Species specificity of ribosomal proteins from chloroplast and cytoplasmic ribosomes of higher plants

BIOCHIMICA ET BIOPHYSICA ACTA

203

BBA 96381

SPECIES SPECIFICITY OF RIBOSOMAL P R O T E I N S FROM CHLOROPLAST AND CYTOPLASMIC RIBOSOMES OF H I G H E R PLANTS E L E C T R O P H O R E T I C STUDIES CI.AUDIO GUALERZI"

AND P I E R O

CAMMARANO

Istit,~¢o Superiore di Sanit& Labovatorio di Biochimica, Rome and C.N.E.N., Laboratorio di Radiobiologia ..Inimale, C.S.N., Casaccia, Rome (Italy) (Received J u l y i 4 t h , I969)

SUMMARY

I. The proteins isolated by the LiCl-urea method from chloroplast ribosomes of several different plants are electrophoretically dissimilar from those extracted in a similar manner from the corresponding cytoplasmic ribosomes. 2. The electrophoretic patterns of proteins of cytoplasmic (8o-S) ribosomes isolated from plants of different subclasses, different order, different genera, exhibit variable degrees of dissimilarity depending on the degree of taxonomic kinship of the plants from which the ribosomes were derived. The electrophoretic patterns of proteins extracted from plants of different varieties and species are identical. 3. The electrophoretic patterns of proteins isolated from the chloroplast (7o-S) ribosomes of plants of different subclasses, of different order and of different genera exhibit a higher degree of electrophoretic dissimilarity than those of the corresponding 8o-S ribosomes; i.e., in general in differently related plants the patterns obtained from cytoplasmic ribosomes tend to be more congruent than those of the 7o-S ribosomes.

INTRODUCTION

Several studies support the view that the protein moiety of both bacterial (7o-S) and animal (8o-S) ribosomes exhibits a certain degree of species-specificity. Thus, it has been shown that (a) ribosomal proteins isolated from the 7o-S particles of several different species of bacteria give rise to highly dissimilar electrophoretic patterns 1, (b) within the vertebrate class, the electrophoretic patterns of ribosomal proteins isolated from 8o-S particles of more or less closely related animal species, although grossly similar, are not entirely congruent 2, and (c) ribosomal proteins from sea urchin embryos of different genera appear electrophoretically dissimilapa. By contrast, the ribosomal proteins from several differentiated tissues of the same animal give rise to identical electrophoretic patterns ~,4. In a preceeding paper s, we have shown that in one plant tissue (Spinach leaves) cytoplasmic and chloroplastic ribosomes are endowed with highly dissimilar protein complements. In this paper we present evidence that the protein moiety of both the • P r e s e n t address: U n i v e r s i t y of P e n n s y l v a n i a . School of Medicine. Dept. of Microbiology, P h i l a d e l p h i a , Pa. i 9 i o 4 (U.S.A.)

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7o-S (chloroplastic) and the 8o-S (cytoplasmic) ribosomes of more or less distantly related plants exhibits species-specific electrophoretic patterns and that the degree of species-specificity correlates grossly with the degree of taxonoinic kinship of the plants from which the ribosomes were derived. It is also shown that in related plants the overall patterns of the 8o-.'4 ribosomal proteins tend to be more conserved than those of the corresponding 7o-S ribosomal proteins. The possible implications of these findings are discussed in relation to ribosome evolution and to site of synthesis of chloroplastic and cytoplasmic ribosomes.

METHODS

Cytoplasmic post-mitochondrial supernatants and highly purified chloroplasts were prepared by the procedure of STUTZ AND NOLL6 from leaves of Spinacia oleracea, Beta vulgaris, Lacluca dioica and Brassica oleracea. Chloroplastic and cytoplasmic ribosomes were isolated and purified as described in detail in a preceeding paper s. For unclear reasons, the 7o-S ribosomes from Brassica species were very scarce and were consistently containined by large amounts of 8o-S (cytoplasmic) ribosomes. Ribosomal proteins were separated from rRNA by the LiCl-urea method as described previously 2,5 and the protein containing extracts were dialyzed 48 h against a IOOOfold excess of o.ooI M Tris-HC1 (pH 7.5, 2o~), 6 M urea and o.oo5 M 2-mercaptoethanol s. As pointed out previously ~, the electrophoretic patterns of the LiCl-ureasoluble proteins were undistinguishable from those of ribosomal proteins extracted with acetic acid according to WALLER AND HARRIS7. Acrylamide-gel electrophoreses were carried out at pH 4.5 in the presence of 8 M urea by the method of LEI~OY et al. s as described in detail elsewherO.

RESULTS

Dissimilarity between chloroplastic and cytoplasmic ribosomes We have shown previously 5 that proteins isolated from chloroplastic (7o-S) and cytoplasmic (8o-S) ribosomes of the same plant (S. oleracea) yield highly dissimilar patterns upon acrylamide-gel electrophoresis. The results of electrophoretic comparisons between ribosomal proteins of chloroplastic and cytoplasmic ribosomes of beet (B. vulgaris) and lettuce (L. dioica), are illustrated in Fig. IA which reproduces typical split-gel patterns. For comparison a typical pattern of ribosomal proteins from chloroplastic and cytoplasmic ribosomes of spinach leaves is also presented. By examination of the gels it can be seen that in all plants tested, the 7o-S and the 8o-S ribosomes yield distinctly dissimilar patterns. In fact, it has been estimated that approx. 6o o,£, of the bands resolved in the. 7o-S side of the gel columns are not matched by a corresponding band on the opposite (8o-S) side (c/. Table l).

Species speci/icity o/ ribosomal proteins/rom 8o-S ribosomes Fig. IB illustrates split-gel comparisons ainong "8o-S" ribosomal proteins of different subclasses (beet or sl)inach vs. lettuce), among plants of different order within the same subclass (beet or spinach vs. Brassica) and among plants of different genera (beet vs. spinach). Fig. IC illustrates split-gel comparisons of 8o-S ribosomal Biochim. Biophys. ,4eta, i 9 9 ( I 9 7 ° ) 2 o 3 - . e l 3

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Fig. i. A. Split-gel comparison of ribosomal proteins from c h l o r o p l a s t i c (7o-S) and cytoplasmic (St~-S) ribosomes of different plants. Sp ~ S'. oleracea; J3eta - 1t. vulgaris; l,ac ~ L. dioica. IL Split-gel comparison o[ ribosomal proteiiis from t/it 8o-S ribo.somes e l spinach (Sp), beet (]¢eta)

and Brassica (l~r) .C. Split-gel comparison of ribosomal proteins from the 8o-S ribosomes of ttr. oleracea (el L 13r. R a p a (Ra), Hr. oh;rac~;a var. botrytix (/lot) anti 13r. oleracea var. c a p i l a l a {Cap) (See top of following page). Biochim.

Biophys. Acta,

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Fig. 3- Split-gel comparisons of ribosomal proteins from the 7o-S ribosomes of spinach (Sp), beet (Beta) and Lettuce (I.ac)

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proteins isolated from plants of different species within the same genus (Br. oleracea vs. Br. rapa) and from plants of different variety within the same species (Br. oleracea var. botrytis vs. Br. oleracea vat. capitata). B y e x a m i n a t i o n of b a n d c o i n c i d e n c e s o n b o t h s i d e s of t h e s p l i t - g e l c o l u m n s ,

it can be seen t h a t the ribosomal proteins of more distantly related plants (such as b e e t a n d l e t t u c e o r s p i n a c h a n d l e t t u c e ) e x h i b i t g r e a t e r d i s s i m i l a r i t i e s t h a n t h o s e of

more closely related plants (such as beet and spinach). Essentially no differences are discernible a m o n g ribosomal proteins from different Brassica species, as well as a m o n g d i f f e r e n t v a r i e t i e s of t h e B r a s s i c a s p e c i e s .

The results of Fig. I(" are more clearly analyzed by examination of typical densitometric recordings illustrated in Fig. 2 which shows the difference a m o n g the -

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Fig. 3. Densitometric recordings of split-gel comparisons between 8o-S proteins of plants of different subclasses (top pattern) and of different genera (bottom pattern). Vertical bars: coinciding bands; open circles: non coinciding bands in the dashed tracing; closed circles: non coinciding bands in the continous tracing. Signs not corresponding to a peak were designed to mark position of bands which were present in the gel but either absent or ill-resolved in the tracing. Tracings were recorded by scanning the negative plate (KODAK o25o ) of a photograph of the stained gel in a Joyce and Loebl microdensitometer.

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8o-S proteins of spinach and lettuce (subclass difference) and between spinach and beet (genus difference). The densitometric recordings of comparisons between Brassica species and varieties are not reproduced, since the various patterns were qualitatively superimposable. Table I shows a tentative estimate of the degree of electrophoretic difference among 8o-S patterns of different plants, based on the recordings of split-gel columns. The degree of dissimilarity between two patterns was estimated as the percent ratio of non-coinciding bands to total resolved bands. The results indicate again that the more distantly related are the plants, the more dissimilar are the electrophoretic patterns of their 8o-S proteins. TABLE DEGREE

1 OF ELECTROPHORETIC

ROPLASTIC

(7o-S)

DISSIMILARITY AMONG RII~OSOMAL PROTEINS

AND CYTOPLASMIC

(8o-S)

I S O L A T E I ) FROM CIILO-

PARTICLES OF DIFFERENTLY RELATED . . . . . . . . . . . . . . . . . . . . . . . .

PLANTS

Ribosome class compared

Plants o[ origin

Taxonomic di[/erence

Degree o! electrophoretic dissimilarity" (%)

8o S-8o S

Br. oleracea-Br, rapa Beta-Spinacia Spinacia- Brassica Spinacia-Lactuca Beta-Lactuca

Species Genus Order Subclass Subclass

None 27 35 56 47

7 ° S-7o S

B e t a -Spinacia Spinacia-Lactuca Beta--Lactuca

Genus Subclass Subclass

48 72 73

7° S - 8 o S

Spinacia Beta Lactuca

65 04 60

" E s t i m a t e d from d e n s i t o m e t r i c recordings of gel p a t t e r n s . T h e degree of electrophoretic d i s s i m i l a r i t y b e t w e e n two s a m p l e s was calculated as t h e p e r c e n t a g e ratio b e t w e e n total noncoinciding b a n d s a n d t o t a l n u m b e r of electrophoretically resolved bands.

Species specificity o~ ribosomal proteins/rom 7o-S ribosomes The gel columns reproduced in Fig. 3 show the results of electrophoretic comparisons between proteins extracted from chloroplastic ribosomes of plants of different subclasses (spinach vs. lettuce and beet vs. lettuce) and of different genera (spinach vs. beet); ribosomal proteins of plants of different species were not compared because of difficulties experienced in isolating sufficiently purified 7o-S ribosomes from Brassica species (c/. METHODS). By observation of band correspondence on both sides of each gel column (Fig. 3), it can be seen that the degree of dissimilarity among the protein patterns of 7o-S ribosomes of differently related plants is far more striking than that observed among the corresponding patterns of 8o-S ribosomes (see Fig. IB). These results are further documented by the densitometric recording of Fig. 4 (which shows tracings of the split-gel comparison between plants of different subclasses (spinach vs. lettuce) and of different genera (spinach vs. beet)). A quantitative estimate of the degree of electrophoretic difference among "7o-S '' proteins (based on typical densitometer tracings) is presented in Table I, which shows that plants of different subclasses Biochim. Biophys. Acta, 199 (197 ° ) 2 o y 2 1 3

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SPECIES-SPECIFICITY OF RIBOSOMAL PROTEINS -

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exhibit approximately 75 % dissimilarity and that plants of different genera yield about 50 % dissimilarity. In the absence of information concerning the 7o-S proteins from plants of different species and varieties we can only conclude that in those plants which have been examined so far the 7o-S patterns are much less congruent than the corresponding 8o-S patterns. DISCUSSION

The following points seem relevant to the interpretation of the results presented here and will be discussed in detail: (a) the evolutionary significance of heterogeneity of ribosomal proteins in differently related plants, (b) the origin of the dissimilarity between tile ribosomal proteins of the 7o-S and tile 8o-S ribosomes in the same plant, and (c) the significance of tile relatively greater stability of the 8o-S patterns as compared to the 7o-S patterns in more or less distantly related plants.

Ribosome heterogeneity in di//erent plants According to speculations on tile evolution of the genetic apparatus (c]. articles by CRICK9 and by ORGEL1°) a primitive ribosome may have consisted entirely of RNA and this primitive core has become progressively perfected with proteins. In its early form the ribosome may ilave been both inefficient and inaccurate. However, it has undoubtly been further perfectionated by natural selection. That accuracy of Biochim. Biophys. ,4cta, x99 (t97 o) 2o3-2t3

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code reading during translation is dependent upon the molecular organization of the ribosome (and not only on the specificity of tRNA and aminoacyl-tRNA synthetases) has been in fact convincingly (lemonstrated by the work of Gol~IXI a..xo KATaJA n and of STAEHFLIN AND MI:SELSON 1''. Firstly and obviously, evolution nlust have perpetuated those protein conlponents which offered the positive advantage of securing both fidelity and efficiency in the translation of the genetic script under several different sets of both intra- and extracellular environmental conditions. In this context it is of interest that (a) the ribosomes of extremely halophilic bacteria are stable in 4 M KCIa"; ihe.v contain a great majority of acidic proteins and a relatively smaller coml)lement of basic proteins ~a, and (b) whereas ribosomes of nwsophilic bacteria exhibit thermal denaturation at about 4 o', those of some thermophilic bacteria become denaturated at temperatures close to 7°° fief. z4). Secondly, evolution may have perpetuated nmtations affecting one or another protein component whenever they were advantageous to correct distorsions of molecular organization caused by attendant nmtations affecting other components of the ribosome. This contention is sustained by the data of RossI,:T axl) GORINI~ showing that (a) particular mutations occurring in the 3o-S subunit of Escherichia coli ribosomes introduce translational ambiguity t)ottl in vivo and in vitro, to a degree comparable to that brought about by streptomycin is, and (b) additional nmtations at the classical streptomycin locus, resulting in restriction of misreading caused by streptomycin, do also counterbalance aml)iguity; that is, a doubly inutated ribosome, both ambiguos and restricted, may function similarly to the wild-type ribosome, in contrast to ribosomes carrying either one or the other mutation ~a. In this connection it is of interest that at least the restrictive mutations seem to involve the protein moiety of the core fraction of the 3o-S subunit lz,a'~. On purely speculative grounds two additional mechanisms should be envisaged as possible ew)lutionary events contributing to the present ribosome heterogeneity: (a) the consolidation of structural alterations of the ribosomes leading to informational suppression an, and (b) the consolidation of ribosome mutations which offered the positiw.' advantage of correcting concurrent changes of components of the synthetic apparatus external to the ribosome (e.g., structural changes of the polymerizing enzymes leading to loss of their ability to fit with the peptide condensing site (c/. article by CIFk'RRI et al.tV)). As mentioned earlier, in E. coli the permission of specific violations of code translation caused by addition of stret)tomycin appears to be strictly dependent on the molecular organization of the 3o-S ribosomal subunit ~1"~2. By analogy it is conceivable that certain mutations, affecting components of the mitochondrial (7o-S) ribosomes of some antibiotic-t)roducing eucaryotes, may have offered the positive advantage of rendering the rit)osomes resistant to the action of the drug produced by the "host cells". Similarly, it appears likely that in plants, chloroplastic (7o-S) ribosonms may have become adapted to unfavorable conditions existing in the cytoplasm of the host cell. Vestiges of factor(s) inhibiting chloroplast protein synthesis are in fact, still found in the cytoplasmic fraction of some higher plants ~8. Nevertheless, it seems conceivable that, during speciation, additional evolutionary events (besides selection by positive adaptive value.) may have participated to some extent in producing such differences as are obserw:d among contemporary ribosomes. Thus, it is plausible that certain mutations affecting details of the primary Biochim. Biophys..4cta, 199 (197o) 2o3-2i 3

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structure of individual ribosomal proteins may have exerted no influence on the molecular organization of the ribosomes if they did not result in any drastic change of the overall shape and charge of the molecule. Such particular genetic accidents (of neutral adaptive value) may have been perpetuated if closely linked to other advantageous genetic traits. In this context, it seems possible that some of those protein components which appear more or less dissimilar in the electrophoretic patterns, may, in fact, represent closely related chemical entities derived one from the other by such simple events as the substitution of few amino acids or the insertion within the chain, of a short modified amino acid sequence. If it were so, the overall structure of the various ribosomal proteins in related plants might be relatively more conserved than would appear by electrophoretic comparisons. Dissimilarity betw,een 7o-.~ and 8o-S ribosomes in the same plant Our results indicate that the 8o-S ribosomal proteins of a given plant are more similar to the corresponding 8o-S ribosomal proteins of a distantly related plant, than to the 7o-S proteins of the same plant from which the 8o-S ribosomes were derived. The evolutionary significance of such striking dissimilarities as are found, may, perhaps, be explained if (a) credit is given to the view that eucaryotic plants are the result of ancient endosymbioses between a photosynthesizing procaryote and a heterotroph protozoan ~9, and if (b) it is assumed that the genome of the extant endosymbiontes (i.e., the photosynthetic plastids of both tile lower and the higher plants) still codes for the synthesis of chloroplast-specific ribosomal proteins. It is now known that both the I6-S and the 23-S rRNA of chloroplastic ribosomes are a product of the plastid genome "°. However, because of the limited dimensions of the DNA of most cytoplasmic organelles, the possibility nmst be taken into account that the synthesis of the several 7o-S proteins may have been deferred to the nuclear genome during the course of a prolonged endosymbiosis. A partial answer to this question may be given by determining the proportion of the total information which would be detracted from the plastid genome if ribosome synthesis were entirely directed by chloroplast DNA. Previous estimates 5, indicate that each chloroplastic (7o-S) ribosome, contains approx. 6o protein molecules of 2.3' lO4 daltons each 2~. If it is assumed that some proteins are reiterated in each ribosome, the number of genes necessary to code for the different kinds of ribosomal proteins would be lower than 6o, although not less than 3o (i.e., the actual number of bands seen upon electrophoresis) 5. Unfortunately, precise estimates of the amount of genetic information which can be encoded in a plastid genome are complicated by the fact that the amount of DNA per plastid varies considerably in different photosynthesizing eucaryotes, ranging from an amount of o.3- I.O. IO-~s g per organelle (as in the very small chlorol)last of Aeetabularia 22.2s) up to as nmch as 4.5" IO-15 g DNA per organelle (as in Chlorella 24 and Euglena 25)'. In addition it has been suggested that the relatively high proportion of DNA found in the chloroplasts of some photosynthesizing organisms could be accounted for by either polyteny or polyploidy 27 .a2. Therefore, although some plants contain a quantity of DNA equal to, or greater than, that of the single chromosome " Tobacco leaves exhibit up to 8-io-tSg D N A p e r p l a s t i d 2e. I n d i r e c t t h a t b e e t a n d s p i n a c h c o n t a i n a b o u t - . i o -t5 g I I N A p e r c h l o r o p l a s t 2s.

estimates

indicate

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of E. coli (7" I°-15 g, or 4" 109 daltons) 33 the actual genetic potentiality of their plastid genome should be reduced in proportion to ploidy. In the absence of certain essential data concerning the degree of ploidy of the plastid DNA of higher plants, no informed guess is possible as to the amount of protein which can be coded for by their chloropla,st genome. Nevertheless, there is convincing evidence (derived from target analysis of ultraviolet "bleaching" curves) that one chloroplast of Euglena (about 4.5" IO-lS g DNA) 2~ originates from the fusion of three proplastids which are present in the dark-grown cells ~. This fact suggests that, in Euglena, each chloroplast contains three distinct DNA units of about 1.5" IO-~5 g each (i.e., three chromosomes of 0. 9' IO9 daltons each). This amount of DNA is of the same order of magnitude as that contained in the minimum-sized ctdoroplast of Acetabularia (o.2.IO9-O.6 . l o 9 daltons). Thus, one might argue that the latter contains an haploid genome consisting of one chromosome of o.2.~o ~0.6. IO9 daltons, and that the plastids of most photosynthesizing eucaryotes contain multiple members of such DNA units ranging in size from 0.2. IO9 to o. 9. I(P daltons. With these considerations in mind, we may then proceed to estimate which proportion of a chrolnosome having the assumed dimensions would be involved in ribosome synthesis postulating that (a) similar to E. coli 3~.3e, each chloroplast chromosome contains ten cistrons* for each of the two inajor components of rRNA, and (b) each chromosome contains fifty different genes, each coding for one ribosomal protein of 23 ooo molecular weight. These numbers of rRNA cistrons and of ribosomal protein genes would constitute DNA segments of respectively 33" IO6 daltons** and 2o. IOe daltons. It follows that, under the assumed conditions, overall ribosome synthesis would involve approx. 2 ° o of one chromosome of o. 9- IO9 daltons (such as might be that of Euglena) or 9 °'o of one chromosome of o.2. IcP daltons (such as might be that of Acetabularia). Therefore, if our assumptions are not too far from real (and provided that ribosomal protein genes are not redundant) one may conclude that ribosome synthesis detracts only a small proportion of the total information encoded in the plastid genome, and, thus, it seems theoretically possible tbat ribosomal proteins are a product of organellar DNA.

Relative stability o~ the 8o-S patterns If one accepts the assumption that chloroplast ribosomal proteins are coded for by plastid DNA, the relatively greater stability of the 8o-S patterns as compared to the extreme variability of the 7o-S patterns might then be easily explained. Since plastids are numerous, selfreplicating and independently mutable 25, these facts alone would allow for more rapid evolutionary changes. For instance, clones of organelles, each characterized by some particular genetic trait, inay accunmlate in the cytoplasmZL Several independent nmtations affecting a given ribosomal protein, may arise in one or another plastid. These mutations (if of either positive or neutral adaptive value) may eventually be perpetuated under conditions favoring tile selection of the clone in which the nmtation has emerged. In this connection it should also • T e n c i s t r o n s f o r e a c h o f t h e t w o m a j o r c o m p o n e n t s of c h l o r o p l a s t o v e r e s t i m a t e d f i g u r e . 13y I i N A - D N A h y b r i d i z a t i o n m e t h o d s it h a s b e e n c h l o r o p l a s t D N A o f T o b a c c o l e a v e s , w i t h a s s u m e d d i m e n s i o n s of 2 . 8 . l o * four cystrons for I6-S ribosomal I{NA and four cistrons for z3-S ribosomal "• B a s e d o n t h e a s s u m p t i o n t h a t o n e 16-S a n d o n e 2 3 - S n a o l e c u l e of I I N A s u m t o r . 6 6 . i o ~ d a l t o n s ~.

Hiochim. Biophys. Acta, t 9 9 / 1 9 7 o ) 2 o 3 - z i 3

r i b o s o m a l R N A is a n e s t i m a t e d z0 t h a t t h e daltons, contains only RNA. chloroplast ribosomal

SPECIES-SPECIFICITY OF RIBOSOMAL PROTEINS

2I 3

be noted that the greater evolutionary versatility of the plastid genome, as compared to the nuclear genome, may have contributed to a certain extent in determining differences between the ribosomal proteins of chloroplastic and cytoplasmic ribosomes in the same plants. Finally, certain limitations to the interpretation of the present results should be pointed out. In fact, although differences in electrophoretic mobilities reveal that proteins are different, dissimilar proteins may migrate identically in the gel and some individual components might not be revealed. Therefore, estimates of dissimilarity, such as those presented here (c/. Table I) are intended to indicate merely electrophoretic heterology. Whether and to which extent the latter correlates with the actual degree of chemical heterology cannot be assessed until individual proteins are isolated and characterized. ACKNOWLEDGMENTS

We are indebted to Dr. Antonio Fantoni for critically reviewing the manuscript and to Mr. Antonino Romeo for his highly skillful technical assistance. REFERENCES 1 2 3 4 5 6 7 8 9 IO ii I2 13 14 15 16 17 18 i9 2o 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38

J. P. WALLER, J. Mol. Biol., io (i964) 319. M. DI GIROLAMO AND P. CAMMARANO, Biochim. Biophys. Acta, 168 (I968) 181. V. MUTOLO, G. GIUDICE, V. I t o e P s AND G. DONATUTI, Biochim. Biophys. Acta, 138 (1967) 214. R. B. I . o w AND I. G. WOOL, Science, 155 {i967) 33 ° . G. f;UALERZI AND P. CAMMARANO, Biochim. Biophys. Acta, I9O (r969) I7o. E. STUTZ AND H. NOLL, Proc. Natl. Acad. Sci. U.S., 57 (I967) 744. J. P. \VALLER AND J. I. HARRIS, Proc. Natl. Acad. Sci. U.S., 47 (I96I) 18. P. S. I,EBOY, E. C. Cox ANB J. G. FLAKS, Proc. Natl. Acad. Sci. U.S., 52 (I964) I367. F. H. C. CRICK, J. Mol. Biol., 38 (I968) 367 . I.. E. ORGEL, J. Mol. Biol., 38 (1968) 381. I.. GORINI AND E. KATAJA, Proc. Natl. Acad. Sci. U.S., 51 (I964) 487 . U. T. STAEtlELIN AND M. MESELSON, J. Mol. Biol., 19 (i966) 207. S. T. BAYLEY, J. Mol. Biol., 15 (1966) 420. M. T..'~IANGIANTINI, G. TECCE, G. TOSCHI AND A. TRENTALANCE, Biochim. Biophys. Acta, lO3 (1965) 252. R. ROSSET AND I.. GORINi, J. Mol. Biol., 39 (1969) 95. P. J. I(EID, E. ORXAS AND T. K. GARTNER, Biochim. Biophys. Res. Commun., 21 (I965) 66. O. CIFFRRI, B. PARISI, A. PERANI AND ~1. GRANDI, J. Mol. Biol., 37 (1968) 529 • •. K. BOARDMAN, I~.. I. B. FRANKI AND S. G. WILDMAN, J. Mol. Biol., 17 ti966) 47 o. L. SAGAN,J. Theoret. Biol., 14 (1967) 225. A. TEWARI AND S. G. WILDMAN, Proc. Natl. Acad. Sci. U.S., 59 (2968) 569 • W. M6LLER AND A. CRAMr)ACH, J. Mol. Biol., 23 (1967) 377. A. GIBOR AND M. IZAWA, Proc. Natl. Acad. Sci. U.S. 5 ° (1963) II64. E. BALTUS AND J. BRACHET, Biochim. Biophys. Acta, 76 (1963) 490. T. IV,'AMURA AND S. KUWASIIIMA, Biochim. Biophys. Acta, 174 (1969) 33 o. A. (_~IBOR AND S. GRANICK, Science, 145 (1964) 890. W. D. COOFER AND H. S. LORING, J. Biol. Chem., 228 (i957) 813S. GRANICK AND A. GIBOR, Progr. Nucleic Acid Res. Mol. Biol., 6 (1967) I43. J. T. O. KIRK AND T. W. GODV¢IN, Biochemistry o/Chloroplasts, Vol. I, Academic Press..New York, 1966, p. 324 . lI. RIS AND IV. PLAUT, J. Cell Biol., 13 (I962) 383 . B. E. S. GUNNING, J. Cell Biol., 24 (I965) 79N. KISLEV, H. SWIFT AND l.. BOGORAD, J. Cell Biol., 25 (I965) 327 . H. I,YMAN, I1. T. EPSTEIN AND J. A. SCHIFF, Biochim. Biophys. Acta. 5 ° ( I 9 6 I ) 3oi. A. D. HERSHEY AND H. E. MELECHEN, Virology, 3 (1957) 207. Y. BEN-SttAUL, J. A. SCHIFF AND It. T. EPSTEIN, Plant Physiol., 39 (1964) 231. S. A. YANKOFSKY AND S. SPIEGELMAN, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1466. D. DUBNAU, J. SMITH, P. MORELL AND J. MARMUR, Proc. Natl. Acad. Sci. U.S., 54 (I965) 49I. 17. E. LOENING, J. Mol. Biol., 38 (I968) 355. S. T. ]3AYLEY, J. Mol. Biol., I6 (1966) 33 o.

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