Protein synthesis in chloroplasts IV. Polypeptides of the chloroplast envelope

143

Biochimica et Biophysica Acta, 378 ( 1 9 7 5 ) 1 4 3 - - 1 5 1 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 98194

PROTEIN SYNTHESIS IN CHLOROPLASTS IV. POLYPEPTIDES OF THE CHLOROPLAST ENVELOPE

K E N N E T H W. J O Y * a n d R. J O H N E L L I S

Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, Warwickshire (U.K.) (Received July 5th, 1974)

Summary Envelope membranes were isolated from washed chloroplasts of pea seedlings. As judged by the protein-to-chlorophyll ratio, average preparations contain less than 8% contamination with internal lameUar membranes. Electrophoresis on sodium dodecylsu!phate polyacrylamide gels shows that the envelope membranes contain at least 25 polypeptides. The molecular weight distribution of the envelope polypeptides is different from that of the lamellar polypeptides, there being more polypeptides of molecular weights above 50 000 in the envelopes. Two envelope polypeptides become labelled when isolated intact chloroplasts are incubated in the light with [3sS] methionine. One of these is similar in molecular weight to the main polypeptide labelled in lamellae, but the other is unique to the envelope fraction. Incorporation of label into both polypeptides is totally light
Introduction Previous papers in this series have shown that isolated intact pea chloroplasts will use light energy to incorporate labelled amino acids into discrete proteins [1,2]. Although chloroplasts contain up to 50% of the total ribosomes

* Permanent address: Biology Department, Carleton University, Ottawa, KIS 5 B6, Canada.

144 in a leaf, the synthetic system of chloroplasts does not appear to produce a large number of chloroplast proteins. Isolated chloroplasts synthesise the large subunit of Fraction I protein, as the sole detectable soluble product [1], and five unidentified proteins of the internal lamellar system of the chloroplast [2 ]. The conclusion that chloroplast ribosomes make a restricted range of proteins is confirmed by inhibitor experiments with intact cells [3]. The development of techniques for the isolation of chloroplast envelopes by Mackender and Leech [4] has opened up possibilities for studying the components of the bounding membrane of chloroplasts. This envelope is involved in controlling the transport of metabolites [5--9] and possibly proteins [10]. Several papers on the lipid composition of chloroplast envelopes have appeared [ 1 1 - - 1 4 ] . In this paper we describe the polypeptide composition of pea chloroplast envelopes, and show that two of these polypeptides are synthesised by chloroplast ribosomes. Materials and Methods In addition to the buffers described in previous Work [1,2], tricine resuspension buffers were used (66 mM tricine--KOH, 50 mM KC1, 6.6 mM MgC12 ) containing various amounts of sucrose, and adjusted to either pH 7.5 or pH 8.3. The pH was adjusted at r o o m temperature, but unless otherwise stated, all other operations were carried out at 0--4°C.

Isolation of chloroplasts Ten-day-old pea seedlings (Pisum sativum, var. Feltham First) were grown and homogenised as described previously [1], except that 20 g leaf tissue was homogenised in 100 ml sucrose isolation medium with t w o bursts of 4 s homogenisation, and the homogenate was squeezed through 2 layers of muslin and then allowed to drain through a single layer of Miracloth (Calbiochem). After centrifugation for 2 min at 2500 × g, the crude chloroplast pellet was resuspended using a small cotton-wool swab in either (a) 10 ml KC1 resuspension medium [1] if it was to be incubated for amino acid incorporation studies, or (b) 26 ml tricine resuspension buffer (pH 8.3) containing 0.4 M sucrose for direct envelope preparation.

Purification of chloroplast envelopes The m e t h o d was modified from the procedures of Mackender and Leech [4] and Poincelot [12]. Crude chloroplasts, either freshly prepared or after incubation, were washed by centrifugation for 7 min at 2750 × g through a layer (about 3.5 cm deep in 50 ml tube) of tricine resuspension buffer (pH 7.5) containing 0.98 M sucrose. The washed chloroplasts were resuspended in 25 ml bursting medium (60 mM tricine--KOH, 25 mM KC1, 3.5 mM MgC12, pH 7.5), allowed to stand for 10 min, and then disrupted with 6 strokes of a Ten-Broek all-glass homogeniser. Lamellae were removed from the suspension b y t w o centrifugations for 3 min each at 3500 × g. A crude envelope preparation was sedimented from the supernatant by centrifugation for 25 min at 38 000 × g. The crude envelopes were resuspended in 7 ml tricine resuspension buffer (pH 7.5) containing 0.4 M sucrose, and layered on a gradient consisting of 7 ml

145 tricine resuspension buffer (pH 7.5) containing 1.05 M sucrose and 2 ml tricine resuspension buffer (pH 7.5) containing 0.81 M sucrose. The gradient was centrifuged for 40 min at 64 000 × g in a swing-out head. A cloudy yellow band containing envelopes appears at the interface b e t w e e n the 1.05 M and 0.81 M sucrose, while most contaminating green lamellae sediment to the b o t t o m of the tube. The envelope layer was carefully removed with a pipette, diluted with an equal volume of tricine resuspension buffer (pH 7.5) and centrifuged at 38 000 × g for 25 min. The envelope pellet was taken up in 0.7 ml tricine resuspension buffer (pH 7.5) or electrophoresis sample buffer [2]. Lamellae were washed twice in tricine resuspension buffer (pH 7.5) and resuspended in 10 ml tricine resuspension buffer (pH 7.5) or electrophoresis sample buffer.

Gel electrophoresis and counting Electrophoretic fractionation of membrane polypeptides on 15% polyacrylamide gels with sodium dodecylsulphate--urea buffers, staining of gels in Coomassie Blue, and measurement of radioactivity in 1 mm gel slices, was carried o u t as described b y Eaglesham and Ellis [2]. Membrane samples were prepared for electrophoresis b y the addition of sodium dodecylsulphate to a final concentration of 2%, followed b y heating in a boiling water bath for 1 min. This procedure results in the disappearance of the Photosystem I protein band [10].

Amino acid incorporation Chloroplasts were prepared as described, b u t with sterilised solutions and glassware. A b o u t 500 pCi L-[ ~ s S] methionine (120--140 Ci/mmole) was added to 10 ml crude chloroplast suspension containing a b o u t 5 mg chlorophyll, which was then incubated in a 50 ml flask at 20°C for 45 min with red light illumination (4000 lux) from underneath. At the end of the incubation, 1 ml saturated [ 3:S] methionine was added, and envelopes and lamellae prepared from the chloroplasts as described, beginning at the wash stage.

In vivo labelling of chloroplast envelope proteins Stems of l l < l a y - o l d pea seedlings were cut off at soil level, and the ends recut under water. Each shoot was placed in a vial containing 10 ml sterile water with 50 /zCi L-[ 3 s S] methionine with or without 2 pg/ml cycloheximide, and left for 45 h under continuous white light at 14 000 lux. After 24 h a b o u t 7 ml of s o l u t i o n h a d been taken up, and the vials were t o p p e d up with water. At 45 h the leaf tissue from five shoots for each treatment was added to 18 g unlabelled leaf material, and chloroplast envelopes were isolated.

Assay methods Protein was estimated b y the Lowry m e t h o d [15] and chlorophyll b y the m e t h o d of Arnon [16]. However, the latter m e t h o d is n o t sensitive enough to detect the small amounts of chlorophyll present in the envelope fractions witho u t destruction of the whole sample. Therefore, direct absorbance readings of the envelope suspension were made in a microcuvette, and the chlorophyll content obtained from the value of A6 ~ 2 n m--As s 0 rim. This m e t h o d was calibrated with dilutions of a lamellar preparation of known chlorophyll con-

146 tent. This method allows measurement of chlorophyll in the range 0.5 to 15 #g/ml, b u t is usable only for preparations in which the chlorophyll is present as lamellar fragments. Fumarase was assayed by following the change in A: 90 , m [17].

Electron microscopy A pellet of purified envelopes was fixed with 5% glutaraldehyde in 0.025 M potassium phosphate (pH 7.4) containing 0.3 M sucrose. The pellet was post-fixed in 1% osmic acid, dehydrated with ethanol, and e m b e d d e d in Spurr's resin. Sections were stained with uranyl acetate and lead citrate, and examined in an A.E.I. Corinth microscope. Results and Discussion

Purification of pea chloroplast envelopes The methods described above were selected after trial of a number of variations and give a yield of a b o u t 350 tzg envelope protein from 20 g leaf tissue. The washed chloroplast fraction contains 35=-40% of the chlorophyll in the total leaf homogenate, and 50--70% of these chloroplasts appear to be intact as judged by their appearance under phase microscopy. Microscopic observations of various fractions during the purification procedure suggest that envelopes are being removed and purified. Light microscopic inspection of the chloroplasts during the bursting stage confirms that the envelopes balloon into halos surrounding the granal mass. In some cases the halos detach and pinch off into a single large vesicle, or burst completely. The preparation of crude envelopes contains a mixture of lamellae, large clear vesicles, and many small fragments. After the final purification step, the pellet consists of small fragments with a few intact lamellae. Electron microscopic examination of sections of pellets of purified envelopes reveals many membrane fragments and single and double layered vesicles (Fig. 1). The ratio of protein/chlorophyll is a useful parameter for judging the purity of envelope preparations if it is assumed that the envelope contains no chlorophyll. This ratio is a b o u t 5.5/1 in lamellar preparations from pea chloroplasts; Mackender and Leech [4] found a ratio of a b o u t 1/1 for lamellae from Vicia faba chloroplasts, so this ratio varies between species. The ratio for purified envelope fractions varied in different experiments and appears to depend on the precision of removal of the cloudy layer from the high speed sucrose gradient. With careful sampling, protein/chlorophyll ratios as high as 180/1 are obtained, although the total yield of material is low. For most experiments with the average yield of 300--400 pg protein, ratios of 65/1 to 85/1 are obtained. These ratios indicate that from 6 to 8% of the preparation consists of contaminating lamellae; Mackender and Leech [4] found 8--16% contamination in envelopes from V. faba chloroplasts, while Poincelot [12] reported that spinach envelope preparations contained only 4% lamellar contamination. Fumarase activity is detectable in the crude chloroplast suspension but n o t in the purified fractions, indicating that the preparations have little contamination with mitochondria. Many of the mitochondria are removed by washing the chloroplasts [4].

147

Fig. 1. E l e c t r o n m i c r o g r a p h o f a s e c t i o n of a p u r i f i e d c h l o r o p l a s t e n v e l o p e p r e p a r a t i o n . T h e m a r k e r e q u a l s 0.25 ~ m .

148 A

15 B

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10 11

16

200

21000L r~ 500E u

14

&

0

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2's

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100 50 25 M o l e c u l e r w e i g h t x 10 -3

Molecular weight x 10 ,3

Fig. 2. S o d i u m d o d e c y l s u l p h a t e p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f l a m e l l a e a n d e n v e l o p e s . I s o l a t e d pea c h l o r o p l a s t s w e r e i n c u b a t e d w i t h light and [ 3 5 S ] m e t h i o n i n e a n d f r a c t i o n a t e d i n t o lamellae and e n v e l o p e s as d e s c r i b e d in Materials and M e t h o d s . Gel A was l o a d e d w i t h S 0 # g lamellar p r o t e i n a n d gel B w i t h 1 0 0 # g e n v e l o p e p r o t e i n . T h e c o n t i n u o u s curve r e p r e s e n t s a b s o r b a n c e at 6 2 0 n m and t h e h i s t o g r a m s h o w s r a d i o a c t i v i t y o f 1 m m gel slices. T h e m o l e c u l a r w e i g h t scale w a s derived f r o m calibrated gels as d e s c r i b e d p r e v i o u s l y [ 2 ] , a n d t h e b a n d s are n u m b e r e d in a c c o r d a n c e w i t h t h e s y s t e m o f E a g l e s h a m a n d Ellis [ 2 ] .

Polypeptide composition of chloroplast envelopes Envelope and lamellar fractions were treated with sodium dodecylsulphate, and the dissociated polypeptides separated by gel electrophoresis. Typical patterns are shown in Fig. 2. The absorbance traces to not show all the visible subtleties of the gels, and only the major stained bands are numbered, following our earlier scheme [ 2 ] . S o m e of the bands, e.g. band 8, contained t w o or more bands which could be resolved by eye. The envelope fraction contains at least 25 stainable bands. Incubation of envelopes with pronase results in loss of all the bands, confirming their protein nature (Fig. 3).

E E

oJ 400

~

200

f

E u

2~

Electrophoretic

8

mobility (cm)

Fig. 3. S o d i u m dodecylsulphate polyacryla.mide gel electrophoresis of a pronase-treated envelope fraction p r e p a r e d f r o m i s o l a t e d c h l o r o p l a s t s a f t e r i n c u b a t i o n w i t h [ 3 5 S ] m e t h l o n i n e . B e f o r e t r e a t m e n t w i t h sod i u m d o d e c y l s u l p h a t e , labelled e n v e l o p e s w e r e i n c u b a t e d w i t h p r o n a s e at 2 0 # g / m l for 2 h at 37°C. T h e c o n t i n u o u s curve r e p r e s e n t s a b s o r b a n c e at 6 2 0 n m , and t h e h i s t o g r a m s h o w s r a d i o a c t i v i t y o f 2 m m gel slices.

149 The polypeptide patterns from envelope and lamellar fractions are markedly different, although some bands are c o m m o n to b o t h fractions. The polypeptides can be divided into three groups: (i) those characteristic of the envelope fraction, especially the slow-running polypeptides of molecular weights above 50 000 (such as bands 1, 4, 5, 6) and band 20; (ii) those characteristic of lamellae, and which are greatly decreased in the envelope fraction, such as bands 7, 11, 16, 19, 21; (iii) bands which appear in b o t h fractions, such as 8, 14 and 15. The relative contribution of various polypeptides to the overall composition of. each fraction was estimated by cutting out and weighing tracings of individual bands, and averaging results of several experiments. It was calculated that lamellar contamination is n o t sufficient to give the a m o u n t of bands 8 and 15 which are found in envelope preparations. Polypeptides of these mobilities are thus characteristic of b o t h fractions, b u t it is n o t known whether polypeptides with the same mobility from both fractions have the same primary structure. The fact that band 8 is labelled in the envelope fraction, b u t n o t in the lamellar fraction (see below), suggests that it contains different components of the same mobility.

In vitro labelling of chloroplast envelope proteins Label from [ a s s ] methionine is incorporated into membrane proteins when chloroplasts are incubated with light as energy source. Examples of the distribution of the label in lamellar and envelope fractions are shown in Fig. 2. As reported previously [2], one major peak of labelled polypeptide is found in lamellae (coincident with band 14), and several minor peaks (Fig. 2A). In contrast, the envelope fraction (Fig. 2B) contains t w o major labelled peaks, one of molecular weight a b o u t 32 000, similar to that in lamellae, and a second peak coincident with band 8 (molecular weight a b o u t 65 000). Calculations show that the labelling in band 14 of the envelope fraction could n o t be accounted for by lamellar contamination, and thus b o t h labelled peaks seem to be characteristic of envelope membranes. Incubation of chloroplasts in the dark gives no incorporation into either envelope or lamellar fractions, indicating that bacterial contamination is n o t responsible for the incorporation. Fig. 3 shows the result when labelled envelopes are incubated with pronase prior to electrophoresis. Both the absorbance trace and the labelled peaks are lost, indicating that the labelled peaks contain protein. Isolated chloroplasts incorporate labelled amino acids n o t only into membrane proteins b u t also into a major soluble protein, the large subunit of Fraction I protein [1]. As this protein is found to run on gel electrophoresis at a b o u t the same position as band 8, it is possible that the labelled peak in this position from the envelope fraction is large subunit trapped in the envelope vesicles. This possibility was ruled out by t w o experiments. Firstly, sonication of the envelope membranes for 20 s, followed by washing, had no effect on the labelling pattern. Secondly, a labelled envelope fraction was analysed electrophoretically after the addition of purified unlabelled large subunit of pea Fraction I protein. The gel was run for twice the usual time, and the result is shown in Fig. 4. The band of added large subunit is clearly separated from peak 8. A small plastic marker was inserted into the intervening space b e t w e e n the bands before the gel was sliced to allow positive identification of this slice during the

150 I

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15

LSU i

1

4

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Electrophocetic mobility (cm)

Fig. 4. C o - e l e c t r o p h o r e s i s of l a b e l l e d c h l o r o p l a s t e n v e l o p e f r a c t i o n w i t h p u r i f i e d large s u b u n i t of F r a c t i o n I p r o t e i n . T h e gel was r u n f o r t w i c e t h e u s u a l t i m e . T h e d o t t e d line i n d i c a t e s t h e 1 m m slice c o n t a i n i n g a m a r k e r i n s e r t e d in t h e gel b e t w e e n b a n d 8 a n d t h e large s u b u n i t b a n d ( L S U ) b e f o r e slicing. S y m b o l s as in Fig. 2. Fig. 5. S o d i u m d o d e c y l s u l p h a t e p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of c h l o r o p l a s t e n v e l o p e s labelled in vivo. D e t a c h e d pea s h o o t s w e r e f e d w i t h [ 35S] m e t h i o n i n e w i t h a n d w i t h o u t c y c l o h e x i m i d e at 2 # g / m l as d e s c r i b e d in Materials a n d M e t h o d s , and e n v e l o p e f r a c t i o n s t h e n prepared. T h e solid h i s t o g r a m r e p r e s e n t s r a d i o a c t i v i t y of i m m gel slices of t h e c o n t r o l , t h e d o t t e d h i s t o g r a m r e p r e s e n t s r a d i o a c t i v i t y of 1 m m gel slices of the f r a c t i o n f r o m the c y c l o h e x i m i d e - t r e a t e d tissue. O t h e r s y m b o l s as in Fig. 2.

counting of the gel. It can be seen that the labelled peak remains coincident with peak 8, and does n o t run with the large subunit of Fraction I protein.

In vivo labelling of chloroplast envelope proteins The simplest interpretation of the results so far described is that only two of the chloroplast envelope polypeptides are synthesised by chloroplast ribosomes. By inference, the remainder are synthesised on cytoplasmic ribosomes. These conclusions are supported by the results of an in vivo inhibitor experiment. Detached pea shoots were supplied with [3SS]methionine in the presence and absence of cycloheximide at 2 pg/ml as described in Materials and Methods. Envelopes were prepared from the labelled leaves, and the labelling of the envelope polypeptides is shown in Fig. 5. In the absence of cycloheximide, label is incorporated into most of the envelope polypeptides. In the presence of cycloheximide much of this incorporation is reduced, and the labelling pattern now resembles that f o u n d in the in vitro experiments, with the exception of an additional minor peak coincident with band 11. Since cycloheximide inhibits protein synthesis by cytoplasmic, but not by chloroplast, ribosomes in this tissue [3], this result reinforces the conclusion reached from the experiments with isolated chloroplasts. The results presented in this paper show that the bounding double membrane of the chloroplast has a complex polypeptide composition which is markedly different from that of the internal lamellae. It seems reasonable to suggest that the polypeptides of the envelope are concerned with the regulation of the transport of m a n y low molecular weight metabolites, as well as proteins synthe-

151 sised on cytoplasmic ribosomes, across the envelope; therefore t h e y may be expected to differ from those of the internal lamellae which are concerned with converting radiant energy into chemical energy. There is electron microscopic evidence which suggests t h a t the internal lamellae are produced by the invagination of the inner membrane of the chloroplast envelope [18]. We conclude t h a t the synthesis of the internal lamellae is n o t simply an extension of the synthesis of the envelope but involves a major change in the types of proteins inserted into the growing membrane. The evidence from both in vivo and in vitro experiments suggests that at least two, and possibly three, of the envelope polypeptides are synthesised on chloroplast ribosomes, but that the remainder are synthesised on cytoplasmic ribosomes. This division of labour resembles the situation for the polypeptides of the internal lamellae, some of which are made inside the chloroplast, and some outside [2,3], and adds more weight to the conclusion that chloroplasts are in no sense a u t o n o m o u s [10]. The question is raised as to whether the envelope polypeptides t h a t are synthesised inside the chloroplast are also encoded in the chloroplast DNA; such a situation would conform to our model for the synthesis of Fraction I protein [19], but the validity of this suggestion can be established only from genetic studies. We suggest as a line of investigation a search among plastome mutants for chloroplasts which have altered or missing envelope polypeptides. Acknowledgements This work was carried out while K.W.J. was on sabbatical leave from Carleton University, and a travel grant from the National Research Council of Canada is gratefully acknowledged. We also t h a n k Elizabeth M. Ballantine for the preparation of electron micrographs and Elizabeth E. Forrester for technical assistance. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Blair, G.E. a n d Ellis, R . J . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 9 , 2 2 3 - - 2 3 4 E a g l e s h a m , A . R . J . a n d Ellis, R.J. ( 1 9 7 4 ) Bioehim. B i o p h y s . A c t a 3 3 5 , 3 9 6 - - 4 0 7 Ellis, R . J . ( 1 9 7 4 ) P h y t o c h e m i s t r y , in t h e press M a c k e n d e r , R . O . a n d Leech, R.M. ( 1 9 7 0 ) N a t u r e 2 2 8 , 1 3 4 7 - - 1 3 4 8 Bassham, J.A., Kirk, M. a n d J e n s e n , R . G . ( 1 9 6 8 ) Biochim. B i o p h y s . A c t a 1 5 3 , 2 1 1 - - 2 1 8 Heldt, H.W. ( 1 9 6 9 ) FEBS Lett. 5, 1 1 - - 1 4 Heldt, H.W. a n d R a p l e y , L. ( 1 9 7 0 ) FEBS Lett. 10, 1 4 3 - - 1 4 8 Heldt, H.W. a n d Sauer, F. ( 1 9 7 1 ) B i o c h i m . B i o p h y s . A c t a 2 3 4 , 8 3 - - 9 1 Werden, K. a n d Heldt, H.W. ( 1 9 7 2 ) Bioehim. Biophys. A c t a 2 8 3 , 4 3 0 - - 4 4 1 Ellis, R . J . , ( 1 9 7 4 ) M e m b r a n e Biogenesis: M i t o e h o n d r i a , C h l o r o p l a s t s a n d Bacteria (A. T z a g o l o f f , ed.), P l e n u m Publishing Co., New Y o r k , i n t h e p r e s s D o u c e , R., Holtz, R.B. a n d Benson, A.A. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 7 2 1 5 - - 7 2 2 2 P o i n c e l o t , R.P. ( 1 9 7 3 ) A r c h . B i o c h e m . B i o p h y s . 1 5 9 , 1 3 4 - - 1 4 2 D o u c e , R. (1974} Science 1 8 3 , 8 5 2 - - 8 5 3 M a c k e n d e r , R . O . a n d Leech, R.M. ( 1 9 7 4 ) P l a n t Physiol. 53, 4 9 6 - - 5 0 2 L o w r y , O . H . , R o s e b r o u g h , N.J., F a r r , A.L. a n d R a n d a l l , R . J . ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 A r n o n , D.I. ( 1 9 4 9 ) P l a n t Physiol. 24, 1 - - 1 5 R a c k e r , E. ( 1 9 5 0 ) B i o c h i m . B i o p h y s . A e t a 4, 2 0 - - 2 7 M u h l e t h a l e r , K. a n d Frey-Wyssling, A. ( 1 9 5 9 ) J. B i o p h y s . B i o c h e m . C y t o l . 6, 5 0 7 - - 5 1 2 Ellis, R . J . ( 1 9 7 4 ) B i o c h e m . Soc. Trans. 2, 1 7 9 - - 1 8 2

152

VOLUME CODING FOR BBA--NUCLEIC ACIDS & PROTEIN SYNTHESIS B B A is p u b l i s h e d of the journal:

according

for 1975

to a volume-numbering

the scheme

(covering

scheme

the volumes

that embraces

375--417)

inside cover of this issue. The seven individual sections are distinguished addition system

to the runs

colour

parallel

SYNTHESIS

section

code,

to the the

therefore,

BIOCHIMICA

PROTEIN

SYNTHESIS

each section overall BBA

is g i v e n its o w n s e q u e n t i a l scheme:

correspondence

for the

is i n d i c a t e d

ET BIOPHYSICA

ACTA,

in

all s e c t i o n s

is t o b e f o u n d

on the

by a colour code. In volume

number.

NUCLEIC

ACIDS

& PROTEIN

the

below.

T h i s i s s u e is,

Vol. 378/1

Table

or BBA--NUCLEIC

This

ACIDS

&

N68/1

Parallel v o l u m e c o d i n g f o r B B A - N u c l e i c A c i d s & P r o t e i n S y n t h e s i s

Biochimica et Biophysica Acta V o l u m e No.

Nucleic Acids & Pro tein S y n thesis V o l u m e No.

B i o c h i m i c a et Biophysica Acta V o l u m e No.

Nucleic Acids & Protein S y n thesis V o l u m e No.

Biochimica et Biophysica Acta V o l u m e No.

Nucleic Acids & Protein Synthesis V o l u m e No.

Vol. Vol. VoL Vol. VoL Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. VoL

N 1 (1960-1961)

Vol. VoL Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. VoL Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. VoL Vol. Vol. Vol. VoL

N24 N25 N26 N27 N28 N29 N30 N31 N32 N33 N34 N35 N36 N37 N38 N39 N40 N41 N42 N43 N44 N45 N46 N47 N46 N49

Vol. VoL Vol. Vol. Vol. Vol. VoL VoL Vol. Vol. VoL Vol. Vol. VoL VoL Vol. Vol. Vol. VoL Vol. Vol. Vol. Vol. Vol. VoL

NSO N51 N52 N53 N54 N55 N56 N57 N58 N59 N60 N61 N62 N63 N64 N65 N66 N67 N68 N69 N70 N71 N72 N73 N74

45/1 ~ = 47/1 ] 49/1 } 51/1 ~ = 53/1 ] 55 = 61 = 68 = 72 = 76 = 80 = 87 = 91 = 95 = 103 = 108 = 114 = 119 = 123 = 129 = 134 = 138 = 142 = 146 = 149 = 155 =

N 2 (1961) N 3 N 4 N 5 N 6 N 7 N 8 N 9 NIO Nll N12 N13 N14 N15 N16 N17 N18 N19 N20 N21 N22 N23

(1962) (1962) (1963) (1963) (1963) (1964) (1964) (1964) (1965) (1965) (1965) (1966) (1966) (1966) (1966) (1967) (1967) (1967) (1967) (1967) (1968)

157 161 166 169 174 179 182 186 190 195 199 204 209 213 217 224 228 232 238 240 246 247 254 259 262 269

= = = = = = = = = = = = = = = = = = = = = = = = = =

(1968) (1968) (1968) (1968) (1969) (1969) (1969) (1969) (1969) (1969) (1970) (1970) (1970) (1970) (1970) (1970) (1971) (1971) (1971) (1971) (1971) (1971) (1971) (1972) (1972) (1972)

272 277 281 287 294 299 308 312 319 324 331 335 340 349 353 361 366 374 378 383 390 395 402 407 414

= = = = = = = = = = = = = = = = = = = = = = = = =

(1972) (1972) (1972) (1972) (1973) (1973) (1973) (1973) (1973) (1973) (1973) (1974) (1974) (1974) (1974) (1974) (1974) (1974) (1975) (1975) (1975) (1975) (1975) (1975) (1975)

A subscription to the NUCLEIC ACIDS & PROTEIN SYNTHESIS section of BBA for 1 9 7 5 ( 7 v o l u m e s ) is D f l . 6 8 6 . 0 0 ( U S $ 2 7 4 . 4 0 ) ( i n c l u d i n g p o s t a g e ) . B a c k v o l u m e s ( a c c o r d i n g to their N numbers) are available: rates will be supplied on request.