SIZE OF POLYRIBOSOMES IN INTACT RETICULOCYTES*BY RICHARD A. RIFKIND,t Lucio LUZZATTO,t AND PAUL A. AIARKS

DEPARTMENT OF MEDICINE, COLUMBIA UNIVERSITYCOLLEGE OF PHYSICIANS AND SURGEONS

Communicated by Dickinson W. Richards, September 8, 1964

Protein biosynthesis, in a wide variety of cells, occurs upon cytoplasmic ribosomeclusters termed polyribosomes.'-7 On the basis of considerations of polyribosomesize and the chain length of the protein product, it has been suggested that the sizeof a polyribosome is principally determined by the length of its associated mRNA.For example, it has been suggested that 5 ribosomes would fit upon a strand ofmRNA of a length appropriate to code for a globin chain, assuming a coding ratioof 3 nucleotides per amino acid.2 The observed high proportion of polyribosoillescontaining 4-6 ribosomes in reticulocytes synthesizing globin is adduced in supportof this hypothesis. It has been previously reported8' 9 that in sectioned intactreticulocytes, examined by electron microscopy, there are recognizable ribosomeclusters containing 2-6 individual ribosomes, correlating closely with similar ob-servations derived from ultracentrifuge studies. Occasionally, fine filaments con-necting the ribosomes were observed. However, the high background density ofhemoglobin in these sections obscures these structures and may, in particular, hidefine filaments connecting adjacent ribosome clusters. The present studies examinein greater detail the ultrastructure of the filament-polyribosome complexes, andevidence is presented that reticulocytes contain polyribosomes containing as manyas 20 individual ribosomes.

Materials and Methods.-Reticulocytes from phenylhydrazine-treated rabbits'0were subjected to gradual osmotic hemolysis according to the method of Danonet al."1 Such hemoglobin-poor reticulocytes were fixed (1% glutaraldehyde fol-lowed by 1% osmium tetroxide; ref. 9), embedded in epoxy resin, sectioned, andexamined in the electron microscope. Reticulocyte lysates were prepared andanalyzed by centrifugation through a sucrose density gradient as previously de-scribed.1 Fractions from these gradients were applied, at suitable dilutions, toformvar-coated electron microscope grids, washed, and fixed with 1% glutaralde-hyde, then washed, dried, stained with uranyl acetate (1% aqueous, for 6-12 hr),and examined in the electron microscope. This method of preparation and fixationof isolated polyribosomes has the advantage of avoiding the adventitious aggrega-tion of ribosomes which was observed after prefixation with either formalin'2 orglutaraldehyde.Results.-A portion of a thin-sectioned reticulocyte from which much of the

hemoglobin has been removed by gradual osmotic hemolysis is illustrated in Figure1. The plasma membrane (pm) is grossly intact; swollen mitochondria (m),vesicles (v), and polyribosomes (r) are apparent within the reticulocyte ghost, Inthese preparations polyribosomes can be seen which consist of 10 or more individualribosomes associated with a fine filament, 2500 A or more in length (Fig. 2A and B).It is apparent that the ribosomes are not generally distributed in a uniform manneralong the length of the filaments. Rather, small clusters of 2-6 ribosomes areseparated by lengths of bare filament. Failure to discern these short bare portionsof filament against the background of hemoglobin probably explains why the large

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U.~~~;

~~ . ~ W

FIG. 1. Electron micrograph of a portion of a reticulocyte after gradual osmotic hemolysis.The plasma membrane (pm), swollen mitochondria (in), vesicles (v), and polyribosomes (r) areapparent. Magfication 48,00X.

FIG. 2. (A and B) Large polyribosomes from the cell illustrated in Fig. 1. Fine filamentsconnecting the ribosomes are indicated by arrows. Magnification 150,000 X.

polyribosomes were not recognized with previously employed techniques 8 9 Sinceeach section is approximately 600 A thick, the chances of visualizing randonmlyoriented structures of this length are extremely poor, and their size is underesti-mlated.8' 9 The fact that filament-associated clusters of this size are encountered in

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CONTROL 0RNAs0.4- 0.4 +DOC *R|A S

J~~~~~~~~~~~~~~~~~~~~~~~~~~~~~JO0 0.2- o02 6 0.2

6

10 20 30 °0 20 30 l0 20 30FRACTION Nu116R FRACTION NURER FRACTIO NJ140R

FIG. 3.-Sucrose gradient analysis of total reticulocyte lysates. One ml of stroma-free lysatewas layered on a 27-ml 15-35% sucrose gradient, prepared in 0.01 M KCI, 0.001 M MgCl2, 0.01 MTris, pH 7.4. Centrifugation was performed for 120 min at 30C at 25,000 rpm in the SW25.1rotor of the Spinco ultracentrifuge, model L2. At the end of the run the gradients were drippedthrough the flow-cell (2-mm light path) of the Gilford multiple absorbance recorder, model 2000.The full scale setting was 0.1 OD units at 260 m1A. Tendrop fractions were collected. (A) con-trol; (B) treated with sodium deoxycholate (5 mg/ml) for 30 see before layering; (C) treatedwith pancreatic ribonuclease (Worthington) (0.25 jug/ml) for 1 hr at 0C before layering. In eachgradient the peak at fraction 32 corresponds to approximately 80S.

these sections suggests that such structures may be relatively frequent in reticulo-cytes.The large polyribosomes, unless damaged in preparation, should be recoverable

from reticulocyte hemolysates following ultracentrifugation through a sucrose den-sity gradient. Examination by electron microscopy of the more rapidly sedi-menting polyribosomes recovered from a sucrose density gradient (Fig. 3A, fraction7) reveals structures containing as many as 20 individual ribosomes (Fig. 4A andB). In this rapidly sedimenting fraction a broad range of polyribosome sizes isobserved, as determined by electron microscopy (Fig. 5A). Over 16 per cent ofthe polyribosomes in this region contain 15-20 individual ribosomes. In additionto 8 per cent of monoribosomes, at least 3 and possibly 4 modal concentrations ofribosomes in polyribosomes of sizes 4-6, 9-10, 14-16, and possibly 18-20, are ap-parent in Figure 5A. The presence of single ribosomes and the smaller polyribo-somes in the rapidly sedimenting fractions indicates that fragmentation of the largerpolyribosomes has occurred during preparation of the grids. Such breakage couldoccur preferentially at the bare, ribosome-free areas of the filaments, to yield clusterscontaining approximately 5 ribosomes and multiples thereof. Fragmentation ofthe larger polyribosomes may occur, as well, during all stages of preparation. If itoccurs prior to centrifugation, it would result in an artifactually high recovery ofsmall polyribosomes of sizes 4-6, which constitute the large population sedimentingin the 170-200S region (Fig. 5B).

In comparison to the large polyribosomes, polyribosomes of sizes 4-6 appear tobe a more stable structure. This is indicated by electron microscopic examinationof the fraction from the sucrose gradient corresponding to the maximum of the opti-cal density curve in the polyribosome region (Fig. 3A, fraction 21). Analysis ofthis fraction reveals that over 70 per cent of the ribosomes are in clusters of 4 or 5

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F ''while less than 1 per cent aremonoribosomes (Fig. 5B). This

2. ~~~~~~~~~homogeneity would suggestlot.E..v that polyribosomes of this size

are relatively resistant to thoseforces which quite readily dis-rupt the larger polyribosomes.

X: i

A..B To ascertain the nature ofFIG. 4. Large polyribosomes recovered by sucrose den the structure or structures

sity gradient centrifugation of reticulocyte hemolysate. maintaining the large ribosomeThese polyribosomes, containing 20 (A )and 17 (B) ribo .rsomes each, were obtained from the material in fraction 7, clusters, reticulocyte lysatesFig. 3A. Magnification 70,000 X. were subjected to treatment

with desoxycholate and ri-bonuclease. There is no apparent effect of desoxycholate upon either the sedi-mentation pattern (Fig. 3B) or appearance of the large polyribosomes in electronmicrographs. Ribonuclease caused essentially a complete dissociation of all classesof polyribosomes to 80S monoribosomes (Fig. 3C). Deoxyribonuclease (10 ,ig/mlat 00 C for 1 hr) or trypsin (0.2 ,ug/ml and 10 gg/ml at 00 C for 30 min) had noeffect on polyribosome structure. Thus, RNA appears necessary to maintain theintegrity of these large polyribosomes, as has previously been demonstrated forother polyribosomes.2-4 6 The resistance of the large polyribosomes to desoxy-cholate suggests that they are not fragments or small portions of endoplasmic re-ticulum.

Discussion. The number of these large polyribosomes in reticulocytes is atpresent unknown. On electron micrographs of intact cells their percentage is un-derestimated, because of the thinness of the sections and because of the presenceof bare portions along the filament connecting them. Quantitation cannot bebased on sucrose gradient analysis, since damage to these structures is possible dur-ing such a fractionation procedure. Therefore, at this time one can only speculateas to the function of these large polyribosomes in reticulocytes. Several possibilitiespresent themselves for consideration. On the one hand, these large aggregatesmay comprise only a minor proportion of the population, concerned with the syn-thesis of nonglobin proteins which constitute some 5-10 per cent of the newly syn-thesized protein of reticulocytes.13 In this case the large size of these polyribo-somes may reflect the synthesis of a larger protein than globin, encoded in a longermessenger RNA strand. The large polyribosomes might also be polycistronic mes-senger RNA-ribosome complexes for the synthesis of several genetically linked pro-teins, as has been proposed in the case of polyribosomes in poliovirus-infectedcells.i4 On the other hand, it is possible that large polyribosomes of sizes 15-20 arequite numerous in the intact reticulocyte. This is suggested by the fact that theyare readily observed in sectioned reticulocyte ghosts and appear to be easily brokenduring recovery from lysates. Their tendency to break into units of approximately5 ribosomes each (Fig. 5A) may well account for the high proportion of polyribo-somes of this size recovered from sucrose density gradients. If the large polyribo-somes are numerous in reticulocytes, it is likely that they are involved in globinsynthesis.

In this case the significance of the large polyribosomes may be that they include a

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A

300

Un

W

0U)0

100

5 1035 20RIBOSOMES/CLUSTER

B

800-

0U)0'400.

RIBOSOMES/ CLUSTER

FIG. 5.-Size distribution of polyribosomes in fractions from the sucrose density gradientillustrated in Fig. 3A, determined by electron microscopy. (A) Fraction 7; 2123ribosomes counted. (B) Fraction 21; 2284 ribosomes counted.

structure which stabilizes the messenger RNA-ribosome complex in a configurationoptimal for its function in protein synthesis.15-17 The chemical nature of this struc-ture is not defined. The present data indicate that the integrity of the large poly-ribosomes is not dependent upon a desoxycholate-sensitive structure but is de-pendent upon RNA.Summary.-Employing techniques which permit electron microscopic observa-

tions of hemoglobin-free reticulocyte ghosts, evidence has been derived for a popula-tion of large polyribosomes containing up to 20 ribosomes. Similar structureshave been recovered from rapidly sedimenting fractions from a sucrose densitygradient of liemolysates of these cells. The large polyribosomes are degraded byribonuclease but not by desoxycholate.

The authors are indebted to the Department of Microbiology and, in particular, to Dr. CounciLman Morgan for making available the facilities of the electron microscope laboratory.

* This investigation was supported, in part, by grants from the U.S. Public Health Service(Al-05493 and GM 07368), and the National Science Foundation (GB-1817).

t Recipient of a Career Scientist Award of the Health Research Council of the City of NewYork under contract number 1-336.

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4 Present address: University of Ibadan, School of Medicine, Ibadan, Nigeria.Marks, P. A., E. R. Burka, and D. Schlessinger, these PROCEEDINGS, 48, 2163 (1962).

2 Warner, J. R., A. Rich, and C. E. Hall, Science, 138, 1399 (1962).Gierer, A., J. Mol. Biol., 6, 148 (1963).

4Gilbert, W., J. Mol. Biol., 6, 374 (1963).6 Schlessinger, D., J. Mol. Biol., 7, 569 (1963).6 Wettstein, F. O., T. Staehelin, and H. Noll, Nature, 197, 430 (1963).7 Watson, J. D., Science, 140, 17 (1963).8 Marks, P. A., R. A. Rifkind, and D. Danon, these PROCEEDINGS, 50, 336 (1963).9 Rifkind, R. A., D. Danon, and P. A. Marks, J. Cell Biol., 22, 599 (1964).10 Borsook, H., C. L. Deasy, A. J. Haagen-Smit, G. Keighley, and P. H. Lowy, J. Biol. Chem.,

196, 669 (1952).11 Danon, D., A. Nevo, and Y. Marikovsky, Bull. Res. Council Israel, 6E, 36 (1956).12 Huxley, H. B., and G. Zubay, J. Mol. Biol., 2, 10 (1960).13 Dintzis, H. M., H. Borsook, and J. Vinograd, in Microsomal Particles and Protein Synthesis

(New York: Pergamon Press, 1959), p. 95.14 Rich, A., S. Penman, Y. Becker, J. Damell, and C. Hall, Science, 142, 1658 (1963).15 Haselkorn, R., and V. A. Fried, these PROCEEDINGS, 51, 308 (1964).16 Schlessinger, D., J. Mol. Biol., in press.17 Luzzatto, L., J. Banks, and P. A. Marks, submitted for publication.

DRIFT INSTABILITIES IN CYLINDRICAL DISCHARGESINVOLVING BOTH IONS AND ELECTRONS

BY JOSEPH SLEPIAN1115 LANCASTER STREET, PITTSBURGH, PENNSYLVANIA

Communicated August 1, 1964

Dr. Wesson in a recent paper1 in Section II takes as basic his equations (1)-(7).This I commend very highly, particularly the use of equations (6) and (7) for thedensity of the electrons and ions as they approach the limiting radius of the dis-charge. But in Section I he borrows from Kadomtsev2 the assumption that theion and electron temperatures are equal. This is unjustifiable however,3 4 andthe assumption that T, = Te should be dropped. Indeed we have that T, = T, = 0for the discharge reaching the outer insulated radius, when nir = ner = n should beused, but indeed we have that nil F ne at points just short of the cylindrical bound-ary.

In the Ionic Centrifuge3' 4with the end-plates kept at high negative voltage andthe cylinder kept at zero voltage, the total current received by the end-plates willbe all electron currents, and therefore limited by space charge. The cylinder willreceive equal amounts in charge of positive and negative ions, plus a small positivecharge equal in amount to the space charge limited electron current received bythe end-plates. This would be an effective means for generating thermonuclearfusion, provided the gas used in the central arc discharge is capable of thermonuclearfusion. When operated with the cylinder open-circuited, the efficiency would below because T = 0 there, in spite of the presence of large ion current at the cylinder.Operation with the cylinder grounded would raise the efficiency by lowering the heatgenerated by the impact of positive ions, even though the number of positive ionswas increased.

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