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Changes in chloroplast ribosomal proteins in a streptomycin-resistant mutant of Euglena gracilis
Plant Science Letters, 5 (1975) 305--311
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CHANGES IN CHLOROPLAST RIBOSOMAL PROTEINS IN A STREPTOMYCIN-RESISTANT MUTANT O F E U G L E N A G R A C I L I S
GEORGES FREYSSINET Di~partement de Biologic Ge.,.. ale et Appliqu$e, Laboratoire Associ(~ au CNRS, Universit$ de Lyon I, 69621 Villeurbanne (France)
(Received May 30th, 1975) (Revision received and accepted August 1st, 1975)
SUMMARY Ribosomal proteins extracted from chloroplasts of wild-type and a streptomycin-resistant mutant of E u g l e n a gracilis were analyzed by two
INTRODUCTION Study of algae mutants resistant to antibiotics provides useful information on the mechanisms which control the formation of chloroplast ribosomes. As in bacteria, such mutants show modifications in the properties of their chloroplast ~ibosomes [1 and refs. cited therein: 2--4]. With Chiamydomonas, these modifications have been shown to affect some proteins of the chloroplast ribosomes [ 5--8]. Schwartzbach and Schiff [2] have demonstrated that chloroplast ribosomes extracted from Sm~BNgL, a Euglena mutant resistant to streptomycin, have lost the ability to bind this antibiotic. I have compared the ribosomal proteins extracted from chloroplasts of both wild-type and Sm~BNgL mutant of Euglena and observed changes in 4 acidic proteins. This suggests that the mutation involves the alteration of a maturation enzyme acting on several proteins rather than a change in genetic code of each of these proteins.
Abbreviation: DSm, dihydrostreptomycin.
306
METHODS
Euglena gracilis Klebs var. BaciUaris Pringsheim, wild-type and SmrBNgL, a mutant resistant to streptomycin [9] were used, Cultures were prepared as already described [ 10]. Chloroplast ribosomes were extracted from once-washed chloroplasts and the 68 S chloroplast monosomes purified by zonal centrifugation [10]. Ribosomal proteins were extracted using the RNAase method [11]. To analyze small amounts of proteins (200/~g) the technique of Kaltschmidt and Wittmann [12] was modified as follows: First dimension by disc gel electrophoresis. Separating gel: pH 8.7, urea 360 g/l, acrylamide 80 g/l, bisacrylamide 3 g/l, EDTA Na2 8 g/l, boric acid 32 g/l, tris 48.6 g/l and TEMED 3 ml/l. Ammonium persulfate, 0.6 g/l was added just before use. Running buffer: pH 8.6, urea 240 g/l, EDTA Na~ 2.4 g/l, boric acid 9 g/l and tris 16 gfl. Gels were polymerized in plexiglass tubes (0.3 cm X 15.0 cm). The technique described by Howard and Traut [13], in which identical amounts of the sample are applied at the top of each of two separate gels, was used. Electrophoresis was carried out at a constant current of 1 mA/gel for about 24 h at 4 °. After electrophoresis, gels were removed from the tubes and dialyzed. Under such conditions, acidic protein migration was less than 5 cm (gel A) and basic protein migration less than 10 cm (gel B). Second dimension by slab gel electrophoresis. Gel solution and running buffer were prepared as described by Kaltschmidt and Wittmann [12]. The apparatus used (Fig. 1) is a modification of that described previously [12]. The gel sheet dimel, sions were 15 X 15 X 0.2 cm; one to six chambers can be employed at the same time. Each chamber was filled with 50 ml of gel solution; the dialyzed 1-D gels were cut, 5 cm long for gel A and 10 cm for gel B and laid on top of the chamber. After polymerization a constant voltage of 5 V/cm was applied for about 24 h at room temperature. After 2 h of elctrophoresis, the buffer was changed and circulation of the new buffer was performed with a peristaltic pump for the remaining time. After electrophoresis the slab was removed and placed into a tray containing a solution of Coomassie brilliant blue 0.025 %, acetic acid 10 % and iso-propylalcohol 25 % for 12 h. Destaining was performed in a 6-1 tray containing methanol 25 % and acetic acid 10 % in water. The destaining solution circulated through activated charcoal. RESULTS
Table I shows that chloroplast ribosomes of wild-type strain are able to bind DSm. With the mutant strain this binding is low and close to the one obtained with cytoplasmic ribosomes extracted from either wild-type or mutant cells.
307
Fig. 1. Two-dimensional electrophoresis apparatus: (A) Photograph of the apparatus installed in the lower buffer vessel; (B) Side view of the block chamber. The technique of assembly is as described previously [12]. The cover which supports the upper electrode was cut in six parts, one for each chamber. The chamber dimensions are 150 x 150 × 2 mm. Cooling is achieved by introducing through the holes a tygon tubing "worm" in which a cold liquid circulates.
TABLE I
[ 3H] DIHYDROSTREPTOMYCIN BINDING TO RIBOSOMES EXTRACTED FROM EUGLENA GRACILI$
Chloroplast and cytoplasmic ribosomes extracted from wild-type and mutant SmrBNgL of Euglena were assayed for their ability to bind [ 3H ] dihydrostreptomycin ([ 3H] DSm) as described previously [ 2 ]. Strains
Ribosomes
cpm/A~60
Chloroplast
2984
Cytoplasmic
316
Chloroplast
410
Cytoplasmic
399
Wild-type
SmrBSgL
308
The electrophoretogram obtained with the wild-type strain yields from 56 to 58 spots (Fig. 2A), spot "a" being subdivided in 3 small spots when the electrophoresis is run for a longer time. Fourteen acidic proteins are numbered; in some cases protein "b" migrates with the basic proteins which implies that its isoelectric point must be close to 8.7. This number is close to that obtained with E. coil ribosomes for which 11 acidic proteins were characterized out of the total 55 spots [14]. It is higher than the one reported for chloroplast ribosomes of Chlamydomonas in which 48 proteins were numbered [ 1]. The pattern thus obtained differs from that previously observed for proteins extracted from cytoplasmic ribosomes of Euglena which yielded 69 spots, among which 2 were acidic proteins [11]. The electrophoretogram obtained with the mutant strain (Fig. 2B) shows some differences from that of wild-type strain: 4 acidic proteins, faintly visible on the patterp, of the wild-type strain (arrows on Fig. 2A) are not
i ~.
.~
I1~'~~ ~ • ~ ~ ~i~¸.
~/ ~
!. )
N _ • •
2A
, ~
6
2B
Fig. 2. Photograph of the two-dimensional electrophoretogram obtained with chloroplast ribosomal proteins of Euglena gracilis. (A) Proteins extracted from wild type cells; (B) Proteins extracted from mutant Smr~BNgL; (C) and (D) Enlargement of the left upper part of the gel from wild-type and the mutant respectively. Arrows indicated position of altered proteins.
309
detected on that of the mutant strain; on the contrary, but only in some cases, the pattern of the mutant strain shows, in the central part of the gel, some additional faintly stained spots (arrows on Fig. 2B). Analyses were also performed with the 30 S and 50 S subunits obtained after dialysis of the chloroplast ribosomes against a 1 mM Mg2+ buffer. The electrophoretograms of the 50 S subunits extracted from both strains show no differences. The patterns obtained with 30 S subunits which contain the majority of the acidic proteins show, between the two strains, the same differences as those reported above for the 68 S monosomes. DISCUSSION
Results on binding assay, Table I, are identical to those previously obtained [ 2 and Schwartzbach, personal communication]. In these conditions, we can therefore suppose that the chloroplast ribosomes analyzed in this study are in a state similar to that of the ribosomes prepared by Schwartzbach and Schiff [2]. These authors were able to bind a maximum of 0.13 molecule of DSm per mole of ribosomes. This value is lower than the one reported for either E. coli ribosomes (1 mole of DSm/mole of ribosomes [15,16] ) or Chlamydomonas chloroplast ribosomes (0.6 mole of DSm/mole of ribosomes [ 4 ] ). This low binding might result either from a ribosome alteration during extraction, as Garvin et al. [ 17 ] found a decrease in the DSm binding after various treatments of ribosomes, or from a heterogeneity in the chloroplast ribosome population. In both cases, the proteins responsible for the antibiotic binding can be supposed to be present in low amounts in the anal.yzed protein preparation. These proteins will be searched for among the faintly stained spots, as the proteins which appear to be altered in the mutant strain belong to these spots precisely. The correlation observed between the lack of affinity for streptomycin and the alteration of several proteins is unusual. Indeed, most frequently, the lack of affinity for antibiotic is connected with a change in only a single defined protein. This observation requires the following comments: (1) In a few cases, a change in ribosomal properties has been shown to be associated with an alteration of several ribosomal components. This has been reported, in E. coil, for a mutant "lir" [18] and a showdomycin-resistant mutant [19]. Furthermore, Mets and Bogorad [6] have found that an intensively stained spot present in the 2-dimensional pattern of the 52 S subunits extracted from wild-type is replaced by 4 faint spots in the 2-dimensional pattern of the 52S subunits extracted from a uniparental heredity mutant of Chlamydomonas resistant to erythromycin. (2) In E. coli, it is well known that several proteins are involved in the streptomycin binding. This binding is lost when one of these proteins is altered [15,20]. (3) Among Chlamydomonas mutants resistant to the same antibiotic it is possible to isolate strains with mendelian heredity and strains with uniparental heredity. This has been obtained, for instance, for mutant~ resistant to streptomycin
310 [4] and to erythromycin [6 ]. This tends to show t h a t a given property is controlled by both nuclear and chloroplast DNA. As it is unlikely that both these DNAs code for the primary structure of the same polypeptide, it can be inferred t h a t the antibiotic binding depends on the combined activity of several polypeptides. One may assume that such a dual control also exists in Euglens. (4) I t is known ~hat, after translation, proteins can be modified, especially by phosphorylation, methylation, acetylation or proteolytic cleavages. Some o f these modifications can induce changes in charge and function [ 21]. It can then be supposed that the mutation of an enzyme responsible for some of these secondary modifications may cause simultaneous changes in several different proteins. Finally, the following hypotheses may be considered: (a) One or several of the altered proteins can play a role in the antibiotic binding. (b) At the site of the genome, the mutation can be punctual; in this case, we can suppose that it either concerns an enzyme of a post-translational maturation step or results in misassembly and loss of other proteins from the ribosomal subunit which can be related to the binding of streptomycin. We can also imagine that the mutation has affected, at the same time, several sites of the genome, thus modifying the primary structure of several of the ribosomal proteins. Presently, it is difficult to make a choice. Experiments, in progress, on various mutants resistant to antibiotics and studies on synthesis of the ribosomal proteins would without any doubt allow an approach to a more complete answer of this particular problem. ACKNOWLEDGEMENTS The author would like to thank Prof. V. Nigon for advice and encouragement throughout this work, Dr..J.A. Schiff who generously provided the Sm~BNgL mutant and Dr. M. Beljanski for helpful information. The technical assistance of Mrs. C. Schwob and P.M. Malet is greatly appreciated. This work was supported by C.E.A, France. REFERENCES
1 M.R. Hanson, J.N. Davidson, L.J. Mets and L. Bogorad, Mol. Gen. Genet., ~~? (1974) 105. 2 S.D. Schwartzbach and J.A. Sehiff, J. Bacteriol., 120 (1974) 334. 3 A. Boschetti, V. Niggli, U. Otz and T. Wiedmer, Physiol. Plant., 31 (1974) 169. 4 A. Boschetti and S. Bogdanov, FEBS Letters, 38 (1974) 19. 5 J.E. Boynton, W.G. Burton, N.W. Gillham and E.H. Harris, Proc. Nail. Acad. Sci. USA, 70 (1973) 3463. 6 L. Mets and L. Bogorad, Proc. Natl. Acad. Sci. USA, 69 (1972) 3779. 7 J.N. Davidson, M.R. Hanson and L. Bogorad, Mol. Gen. Genet., 132 (1974) 119. 8 N. Ohta, M. Inouye and R. Sager, Fed. Proc., 33 (1974) 1584.
311 9 10 11 12. 13 14 15 16 17 18 19 20 21
J. Diamond and J.A. Schiff, Plant Sci. Letters, 3 (1974) 289. G. Freyssinet, 1~oc. Int. Cong. Photosynthesis, 3 (1974) 1731. G. Freyssinet and J.A. Schiff, Plant Physiol., 54 (1974) 543. E. Kaltschmidt and H.G. Wittmann, Anal. Biochem., 36 (1970) 401. G.A. Howard and R.R. Traut, FEBS Letters, 29 (1973) 177. E. Kaltschmidt, Anal. Biochem., 43 (1971) 25. F.N. Chang and J.G. Fiaks, Proc. Natl. Acad. Sci. USA, 67 (1970) 1321. H. Kaji and Y. Tamaka, J. Mol. Biol., 32 (1968) 221. R.T. Garvin, D.K. Biswas and L. Gorini, Proc. Natl. Acad. Sci. USA, 71 (1974) 3814. J. Krembel and D. Apirion, J. Mol. Biol., 33 (1968) 363. M. Beljanski, P. Bourgarel and M. Beljanski, Proc. Natl. Acad. Sci. USA, 68 (1971) 491. G. Schreiner and K.H. Nierhaus, J. Mol. Biol., 81 (1973) 71. F.N. Chang, C.N. Chang and W.K. Paik, J. Bacteriol., 120 (1974) 651.
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CHANGES IN CHLOROPLAST RIBOSOMAL PROTEINS IN A STREPTOMYCIN-RESISTANT MUTANT O F E U G L E N A G R A C I L I S
GEORGES FREYSSINET Di~partement de Biologic Ge.,.. ale et Appliqu$e, Laboratoire Associ(~ au CNRS, Universit$ de Lyon I, 69621 Villeurbanne (France)
(Received May 30th, 1975) (Revision received and accepted August 1st, 1975)
SUMMARY Ribosomal proteins extracted from chloroplasts of wild-type and a streptomycin-resistant mutant of E u g l e n a gracilis were analyzed by two
INTRODUCTION Study of algae mutants resistant to antibiotics provides useful information on the mechanisms which control the formation of chloroplast ribosomes. As in bacteria, such mutants show modifications in the properties of their chloroplast ~ibosomes [1 and refs. cited therein: 2--4]. With Chiamydomonas, these modifications have been shown to affect some proteins of the chloroplast ribosomes [ 5--8]. Schwartzbach and Schiff [2] have demonstrated that chloroplast ribosomes extracted from Sm~BNgL, a Euglena mutant resistant to streptomycin, have lost the ability to bind this antibiotic. I have compared the ribosomal proteins extracted from chloroplasts of both wild-type and Sm~BNgL mutant of Euglena and observed changes in 4 acidic proteins. This suggests that the mutation involves the alteration of a maturation enzyme acting on several proteins rather than a change in genetic code of each of these proteins.
Abbreviation: DSm, dihydrostreptomycin.
306
METHODS
Euglena gracilis Klebs var. BaciUaris Pringsheim, wild-type and SmrBNgL, a mutant resistant to streptomycin [9] were used, Cultures were prepared as already described [ 10]. Chloroplast ribosomes were extracted from once-washed chloroplasts and the 68 S chloroplast monosomes purified by zonal centrifugation [10]. Ribosomal proteins were extracted using the RNAase method [11]. To analyze small amounts of proteins (200/~g) the technique of Kaltschmidt and Wittmann [12] was modified as follows: First dimension by disc gel electrophoresis. Separating gel: pH 8.7, urea 360 g/l, acrylamide 80 g/l, bisacrylamide 3 g/l, EDTA Na2 8 g/l, boric acid 32 g/l, tris 48.6 g/l and TEMED 3 ml/l. Ammonium persulfate, 0.6 g/l was added just before use. Running buffer: pH 8.6, urea 240 g/l, EDTA Na~ 2.4 g/l, boric acid 9 g/l and tris 16 gfl. Gels were polymerized in plexiglass tubes (0.3 cm X 15.0 cm). The technique described by Howard and Traut [13], in which identical amounts of the sample are applied at the top of each of two separate gels, was used. Electrophoresis was carried out at a constant current of 1 mA/gel for about 24 h at 4 °. After electrophoresis, gels were removed from the tubes and dialyzed. Under such conditions, acidic protein migration was less than 5 cm (gel A) and basic protein migration less than 10 cm (gel B). Second dimension by slab gel electrophoresis. Gel solution and running buffer were prepared as described by Kaltschmidt and Wittmann [12]. The apparatus used (Fig. 1) is a modification of that described previously [12]. The gel sheet dimel, sions were 15 X 15 X 0.2 cm; one to six chambers can be employed at the same time. Each chamber was filled with 50 ml of gel solution; the dialyzed 1-D gels were cut, 5 cm long for gel A and 10 cm for gel B and laid on top of the chamber. After polymerization a constant voltage of 5 V/cm was applied for about 24 h at room temperature. After 2 h of elctrophoresis, the buffer was changed and circulation of the new buffer was performed with a peristaltic pump for the remaining time. After electrophoresis the slab was removed and placed into a tray containing a solution of Coomassie brilliant blue 0.025 %, acetic acid 10 % and iso-propylalcohol 25 % for 12 h. Destaining was performed in a 6-1 tray containing methanol 25 % and acetic acid 10 % in water. The destaining solution circulated through activated charcoal. RESULTS
Table I shows that chloroplast ribosomes of wild-type strain are able to bind DSm. With the mutant strain this binding is low and close to the one obtained with cytoplasmic ribosomes extracted from either wild-type or mutant cells.
307
Fig. 1. Two-dimensional electrophoresis apparatus: (A) Photograph of the apparatus installed in the lower buffer vessel; (B) Side view of the block chamber. The technique of assembly is as described previously [12]. The cover which supports the upper electrode was cut in six parts, one for each chamber. The chamber dimensions are 150 x 150 × 2 mm. Cooling is achieved by introducing through the holes a tygon tubing "worm" in which a cold liquid circulates.
TABLE I
[ 3H] DIHYDROSTREPTOMYCIN BINDING TO RIBOSOMES EXTRACTED FROM EUGLENA GRACILI$
Chloroplast and cytoplasmic ribosomes extracted from wild-type and mutant SmrBNgL of Euglena were assayed for their ability to bind [ 3H ] dihydrostreptomycin ([ 3H] DSm) as described previously [ 2 ]. Strains
Ribosomes
cpm/A~60
Chloroplast
2984
Cytoplasmic
316
Chloroplast
410
Cytoplasmic
399
Wild-type
SmrBSgL
308
The electrophoretogram obtained with the wild-type strain yields from 56 to 58 spots (Fig. 2A), spot "a" being subdivided in 3 small spots when the electrophoresis is run for a longer time. Fourteen acidic proteins are numbered; in some cases protein "b" migrates with the basic proteins which implies that its isoelectric point must be close to 8.7. This number is close to that obtained with E. coil ribosomes for which 11 acidic proteins were characterized out of the total 55 spots [14]. It is higher than the one reported for chloroplast ribosomes of Chlamydomonas in which 48 proteins were numbered [ 1]. The pattern thus obtained differs from that previously observed for proteins extracted from cytoplasmic ribosomes of Euglena which yielded 69 spots, among which 2 were acidic proteins [11]. The electrophoretogram obtained with the mutant strain (Fig. 2B) shows some differences from that of wild-type strain: 4 acidic proteins, faintly visible on the patterp, of the wild-type strain (arrows on Fig. 2A) are not
i ~.
.~
I1~'~~ ~ • ~ ~ ~i~¸.
~/ ~
!. )
N _ • •
2A
, ~
6
2B
Fig. 2. Photograph of the two-dimensional electrophoretogram obtained with chloroplast ribosomal proteins of Euglena gracilis. (A) Proteins extracted from wild type cells; (B) Proteins extracted from mutant Smr~BNgL; (C) and (D) Enlargement of the left upper part of the gel from wild-type and the mutant respectively. Arrows indicated position of altered proteins.
309
detected on that of the mutant strain; on the contrary, but only in some cases, the pattern of the mutant strain shows, in the central part of the gel, some additional faintly stained spots (arrows on Fig. 2B). Analyses were also performed with the 30 S and 50 S subunits obtained after dialysis of the chloroplast ribosomes against a 1 mM Mg2+ buffer. The electrophoretograms of the 50 S subunits extracted from both strains show no differences. The patterns obtained with 30 S subunits which contain the majority of the acidic proteins show, between the two strains, the same differences as those reported above for the 68 S monosomes. DISCUSSION
Results on binding assay, Table I, are identical to those previously obtained [ 2 and Schwartzbach, personal communication]. In these conditions, we can therefore suppose that the chloroplast ribosomes analyzed in this study are in a state similar to that of the ribosomes prepared by Schwartzbach and Schiff [2]. These authors were able to bind a maximum of 0.13 molecule of DSm per mole of ribosomes. This value is lower than the one reported for either E. coli ribosomes (1 mole of DSm/mole of ribosomes [15,16] ) or Chlamydomonas chloroplast ribosomes (0.6 mole of DSm/mole of ribosomes [ 4 ] ). This low binding might result either from a ribosome alteration during extraction, as Garvin et al. [ 17 ] found a decrease in the DSm binding after various treatments of ribosomes, or from a heterogeneity in the chloroplast ribosome population. In both cases, the proteins responsible for the antibiotic binding can be supposed to be present in low amounts in the anal.yzed protein preparation. These proteins will be searched for among the faintly stained spots, as the proteins which appear to be altered in the mutant strain belong to these spots precisely. The correlation observed between the lack of affinity for streptomycin and the alteration of several proteins is unusual. Indeed, most frequently, the lack of affinity for antibiotic is connected with a change in only a single defined protein. This observation requires the following comments: (1) In a few cases, a change in ribosomal properties has been shown to be associated with an alteration of several ribosomal components. This has been reported, in E. coil, for a mutant "lir" [18] and a showdomycin-resistant mutant [19]. Furthermore, Mets and Bogorad [6] have found that an intensively stained spot present in the 2-dimensional pattern of the 52 S subunits extracted from wild-type is replaced by 4 faint spots in the 2-dimensional pattern of the 52S subunits extracted from a uniparental heredity mutant of Chlamydomonas resistant to erythromycin. (2) In E. coli, it is well known that several proteins are involved in the streptomycin binding. This binding is lost when one of these proteins is altered [15,20]. (3) Among Chlamydomonas mutants resistant to the same antibiotic it is possible to isolate strains with mendelian heredity and strains with uniparental heredity. This has been obtained, for instance, for mutant~ resistant to streptomycin
310 [4] and to erythromycin [6 ]. This tends to show t h a t a given property is controlled by both nuclear and chloroplast DNA. As it is unlikely that both these DNAs code for the primary structure of the same polypeptide, it can be inferred t h a t the antibiotic binding depends on the combined activity of several polypeptides. One may assume that such a dual control also exists in Euglens. (4) I t is known ~hat, after translation, proteins can be modified, especially by phosphorylation, methylation, acetylation or proteolytic cleavages. Some o f these modifications can induce changes in charge and function [ 21]. It can then be supposed that the mutation of an enzyme responsible for some of these secondary modifications may cause simultaneous changes in several different proteins. Finally, the following hypotheses may be considered: (a) One or several of the altered proteins can play a role in the antibiotic binding. (b) At the site of the genome, the mutation can be punctual; in this case, we can suppose that it either concerns an enzyme of a post-translational maturation step or results in misassembly and loss of other proteins from the ribosomal subunit which can be related to the binding of streptomycin. We can also imagine that the mutation has affected, at the same time, several sites of the genome, thus modifying the primary structure of several of the ribosomal proteins. Presently, it is difficult to make a choice. Experiments, in progress, on various mutants resistant to antibiotics and studies on synthesis of the ribosomal proteins would without any doubt allow an approach to a more complete answer of this particular problem. ACKNOWLEDGEMENTS The author would like to thank Prof. V. Nigon for advice and encouragement throughout this work, Dr..J.A. Schiff who generously provided the Sm~BNgL mutant and Dr. M. Beljanski for helpful information. The technical assistance of Mrs. C. Schwob and P.M. Malet is greatly appreciated. This work was supported by C.E.A, France. REFERENCES
1 M.R. Hanson, J.N. Davidson, L.J. Mets and L. Bogorad, Mol. Gen. Genet., ~~? (1974) 105. 2 S.D. Schwartzbach and J.A. Sehiff, J. Bacteriol., 120 (1974) 334. 3 A. Boschetti, V. Niggli, U. Otz and T. Wiedmer, Physiol. Plant., 31 (1974) 169. 4 A. Boschetti and S. Bogdanov, FEBS Letters, 38 (1974) 19. 5 J.E. Boynton, W.G. Burton, N.W. Gillham and E.H. Harris, Proc. Nail. Acad. Sci. USA, 70 (1973) 3463. 6 L. Mets and L. Bogorad, Proc. Natl. Acad. Sci. USA, 69 (1972) 3779. 7 J.N. Davidson, M.R. Hanson and L. Bogorad, Mol. Gen. Genet., 132 (1974) 119. 8 N. Ohta, M. Inouye and R. Sager, Fed. Proc., 33 (1974) 1584.
311 9 10 11 12. 13 14 15 16 17 18 19 20 21
J. Diamond and J.A. Schiff, Plant Sci. Letters, 3 (1974) 289. G. Freyssinet, 1~oc. Int. Cong. Photosynthesis, 3 (1974) 1731. G. Freyssinet and J.A. Schiff, Plant Physiol., 54 (1974) 543. E. Kaltschmidt and H.G. Wittmann, Anal. Biochem., 36 (1970) 401. G.A. Howard and R.R. Traut, FEBS Letters, 29 (1973) 177. E. Kaltschmidt, Anal. Biochem., 43 (1971) 25. F.N. Chang and J.G. Fiaks, Proc. Natl. Acad. Sci. USA, 67 (1970) 1321. H. Kaji and Y. Tamaka, J. Mol. Biol., 32 (1968) 221. R.T. Garvin, D.K. Biswas and L. Gorini, Proc. Natl. Acad. Sci. USA, 71 (1974) 3814. J. Krembel and D. Apirion, J. Mol. Biol., 33 (1968) 363. M. Beljanski, P. Bourgarel and M. Beljanski, Proc. Natl. Acad. Sci. USA, 68 (1971) 491. G. Schreiner and K.H. Nierhaus, J. Mol. Biol., 81 (1973) 71. F.N. Chang, C.N. Chang and W.K. Paik, J. Bacteriol., 120 (1974) 651.