivo data, and, in the case of 353, there were puzzling losses of some cross-resistances. These discrepancies were absent, however, when the ribosomes were tested for antibiotic response in protein-synthesizing activity directed by RNA from phage MS2 (Reijnders, L., unpublished observations). It is striking that, of all the mutants examined by us, none has been found to be resistant as a result of an altered mitochondrial membrane. The isolation of such a mutant was recently reported by Bunn et aL 9. This mutant, selected for mikamycin resistance, was found to be cross-resistant to a wide range of unrelated compounds, including chloramphenicol, lincomycin, carbomycin, tetracycline and oligomycin. The genetics of this mutant, originally thought to be cytoplasmic, have since been found to be complex TM. Moreover, only indirect arguments were used to classify this mutant as a membrane mutant. In our opinion the only unambiguous characterization of such a mutant will come from a study of the isolated mitochondrial ribosomes. Although in bacteria, resistance to erythromycin has been shown to be determined by a mutated protein of the 50-S subunit 10.11, we have not been able to detect any convincing difference in the composition of the proteins of the large ribosomal subunit from wild-type and several mutant strains, either visually or using doublelabel techniques in combination with acrylamide gel electrophoresis in urea at both pH 4.5 and pH 8.7. The negative evidence that we have presented on this point does not exclude the possibility of amino acid substitutions in a ribosomal protein without change in charge. However, we consider this unlikely since in bacteria such mutants are only weakly resistant 15. We have previously considered the possibility that antibiotic resistance in these mutants is determined by r R N A ~6. This alternative has the attraction that it localizes the change in a component already shown to be specified by mtDNA 17. rRNA mutations conferring antibiotic resistance in bacteria are so far unknown, but genetically, mitochondria differ from bacteria in two important respects. A bacterial genome usually possesses several cistrons specifying r R N A in a single molecule of DNA; mtDNA, in contrast, has only one cistron for each rRNA per molecule ~7. There are, of course, many m t D N A molecules in one cell, but it is conceivable that each molecule constitutes an independent unit for mutation and segregation. Since resistance to the antibiotics involved is recessive to sensitivity, bacterial mutants in rRNA will not readily be detected. However, a mitochondrion with a full complement of resistant ribosomes can arise as a result of a single mutation and, under selective conditions, replace the original antibiotic sensitive population. ACKNOWLEDGEMENTS L.A.G. is grateful to EMBO for the award of a short-term fellowship which made possible a visit to the Centre de G6n6tique Mol6culaire du C.N.R.S., Gif-surYvette, France. Part of the work was carried out during tenure by L.A.G. of a fellowship in the European Programme of the Royal Society of Great Britain. The work at Gif-sur-Yvette has been supported by a grant from DGRST, No. 6600168. This work was also supported in part by a grant to P.B. from the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
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REFERENCES 1 Coen, D., Deutsch, J., Netter, P., Petrochilo, E. and Slonimski, P. P. (1970) in Control of Organelle Development (Miller, P. L., ed.), pp. 449-496, Cambridge University Press, Cambridge 2 Bolotin, M., Coen, D., Deutsch, J., Dujon, B., Netter, P., Petrochilo E. and Slonimski, P. P. (1971) Bull. Inst. Pasteur 69, 215-239 3 Netter, P., Petrochilo, E., Slonimski, P. P., Bolotin, M., Coen, D., Deutsch, J. and Dujon, B. (1973) Genetics, in the press 4 Slonimski, P. P., Perrodin, G., Bolotin, M., Coen, D., Deutsch, J., Dujon, B., Netter, P. and Petrochilo, E. (1973) Biochimie, in the press 5 Grivell, L. A., Netter, P. and Slonimski, P. P. (1972) Abstract 8th FEBS Meeting, Amsterdam, No. 623 6 Grivell, L. A., Reijnders, L. and Borst, P. (1971) Biochim. Biophys. Acta 247, 91-103 7 Grivell, L. A., Reijnders, L. and De Vries, H. (1971) FEBS Lett. 16, 159-163 8 Traub, P., Mizushima, S., Lowry, C. V. and Nomura, M. (1971) in Methods in Enzymolo#y (Colowick, S. P. and Kaplan, N. O., eds), Vol. 20, pp. 391-407, Academic Press, New York 9 Bunn, C. L., Mitchell, C. I-[., Lukins, H. B. and Linnane, A. W. (1970) Proc. Natl. Acad. Sci. U.S. 67, 1233-1240 10 Otaka, E., Teraoka, H., Tamaki, M., Tanaka, K. and Osawa, S. (1970) J. Mol. Biol., 48,499-510 11 Smith, I., Goldthwaite, C. and Dubnau, D. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 85-89 12 Funatsu, G. and Wittmann, H. G. (1972) J. MoL Biol. 68, 547-550 13 Apirion, D. (1967) J. Mol. Biol. 30, 255-275 14 Linnane, A. W., Haslam, J. M., Lukins, H. B. and Nagley, P. (1972) Annu. Rev. Microbiol. 26, 163-198 15 Tanaka, K., Tamaki, M., Itoh, T., Otaka, E. and Osawa, S. (1971) Mol. Gen. Genet. 114, 23-30 16 Borst, P. (1972) Ann. Rev. Biochem. 41,333-376 17 Reijnders, L., Kleisen, C. M., Grivell, L. A. and Borst, P. (1972) Biochim. Biophys. Acta 272, 396-407
M I T O C H O N D R I A L A N T I B I O T I C R E S I S T A N C E I N YEAST: RIBOSOMAl_, M U T A N T S R E S I S T A N T TO C H L O R A M P H E N I C O L , E R Y T H R O M Y C I N A N D SPIRAMYCIN
L. A. GRIVELL*, P. NETTER**, P. BORST* and P. P. SLONIMSKI**
Section for Medical Enzymolooy*, Laboratory of Biochemistry, University of Amsterdant, Eerste Constantijn Huygensstraat 20, Amsterdam (The Netherlands) and Centre de G~ndtique Mol~culaire du C.N.R.S. ~*, 91-Gif-sur-Yvette (France)
(Received January 25th, 1973)
SUMMARY 1. Mutants of Saccharomyces cerevisiae, resistant to chloramphenicol, erythromycin and/or spiramycin as a result of a mutation in m t D N A have been characterized biochemically. 2. Three erythromycin-resistant mutants, mapping at two distinct loci on a linkage map of mitochondrial genes, have been examined. Mutants 354 and 514, highly resistant in vivo, possess altered mitochondrial ribosomes. Besides being genetically distinct, these two mutants can be distinguished on the basis of their response to lincomycin and in their quantitative response to erythromycin, carbomycin and spiramycin. Weakly resistant mutant 553 is also a ribosomal one. 3. Chloramphenicol-resistant mutants 321 and 323 also possess altered mitochondrial ribosomes. 4. Acrylamide gel electrophoresis of proteins from the 50-S mitochondrial ribosomal subunit of the wild type and several of the mutants has failed to reveal differences in composition. The possibility that resistance is determined by ribosomal R N A is discussed.
INTRODUCTION A number of mutants of Saccharomyces cerevisiae, resistant to a series of antibacterial antibiotics as a result of a mutation in m t D N A have recently been isolated and characterized genetically 1-3 and physiologically4. We describe in this paper the biochemical characterization of these mutants and first attempts to identify the component responsible for resistance. A preliminary account of this work has appeared in a recent abstract 5. METHODS AND MATER[ALS lsochromosomal diploid strains were used for all experiments. Their construction, genetic and physiological characterization are described elsewhere 3'4. These
YEAST MITOCHONDRIAL RIBOSOMAL MUTANTS
359
strains are: PS30 carrying the mitochondrial mutation CAR21", PS40, C R323," PS136, ER553," IL249, E R354,"IL246, E R353,"IL257, SR352," ILl7, E~I 4. 1L46 is the standard reference strain sensitive to the antibiotics. They were grown on the appropriate antibiotic-containing glycerol medium 3, before being used to produce pre-inocula for large-scale preparations. Methods for further growth of cells, isolation of mitochondria and purification of mitochondrial ribosomes have been described previously 6, as have also procedures for assay of mitochondrial protein synthesis in vitro and ribosomal peptidyl transferase activity 7.
Ribosomal subunit preparation 50-S ribosomal subunits were obtained by centrifugation of mitochondrial 74-S monosomes through 15-30 % (w/w) isokinetic sucrose gradients containing 500 mM NH4CI, 10 mM Tris-HCl (pH 7.5), 10 mM magnesium acetate, 6 mM 2-mercaptoethanol (Spinco SW-41 rotor, 5 h at 41 000 rev./min and at 4 °C) and pooling of appropriate fractions. Subunits were conveniently recovered by polyethyleneglycol precipitation. This was carried out by addition of solid carbowax 6000 to a final concentration of 10 % (w/v) and allowing the suspension to stand at 0 °C overnight. The subunits were then recovered by centrifugation at 15 000 × 9 and 2 °C for 10 rain. Acrylamide gel electrophoresis Subunits were taken up in a buffer containing 0.03 M methylamine-acetate, 6 mM urea, 3 mM 2-mercaptoethanol and 0.25 mM EDTA (pH 5.6) at a concentration corresponding to 20 A260 nm units per ml. T 1 and pancreatic ribonucleases (previously heated at 70 °C for 10 rain) were then added to final concentrations of 50 units and 10 pg/ml, respectively, and RNA was digested by incubation of the mixture at 37 °C for 45 rain. 0.1-ml samples, containing protein derived from about 2 A26 on,, units of ribosomes and 0.0005 % basic fuchsin as tracking dye were applied to 10 % acrylamide gels containing 8 M urea (pH 4.5) 8. Gels were contained in perspex tubes of internal diameter 0.6 cm and length 13 era. Electrophoresis was at 2.5 mA per tube and 4 °C for about 5 h. After removal from the tubes, gels were fixed for 30 min in about 20 vols 12.5 % (w/v) trichloroacetic acid and stained overnight in a 0.05 % solution of Coomassie Brilliant Blue in the same solvent. Destaining was by several rinses in 12.5 % trichloroacetic acid. Gels containing radioactively labelled proteins were stained, frozen in solid CO2 and cut in 1-mm slices by means of a mechanical gel slicer (Mickle Engineering Co., Surrey). Slices were treated with 0.5 ml of a 9 : 1 (v/v) mixture of Nuclear Chicago Solubilizer and water at 50 °C for 16 h. Distribution of radioactivity was determined in a PPO-POPOP-toluene scintillator, with a Nuclear Chicago Unilux III liquid scintillation counter. Radioactive labellin 9 of mitoehondrial ribosomal proteins Cells were grown for approximately 8 generations in a minimal medium composed of 0.67 % (w/v) Difco yeast nitrogen base (without amino acids), 0.1% glucose and 2 % potassium lactate (pH 4.5). 4,5-[3H]Leucine (specific activity 36 Ci/mmole) or [U-14C]leucine (specific activity 331 Cifmole; Amersham) were present at con-
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L.A. GRIVELL et al.
centrations o f 0.5 mCi and 0.05 mCi per 1, respectively. G r o w t h was stopped by the addition o f [12C]leucine (1 mg/ml) and rapid chilling to 0 °C. Equal quantities o f differentially labelled m u t a n t and wild-type strains were then mixed and harvested. All subsequent steps in the isolation o f mitochondrial ribosomal proteins were carried out on the mixture. Chloramphenicol was obtained f r o m Roussel or Brocades; erythromycin (free base) f r o m Roussel or Sigma; spiramycin (free base) f r o m Rh6ne-Poulenc, lincomycin f r o m U p j o h n and c a r b o m y c i n f r o m Pfizer. RESULTS Characterization o f antibiotic-resistant mutants in vivo
Table I summarizes relevant properties o f mutants selected for further biochemical study. Full physiological characterization will be described in a separate article 4. TABLE I PROPERTIES OF MITOCHONDR1AL ANTIBIOTIC-RESISTANT MUTANTS: EFFECTS OF CHLORAMPHENICOL, ERYTHROMYCIN AND SPIRAMYCIN ON THE RATE OF GROWTH DURING EXPONENTIAL PHASE Resistance is expressed in terms of the concentration of antibiotic (in mg/ml) required to inhibit by 25 and 50 %, respectively, the rate of growth during exponential phase of cells in liquid, glycerolcontaining media. All mutants grew with similar generation times in media lacking antibiotics 4. Data taken from ref. 4. Inhibition (%)
Wild type CRaZl
cR323 ERs14
ER354 ER553
Chloramphenicol
Erythromycin
Spiramycin
25
50
25
50
25
50
1.1 4.0 3.6 1.0 0.4 0.59
2.4 3> 4 :> 4 2.4 1.2 2.0
0.025 0.039 0.063 ~> 5 3> 5 0.58
0.046 0.078 0.14 ~ 5 3> 5 4.2
0.22 0.8 0.76 ~ 8 4.4 0.2
0.39 1.4 1.5 ~- 8 6.7 0.31
Mutants selected for resistance to erythromycin fall into two distinct classes. First, as exemplified by 514 and 354, a class highly resistant to both erythromycin and spiramycin. 514 is as sensitive as wild type to chloramphenicol while 354 is slightly more sensitive than wild type. Second, exemplified by 553, a class weakly erythromycin resistant and sensitive to chloramphenicol and spiramycin. Mutants 321 and 323, selected for resistance to chloramphenicol, are also slightly cross-resistant to erythromycin and spiramycin. The exact extent o f resistance o f these strains to chloramphenicol is largely masked in vivo, by insensitivity o f the wild type to this antibiotic. This is evidently a result o f impermeability o f the cell membrane, since protein synthesis by isolated mitochondria o f the wild type is very sensitive (see Table II).
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Observed cross-resistances result from the mutation responsible for the primary resistance to the selecting antibiotic, since all characteristics are inseparable in genetic crosses 3. These mutants have been assigned by recombination to positions on a linkage map of mitochondrial D N A (Fig. 1). Chloramphenicol-resistant mutations map at a locus very close to the gene influencing the polar recombination of mitochondrial genes in o~+ ×co- crosses, while erythromycin-resistant mutations occur at two other distinct loci. Mutants of widely differing phenotypes map at each of these loci. Mutants mapping at the same locus are homo- or possibly hetero-alleles, while those mapping at distinct loci are clearly non-alleles.
(d cR21 ER54 cR23 EgRS3
5R52 ERE~
ER~4 Fig. 1. Linkage m a p o f mitochondrial antibiotic-resistance genes. ( F r o m rel: 3). T A B L E II I N H I B I T I O N BY A N T I B I O T I C S O F P R O T E I N S Y N T H E S I S BY M I T O C H O N D R I A LATED FROM WILD-TYPE AND ANTIBIOTIC-RESISTANT STRAINS
ISO-
M i t o c h o n d r i a were incubated at a concentration o f 1-2 m g p r o t e i n / m l u n d e r conditions described previously 5. 15-30 pmoles L-[14C]leucine (specific activity 62 Ci/mole) were incorporated per m g p r o t e i n in 30 m i n at 30 °C. A linear rate o f i n c o r p o r a t i o n is observed in control incubations u n d e r these conditions (Grivell, L. A., u n p u b l i s h e d observations). Figures are the m e a n o f two or in s o m e cases three determinations with separate m i t o c h o n d r i a l preparations.
% Activity remaining
C h l o r a m p h e n i c o l (/zg/ml) 10 25 50 100 E r y t h r o m y c i n Q~g/ml) 0.4 1
10 100 250 L i n c o m y c i n (/~g/ml) 2 20 200 400
ER354
ER553
22 13 10 9
18 11 5 3
16 4 5 2
94 89 102 103 98
93 89 87 89 65
75 -34 28
85 80 36 22
95 78 59 49
52 48 10 5
60 30 3 2
86 53 16
88 78 30
81 23 6
20 9 10
Wild type
cR321
cR323
22 11 6 5
69 54 38 35
94 90 80 74
19 16 I1 10
38 33 27 16
55 47 35 24
- -
- -
43 35 6 4
52 81 41 26
33 9 4
76 31 15
ERs 1,,
- -
Spiramycin (/tg/ml) 1
10 100
L . A . G R I V E L L et al.
362
Biochemical characterization Resistance of mitochondrial protein synthesis to antibiotics might result either from impermeability of the mitochondrial membrane or from a change in the mitochondrial ribosome itself. A form of detoxification mechanism is a third, but unlikely possibility. A comparison of the response to antibiotics of intact, isolated mitochondria with that of purified mitochondrial ribosomes should reveal which of the mutants possess altered mitochondrial ribosomes. Chloramphenicol, erythromycin, spiramycin and lincomycin were, therefore, tested for their effects on protein synthesis by isolated mitochondria (Table II) and on the peptidyl transferase activity of mitochondrial ribosomes (Table III). In the latter assay, response to carbomycin was also tested. Sensitivity to chloramphenicol,
T A B L E III EFFECTS OF ANTIBIOTICS ON THE SYNTHESIS OF ACETYL- [aH]LEUCYL-PU ROMYCIN BY M I T O C I - [ O N D R I A L R I B O S O M E S I S O L A T E D F R O M W I L D - T Y P E A N D A N T I B I O T I C RESISTANT STRAINS Ribosomal peptidyl transferase activity was assayed by a modified fragment reaction using acetyl[3H]leucyl-tRNA from E. coli (specific activity 10 Ci/mmole; 5000 cpm/assay). The reaction mixture (0.15 ml after addition o f ethanol) contained the equivalent of between 0.6 and 1.0 A26o nm units o f mitochondrial ribosomes. 100 % reaction corresponds to between 1500 and 4600 cpm. The results are the mean of up to four determinations with two separate ribosomal preparations. They have been corrected for blanks without ribosomes (approx. 250 cpm). The extent of the reaction in the absence o f puromycin was never greater than 2 % and usually zero.
% Activity remaininy
cR321
Wild type Chloramphenicol (pg/ml) 26 66 132 Erythromycin (ttg/ml) 6 13 20
cR323
ERs14
ERasa
--
22 12 10
65 48 34
107 102 106
--
I10 113
120 115
167 165
-
-
-
-
ERSS3
4
--
--
5 4 2
113 -115
113 -107
118 -113
7 7 10
11 14 21
57 67 85
6
Chloramphenicol (66/~g/ml) ÷ Erythromycin (/~g/ml) 6 13 20 Lincomycin (~g/ml) 5 50
25 5
88 35
94 33
84 60
3 1
5 2
Spiramycin (/~g/ml) 66 132 198
38 27 23
54 49 49
61 59 58
92 106 94
82 92 86
56 56 44
Carbomycin (ktg/ml) 66 132
16 13
13
84 78
68 55
25 23
88 85
90 92 -
157 174
-
-
-
23 -
-
-
-
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spiramycin, lincomycin and carbomycin was measured directly, while response to erythromycin was determined by the ability of this antibiotic to displace chloramphenicol from erythromycin-sensitive, but not -resistant ribosomes. Table IV summarizes data from the two assays. TABLE IV A COMPARISON OF THE RESPONSE OF INTACT MITOCHONDR1A A N D ISOLATED RIBOSOMES TO ANTIBIOTICS
Phenotype intact mitochondria/isolated ribosomes Chloramphenicol CSEs
Erythromycin
Lincomycin
Spiramycin S
S
S
S
g
g
~
CR321
2R 2R
R 9.
R ~
R
CR323
3R 3R
R ~
R R
R
ER51,,
S
3R 3R
2R 2R
2R 2R
ERas,,
S
3R 3R
S S
R 2R
ER553
S
R
S
S
For chloramphenicol-resistant mutants 321 and 323, activities of both intact mitochondria and purified ribosomes are highly resistant to chloramphenicol and moderately resistant to spiramycin and lincomycin. The two mutations were easily distinguishable by their quantitative response to the antibiotics. Since the test of ribosomal erythromycin sensitivity requires a chloramphenicol-sensitive ribosome, the slight erythromycin resistance of intact mitochondria has not yet been verified at the ribosomal level. Antibiotic resistance in these strains is, however, clearly a result of the possession of altered mitochondrial ribosomes. Similarly, erythromycin-resistant mutations 354, 514 and 553 also affect the mitochondrial ribosome. Mutants 354 and 514, besides being genetically distinct, differ in their response to lincomycin in that 514 is resistant and 354 highly sensitive to this antibiotic. Quantitative differences in the levels of resistance to erythromycin, spiramycin and carbomycin are also evident. Mutant 553, as might be predicted from the low levels of resistance exhibited in vivo, yields mitochondria and ribosomes with low resistance to erythromycin, although the anomalous dose response of this strain in vivo (see ref. 4) is not seen in vitro. The significance of the slight resistance of isolated ribosomes to spiramycin is not clear. By the criteria of Bunn et al. 9 this strain would qualify as a membrane mutant, since it yields "sensitive" mitochondria (i.e. > 60 o/inhibited at a concentration of < 0.1 mM antibiotic). /o
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The component altered in antibiotic-resistant ribosomes Resistance to erythromycin in Escherichia coli or Bacillus subtilis is accompanied by a change in one protein of the large ribosomal subunit, detectable by its altered mobility during chromatography on carboxymethyl cellulose or gel electrophoresis in urea at pH 4.5 (refs. 10 and 11). Fig. 2 compares separations of proteins from the 50-S mitochondrial ribosomal subunits of wild-type and mutant strains 354 and 514, obtained by electrophoresis in urea-acrylamide gels at pH 4.5. Despite reproducible resolution of the proteins in up to 28 components, no difference in band pattern can be detected. Similar results were obtained for mutants 321 and 323 (profiles not shown). Separations run at pH 8.7 also failed to reveal any differences between these mutants and the wild type.
Fig. 2. 50-S ribosomal proteins from wild-type and erythromycin-resistant mutants. See Methods and Materials for details of sample preparation and electrophoresis.
In order to increase the sensitivity of the method, separations at pH 4.5 were repeated using differentially-labelled mixtures of wild-type and mutant 50-S ribosomal proteins. These were obtained by growth of the strains for several generations in the presence of [3H]- or [14C]leucine and mixing of mutant with wild-type cells directly before harvesting. The results of such an experiment with chloramphenicol-resistant mutant 323 are displayed in Fig. 3. For comparison, differentially-labelled mixtures of wild-type cells and of wild-type plus mutant cells with labels reversed were also analysed (results not shown). The 14C/3H ratio throughout the gel does not deviate significantly from the mean, implying that the 50-S ribosomal proteins of mutant 323 are not detectably different from those of the wild type. Similar results were obtained for erythromycin-resistant mutant 514 (experiment not shown).
YEAST M I T O C H O N D R I A L R I B O S O M A L M U T A N T S
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b
46
64
~
2
8 .,-~4
C~23 (3HI + C~[ 14c1
,%.,i 0
20
4~
'a 40
60
80 ~ '120 Distence migreted (mm)
Fig. 3. Gel analysis of a differentially labelled mixture of 50-S ribosomal proteins from wild-type a n d chloramphenicol-resistant strain 323. See Methods and Materials. - - - - - - , 14C; - - - , 3H. DISCUSSION
We find that several genetically distinguishable mutants of m t D N A in yeast are resistant to chloramphenicol, erythromycin and/or spiramycin as the result of the possession of altered mitochondrial ribosomes. The mutants exhibit widely differing phenotypes and in several cases are cross-resistant to other antibiotics with related modes of action (e.g. lincomycin, carbomycin). It was initially surprising to find mutants with such widely different biochemical characteristics mapping at the same locus, e. g. 354 and 553. Such phenotypic variation, not separable by genetic recombination, may originate from various substitutions of a single nucleotide at the same position as observed for mutants of E. coli resistant to, or dependent on, streptomycin 12. It may also be that the genetic techniques used at present are incapable of the necessary resolution in the case of mutations resulting from substitutions at closely adjacent positions. In contrast to corresponding bacterial mutants 1°, none of the mitochondrial mutations seem to have deleterious effects on mitochondrial ribosomal function, detectable either by reduced growth rate in the absence of antibiotics 4, or function in vitro (Tables II and III). Furthermore, mutants 354 and 514, primarily resistant to erythromycin, are many times more resistant than corresponding bacterial ribosomal mutants 13. As such, they may be representatives of novel classes of ribosomal mutations. A cytoplasmic erythromycin-resistant mutant was previously shown by us to possess altered mitochondrial ribosomes 7. This mutant resembled 514 in its high resistance to erythromycin and cross-resistance to lincomycin. 514 is in fact the type of erythromycin-resistant mutant arising, most frequently, spontaneously 3. Use of the peptidyl transferase assay has permitted rapid routine screening of ribosomes from many mutants. The assay may be limited, however, in its ability to pick up all types of antibiotic-resistant mutants. Erythromycin-resistant mutant 353, and spiramycin-resistant mutant 352, both mapping at the same locus as 514, were also studied using this technique (results not shown). Although both mutants yielded
366
L.A. GR1VELL et al.
antibiotic-resistant ribosomes, the levels of resistance observed were lower than predicted from in vivo data, and, in the case of 353, there were puzzling losses of some cross-resistances. These discrepancies were absent, however, when the ribosomes were tested for antibiotic response in protein-synthesizing activity directed by RNA from phage MS2 (Reijnders, L., unpublished observations). It is striking that, of all the mutants examined by us, none has been found to be resistant as a result of an altered mitochondrial membrane. The isolation of such a mutant was recently reported by Bunn et aL 9. This mutant, selected for mikamycin resistance, was found to be cross-resistant to a wide range of unrelated compounds, including chloramphenicol, lincomycin, carbomycin, tetracycline and oligomycin. The genetics of this mutant, originally thought to be cytoplasmic, have since been found to be complex TM. Moreover, only indirect arguments were used to classify this mutant as a membrane mutant. In our opinion the only unambiguous characterization of such a mutant will come from a study of the isolated mitochondrial ribosomes. Although in bacteria, resistance to erythromycin has been shown to be determined by a mutated protein of the 50-S subunit 10.11, we have not been able to detect any convincing difference in the composition of the proteins of the large ribosomal subunit from wild-type and several mutant strains, either visually or using doublelabel techniques in combination with acrylamide gel electrophoresis in urea at both pH 4.5 and pH 8.7. The negative evidence that we have presented on this point does not exclude the possibility of amino acid substitutions in a ribosomal protein without change in charge. However, we consider this unlikely since in bacteria such mutants are only weakly resistant 15. We have previously considered the possibility that antibiotic resistance in these mutants is determined by r R N A ~6. This alternative has the attraction that it localizes the change in a component already shown to be specified by mtDNA 17. rRNA mutations conferring antibiotic resistance in bacteria are so far unknown, but genetically, mitochondria differ from bacteria in two important respects. A bacterial genome usually possesses several cistrons specifying r R N A in a single molecule of DNA; mtDNA, in contrast, has only one cistron for each rRNA per molecule ~7. There are, of course, many m t D N A molecules in one cell, but it is conceivable that each molecule constitutes an independent unit for mutation and segregation. Since resistance to the antibiotics involved is recessive to sensitivity, bacterial mutants in rRNA will not readily be detected. However, a mitochondrion with a full complement of resistant ribosomes can arise as a result of a single mutation and, under selective conditions, replace the original antibiotic sensitive population. ACKNOWLEDGEMENTS L.A.G. is grateful to EMBO for the award of a short-term fellowship which made possible a visit to the Centre de G6n6tique Mol6culaire du C.N.R.S., Gif-surYvette, France. Part of the work was carried out during tenure by L.A.G. of a fellowship in the European Programme of the Royal Society of Great Britain. The work at Gif-sur-Yvette has been supported by a grant from DGRST, No. 6600168. This work was also supported in part by a grant to P.B. from the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
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REFERENCES 1 Coen, D., Deutsch, J., Netter, P., Petrochilo, E. and Slonimski, P. P. (1970) in Control of Organelle Development (Miller, P. L., ed.), pp. 449-496, Cambridge University Press, Cambridge 2 Bolotin, M., Coen, D., Deutsch, J., Dujon, B., Netter, P., Petrochilo E. and Slonimski, P. P. (1971) Bull. Inst. Pasteur 69, 215-239 3 Netter, P., Petrochilo, E., Slonimski, P. P., Bolotin, M., Coen, D., Deutsch, J. and Dujon, B. (1973) Genetics, in the press 4 Slonimski, P. P., Perrodin, G., Bolotin, M., Coen, D., Deutsch, J., Dujon, B., Netter, P. and Petrochilo, E. (1973) Biochimie, in the press 5 Grivell, L. A., Netter, P. and Slonimski, P. P. (1972) Abstract 8th FEBS Meeting, Amsterdam, No. 623 6 Grivell, L. A., Reijnders, L. and Borst, P. (1971) Biochim. Biophys. Acta 247, 91-103 7 Grivell, L. A., Reijnders, L. and De Vries, H. (1971) FEBS Lett. 16, 159-163 8 Traub, P., Mizushima, S., Lowry, C. V. and Nomura, M. (1971) in Methods in Enzymolo#y (Colowick, S. P. and Kaplan, N. O., eds), Vol. 20, pp. 391-407, Academic Press, New York 9 Bunn, C. L., Mitchell, C. I-[., Lukins, H. B. and Linnane, A. W. (1970) Proc. Natl. Acad. Sci. U.S. 67, 1233-1240 10 Otaka, E., Teraoka, H., Tamaki, M., Tanaka, K. and Osawa, S. (1970) J. Mol. Biol., 48,499-510 11 Smith, I., Goldthwaite, C. and Dubnau, D. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 85-89 12 Funatsu, G. and Wittmann, H. G. (1972) J. MoL Biol. 68, 547-550 13 Apirion, D. (1967) J. Mol. Biol. 30, 255-275 14 Linnane, A. W., Haslam, J. M., Lukins, H. B. and Nagley, P. (1972) Annu. Rev. Microbiol. 26, 163-198 15 Tanaka, K., Tamaki, M., Itoh, T., Otaka, E. and Osawa, S. (1971) Mol. Gen. Genet. 114, 23-30 16 Borst, P. (1972) Ann. Rev. Biochem. 41,333-376 17 Reijnders, L., Kleisen, C. M., Grivell, L. A. and Borst, P. (1972) Biochim. Biophys. Acta 272, 396-407