Messenger RNA in HeLa Cells :

Transcription

1 ur. J. Biohem. 17 (1970) Messenger RNA in HeLa Cells : An nvestigation of Free and Polyribosome-Bound Cytoplasmi Messenger Ribonuleoprotein Partiles by Kineti Labelling and letron Mirosopy Georges SPOHR, Niole GRANBOULAN~, Carlos MORL, end Klaus SCHRRR Departement de Biologie Molbulaire, nstitut Suisse de Reherhes xperimentales sur le Caner, Lausanne ; Centre de Mirosopie letronique, Universite de Lausanne and Laboratoire de Mirosopie letronique, nstitut de Reherhe Sientifique sur le Caner, Centre National de la Reherhe Sientifique, Villejuif (Reeived June 8/August 8,1970) Messenger RNA (mrna) and messenger like RNA (mlrna) were investigated in the ytoplasm of HeLa ells while ribosomal RNA synthesis was arrested. Under these onditions, funtional mrna assoiated in polyribosomes and ytoplasmi free mlrna are formed and an be labelled seletively to steady state. All ytoplasmi non-ribosomal RNA sedimenting at more than 6-7 S exists in the form of ribonuleoprotein omplexes whih pre-exist in the ell, and are stable upon ell lysis, sedimentation and (after fixation) CsCl density gradient analysis. The funtional, true mrna is ontained in a omplex of mrna and protein whih bands in assoiation with ribosomes (e = 1.52 to 1.60 g/m3) in CsCl density gradients or, released by DTA, at its own intrinsi density of g/m3. The ytoplasmi free mlrna bands as a partile of mlrna-ontaining ribonuleoprotein at an idential low density. The moleular weight spetrum of mrna is idential to that of mlrna and the sedimentation pattern of the mrna-protein omplex released from polyribosomes is similar to that of the free mlrna - protein omplex. The physio-hemial separation of mrna - and mlrna - protein omplexes allowed us to follow their relative kinetis of synthesis and deay. ah type of ribonuleoprotein obeys a different, stritly time-dependent pattern. Label enters the pool of free mlrna - protein omplexes first and may, in a pulse-hase experiment, be partially hased into polyribosomes. At steady state (6 h) /, of the labelled RNA remains in the form of free mlrna-protein partiles. These annot be hased into polyribosomes, the kinetis of mrna. and mlrna- protein omplexes deay following idential patterns. These findings are in agreement with a model aording to whih mlrna from the nuleus first joins the pool of free ribonuleoprotein. Then, the ativated mrna- protein omplexes attah to ribosomal subunits and form polyribosomes whereas inativated mlrna * protein omplexes remain free in the ytoplasm. n order to further strengthen the evidene in favour of the real existene of mrna- and mlrna * protein omplexes in the ells, the orresponding frations from sedimentation or CsCl density gradients were observed in the eletron mirosope. By this method it was possible to see small ytoplasmi partiles whih have not before been identified. The rounded strutures, seem to onsist of the oiling of a 35 8 wide pearl- with diameters ranging from 100 h to 200 8, like hain whih may also be identsed in polyribosomes. The frequeny of these partiles is highest in the mlrna. protein band (e = g/ms). Thus they may orrespond to the mrna - protein omplex. However sine they share some morphologial features with other known biologial strutures the evidene is not onlusive. t We are desolated to announe to our friends and ollegues that Niole Granbouland died in an aident shortly before the ompletion of this manusript. We dediate this paper to her memory. Unusuul Abbreviations. mrna, messenger RNA (this term is restrited to funtional mrna isolated from ative PolPibosomes) ; mlrna, messenger-like RNA; rrna, ribosomal RNA; Azso unit, the quantity of material ontained in 1 ml of a solution whih has an absorbane of 1 at 260 nm, when measured in a l-m path length ell. The existene in animal ells of messenger ribonuleoprotein partiles, formed by the assoiation of nasent and funtional messenger RNA with speifi protein, has long been onsidered possible on theoretial grounds. t was an enigma how messenger RNA in eukarioti ells is proteted against non-speifi degradation on its way from the hromatin to the polyribosome~ espeia y in suh as avian erythroblasts, whih are rih in ribonuleases.

2 Vo1.17, No.2,1970 G. SPOHR, N. GRANBOULAN, C. MORL, and K. SCHRRR 297 Furthermore, the existene of messenger RNA speies with different half lives in the same ell led to the postulation of fators onfering speifi stability to partiular types of messenger [l]. The demonstration that messenger-like RNA (mlrna) (f. [2-41) forms omplexes with proteins in the nuleus [5] and in the ytoplasm [6] not assoiated with ribosomal partiles was the first experimental evidene for the existene of suh strutures. However, onlusive evidene was laking that true messenger RNA was present in these omplexes. The experiments of Perry and Kelley [7], Henshaw [8], Burny et al. [9] and Cartouzou et al. [lo] demonstrated similar ribonuleoprotein omplexes in funtional polyribosomes and thus gave the first hint to the existene of speifi ribonuleoprotein partiles involving true mrna. However the interpretation of these data beame unertain sine the disovery by Girard and Baltimore [ 111 of non-speifi RNA-protein assoiations in ytoplasmi extrats of animal ells. Control experiments arried out in our laboratory showed that the formation of artifiial omplexes between RNA and proteins of ytoplasmi extrats of HeLa ells may indeed our but depend on the kind of purified RNA added. Ribosomal RNA forms few suh artifats but messenger-like RNA frations engage in them to a large extent. However, theoretial arguments were still in favour of the existene of messenger-protein omplexes. Their supposed funtion in messenger stability and possibly regulation, as well as the experimental evidene in favour of the existene of mrna - protein omplexes enouraged us to find other lines of evidene for their real existene and funtions. A reproduible, time dependent pattern of synthesis and deay of partiular ribonuleoprotein partiles exluding random aggregations would be a strong argument in favour of their real existene. Thus, we hose the possibility to distinguish artifats from reality by kineti labelling. Furthermore, we deided to attempt the diret demonstration of these partiles by eletron mirosopy. t was possible to demonstrate a reproduible pattern of labelling and deay of free and polyribosome-bound messenger ribonuleoprotein partiles in our experiments. They show the existene of two lasses of mrna-protein partiles in the ytoplasm : those whih engage with polyribosomes having passed through the pool of free partiles and another lass whih remains always free in the ytoplasm, apparently unable to attah to ribosomes and thus to express its message. By eletron mirosopy we have found a hitherto unidentified ytoplasmi struture whih, although sharing some morphologial features with other biologial entities may possibly represent the mrna * protein omplex. MATRAL AND MTHODS Substanes Surose : RNAse free preparations from Mann Researh Laboratories (U.S.A.). Atinomyin D : two preparations graiously given by RhBne- Poulen (Prane) and by Merk, Sharp and Dohme (U.S.A.) were used. Sodium deoxyholate: from Fluka (Switzerland). t was purified from an ethanol solution by preipitation with hexane or a hexanel aetone mixture. Sodium dodeylsulfate : from Serva (Germany). Triton X-100: from Mann Researh Laboratories (U.S.A.). [3H]Uridine : from the Radiohemial Center (Amersham, ngland) with a speifi ativity of more than 20 C/mmole. Media for tissue ulture: from Gibo (U.S.A.). Solutions Lysis buffer: 0.01 M KC, M MgC,, 0.005M 2-meraptoethanol, 0.01 M triethanolamine, ph 7.4. Suspension buffer: 0.01 M KC1, M MgCl,, 0.01 M triethanolamine ph 7.4. Gradient buffer: 0.01 M triethanolamine, 0.01 M NaC1, M MgCl,. Fixation buffer: 0.01 M KC, M MgClz, 0.03 Triethanolamine ph 7.8. Dialysis buffer: 0.01 M KC1, M MgCl,, 0.01 Triethanolamine ph 7.8. Surose gradients where not speified /,, surose in suspension buffer. The perentage of surose solutions are always given in weight per volume. MTHODS Cell Culture and Labelling Tehniques HeLa ells (lone S,) were grown in suspension with a generation time of about 24h in agle's spinner medium, supplemented with loo/, alf serum. Atinomyin D was added at a onentration of 0.05 pglml 30 min prior to labelling [12]. Where not speified, tritiated uridine was added diluted to a final onentration of 0.5 pm. Cell Frationation Labelling was stopped by the addition of about one third the volume of frozen (- 20") arle's saline (without magnesium and alium). The ells were entrifuged at 280 x g for 5 min, washed first with 20 times their volume of arle's saline and then with 20 volumes of isotoni surose in lysis buffer. Then the washed ells were suspended in 8 times their volume of hypotoni lysis buffer. After 3 min of swelling, Triton X-100 was added to a onentration of 0.25O/,; 2 min later isotoniity was restored by the addition of 2 M surose (in lysis

3 298 Messenger RNA in HeLa Cells ur. J. Biohem. buffer) to 0.25 M; 10 min after suspension, the ells were disrupted by 6-12 measured strokes of a tight Doune glass homogenizer. n order to keep nulear breakage negligible, homogenization was stopped when about 70 of the nulei were free. Unbroken ells, nulei, mitohondria and lysosomes were sedimented for 20 min at xg. The postmitohondrial supernatant (S-10000) was adjusted to a onentration of 0.5OlO sodium deoxyholate and immediately layered on a surose gradient. Alternatively, all ytoplasmi ribosomal and messenger ribonuleoprotein partiles were sedimented for 3 h at xg through a ushion of 15 /0 surose in suspension buffer into the "ytoplasmi partile pellet". Under these onditions more than 9001, of the labelled RNA heavier than 6s sediments assoiated with proteins. This pellet was resuspended in 0.25 M surose in suspension buffer with the help of a small Doune homogenizer. The suspension was larified by a 15 min entrifugation at 4500 xg. All operations were performed at temperatures between 0" and 4". Caesium Chloride and Density-Gradient Centrifugation Buoyant density entrifugation was arried out in the presene of formaldehyde aording to Spirin et al. [13,14]. Usually 2 to 4 units of absorbane at 260 nm of partiles were adjusted to Sol0 formaldehyde in 1.0 ml fixation buffer and kept at 4" for at least 20 h. Subsequently, the sample was dialysed exhaustively against dialysis buffer ontaining formaldehyde at 1 "0. The dialysed sample, in a final volume of 1.6 ml, was adjusted with solid CsCl to a density of e = 1.4Og/ m3 and the solution was layered over 1.9 ml of CsCl at a density of 1.60 in fixation buffer. Centrifugation was for 24 to 48 h at rev./min in the SW-56 Spino Rotor. Alternatively, the Spino SW-65 Rotor was used. n this ase, heavy and light solutions (2ml eah) had a density of 1.65 and 1.30 g/m3, respetively. Centrifugation was for 18 h at rev./min. 6 drop-frations were olleted from the bottom of the tubes and absorbane was determined at 260nm; the density of every 5th fration was determined by refrative index and orreted for the error indued by formaldehyde. General Methods RNA extration, surose gradient analysis and radioativity assays were arried out aording to Sherrer [M. letron Mirosopy Tehniques Gradient samples (surose or aesium hloride) were treated with 6O0 formaldehyde in fixation buffer (if not already fixed) and prepared for eletron mirosopy as follows : one droplet was deposited on a grid overed with it thin ollodion membrane stabilized by a arbon film. After about 15 se the exess of liquid was removed by absorption with a filter paper. The grid was immediately rinsed with the buffer used for the preparation of the samples (without surose or CsC1). Without allowing it to dry, the material was negatively stained with a freshly prepared aqueous solution of uranyl aetate (0.5 Ole), and then immediately examined in the eletron mirosope. As known, when the onentration of the biologial material on the grid is very low, the stain obtained is positive. n some ases sodium siliotungstate (1 in distilled water ontaining Mg++ at the same molarity as the buffer) was also used. For shadowing, the exess buffer used to rinse the grids was removed with it filter paper and the grids were dried in air. Then they were shadowed with platinum-arbon at an angle of 7" by rotation under a vauum of 5 x mm Hg. All speimens were examined with a Hitahi letron Mirosope at a nominal magnifiation of for stained preparations and or for shadowed grids. RSULTS The nflux of narna to Cytoplasm and Polyribosomes under Conditions of Arrested rrna Synthesis n order to follow the influx of messenger-like RNA to the ytoplasm in the absene of ribosomal RNA synthesis we took advantage of the observation of Perry [ and Georgiev et al. [18] that low doses of atinomyin D speifially inhibit 45 S RNA formation in the nuleolus without seriously affeting mlrna synthesis in the nuleoplasm. xploratory experiments showed that the dose of 0.05 pg/ml atinomyin D, lose to that hosen by Penman et al. [12], was optimal for our purpose. arlier reports in the literature [19] laim that the rate of protein synthesis is not affeted under these onditions. We found that it drops slowly to about 6001, of the initial rate after 8 h of exposure (Fig.1). Sine the half lives of polyribosomes and mrna in HeLa ells after omplete inhibition of RNA synthesis by a high atinomyin dose are about 3 h [20] it is evident that under our onditions new messenger RNA must reah the polyribosomes and be translated in order to maintain this level of protein synthesis. This labelling system is adequate to our purpose, sine our main goal is to investigate the pattern of influx of mrna into the various ribo-

4 Vol. 17, No.2, 1970 G. SPOHR, N. GRANBOULAN, C. MORL, and K. SCHRRR 299! Time (h) Fig. 1. Rate of protein synthesis in HeLa ells exposed to a low dose of atinomyin D. A ell suspension (34 x lo4 ells/ml, 500 ml) in omplete medium was treated with atinomyin (O.O5.pg/ml). At eah time point a 25 ml aliquot was removed and inubated for 30min with a mixture of l4c-labe1led amino aids, 1 pc eah of valine (260 mc/mmole), leuine (311 mc/mmole), and isoleuine (308 mc/mmole). Labelling was stopped by addition of rushed frozen arle s saline, the ells were entrifuged at 280xg and washed twie with old arle s saline. The ell pellet was suspended in hypotoni buffer and lysed by homogenization. The lysate was inubated with DNAse (20 pg/ml, 20, 15 min) to redue visosity, and lysis was ompleted by the addition of sodiumdodeylsulfate. To ontrol self-absorption, 3 aliquots of different volumes were preipitated in 10 O/,, trihloroaeti aid. Radioativity was determined as desribed in methods and normalized relative to absorbane at 260 nm. 0, atinomyin treated ultures; +, ontrol ulture nuleoprotein frations in a given physiologial ondition, and also to investigate the possibility of a regulatory disrimination between messenger types before their entry into polyribosomes. n addition these experiments show that rrna synthesis and the formation of new ribosomal subunits are not neessary for the transport of mrna from the nuleus to the ytoplasmi polyribosomes as it was proposed by Joklik and Beker [21] and reently by Sidebottom and Harris [22]. Pree and Polyribosome-Bound Cytoplasmi Messenger Ribonukoprotein Partiles The experiment shown in Fig. 2 demonstrates the existene of free and polyribosome-bound messenger ribonuleoprotein partiles. Furthermore it shows that it is possible to distinguish free messenger ribonuleoprotein partiles from those bound in polyribosomes, sine little ross ontamination ours in a CsCl density gradient. The uridine labelled mrna assoiated with polyribosomes bands after fixation exlusively in the area of ribosomal material (e = g/m3). But labelled ytoplasmi mlrna not assoiated with polyribosomes, osedimenting in surose gradients with monoribosomes and subribosomal partiles, bands in CsCl exlusively at a density lower than 1.48 g/m3, outside the band of ribosomal material. HeLa ells were labelled with uridine for 3.5 h in the presene of atinomyin as desribed in Material and Methods. Polyribosomes and monoribosomes of the ytoplasmi extrat were pelleted by a 45min entrifugation at x g through a ushion of 15 surose. The redissolved pellet was frationated on a surose gradient aording to Fig.2A. The fration of polyribosomes ontaining 3 to about 30 ribosomal units was sedimented quantitatively and resuspended. One half of this polyribosome suspension was fixed immediately with formaldehyde (Fig. 2B), the other half was fixed after addition of DTA (Fig.2C) and both were banded in CsC1. t is obvious that the labelled RNA bound to polyribosomes bands exlusively in an area with the ribosomal material at a density of g/m3 with little or no ontamination in the zone of free messenger-like ribonuleoprotein partiles (e = 1.45 g/m3). The release of messenger RNA from polyribosomes by DTA (Fig.2C) results in a quantitative shift of the polyribosome bound label to the area of free messenger ribonuleoprotein partiles with a density of about 1.45 g/m3. No label remains assoiated with the 1.57 g/m3 density band. This result represents a onfirmation of the experiments of Perry and Kelley [7], Henshaw [8] and Cartouzou et al. [lo], indiating that the mrna in animal polyribosomes may not be free but is assoiated with proteins. The density of 1.45 g/m3 orresponds to about 75O/, protein and 25O/, RNA in the omplex. To demonstrate the existene of free messengerlike ribonuleoprotein partiles, the partiles remaining in the polyribosome supernatant were pelleted by 2 h entrifugation at xg. The resuspended pellet was fixed with formaldehyde without DTA treatment and banded in a CsCl gradient as shown in Fig.2D. This fration, whih is slightly ontaminated with monoribosomes, but does not ontain polyribosomes, shows the wider absorbane distribution harateristi for native ribosomal subunits. These are ompletely unlabelled in our experiment. However, the labelled RNA, whih orresponds to messenger-like frations, bands under these onditions at a density of 1.45 g/m3 and thus in the same area as the messenger ribonuleoprotein partiles released from polyribosomes (Fig. 2 C). The density of this fration orresponds to that of the partiles, termed informosomes [23], isolated by Spirin and Nemer [6] from sea urhin embryos, and by Spirin et al. [41 from fish embryos. The radioativity liberated from polyribosomes by DTA treatment (Fig.2C) must be attributed to funtional messenger RNA assoiated with proteins. This follows from the fat that protein synthesis

5 300 Messenger RNA in HeLa Cells ur. J. Biohem. 0.5 a A - N m 2 m e a 9 1 t ttorn Frations top Frations Polyribosornes + DTA N m p0 m f! Frations. Fig.2. Density profiles of polyribosome bound messenger ribonwleoprotein and of free ytoplasmi messenger-like ribonwleoprotein: (A) Cells were labelled for 3.5 h (200 pc [3H]uridine diluted to 0.05 pm, 29 x lo4 ells/ml, 800 ml, atinomyin 0.05 pg/ml) and the postmitohondrial ytoplasmi extrat prepared aording to methods. The polyribosomal pellet obtained by entrifugation (30 min, xg) through a 15O/, surose ushion in buffer was resuspended and frationated on a surose gradient (15 to 30 /,, rev./min, 120 min, 2O, Spino SW 27). (B) and (C) Polyribosomes were pooled aording to (A) and olleted by entrifugation (2 h, xg), the pellet was resuspended, fixed and analysed on a CsCl density gradient aording to methods (50000 rev./min, 18 h, 4O, Spino SW 65). (B): without DTA, (C): treated before fixation with DTA (20 pmoles/mg ribosomes). (D): the supernatent of the polyribosomes fration prepared for (A) was sedimented by entrifugation (2 h, 36000Oxg) and the pellet was fixed and analysed on CsCl gradients aording to (B). 0, absorbane at 260 nm; 0, rh]uridine ativity ontinues under the given experimental onditions at a rate superior to that attributable to pre-existing mrna. Thus newly formed messenger RNA whih an be represented in our experiment only by the labelled RNA must assoiate with polyribosomes. The attribution of messenger quality to the RNA ontained in the frations of ytoplasmi free messenger-like ribonuleoprotein is more questionable. However there are lines of evidene onfirming suh an assumption. We have been able to demonstrate that analogous frations isolated from HeLa ells ontain biologial ativity in protein synthesis in vitro [24]. n another experimental approah we ould demonstrate by moleular hybridization that ompetition takes plae between the RNA from polyribosome-bound and free ytoplasmi ribonuleoprotein frations labelled under analogous onditions [25]. However, there is no evidene that all the label in the free messenger ribonuleoprotein fration represents true mrna. Thus, we will ontinue to term this fration messenger-like RNA (mlrna). Before analysing in more detail the messenger ribonuleoprotein partiles, we have to onfront two possibilities of artifats whih ould falsify our

6 Vol. 17, No. 2, 1970 G. SPOHR, N. GRANBOULAN, C. MORL, and K. SCHRRR 301 a L Density (9/m3) Density (9/m3) Density (9/m3) Fig.3. Buoyant density profiles of pure polyribosomes (A) and of ytoplasmi partile frations, pure (B) or ontaminated (C) with nulear ribonuleoprotein partiles. (A) Purified polyribosomes free of ytoplaami or nulear partiles. Cells were labelled for 6 h (200 pc [3H]uridine, 34 x lo" ells/ml, 200 ml, 0.05 pg/ml atinomyin) and a ytoplasmi extrat was prepared aording to Methods. After surose gradient frationation, the polyribosome area was pooled (f. Fig. 2) sedimented (3 h, xg), resuspended, prepared for CsCl density entrifugation and banded aording to Methods (32000 rev./min, 24 h, 2O, Spino SW 56). (B) Cytoplasmi partile fration. Cells were labelled for 6 h (100 pc [3H]uridine, 33 x lo4 ells/ml, 250 ml, 0.05 pg/ml atinomyin) and a ytoplasmi extrat was prepared, the pellet obtained by entrifugation (3 h, xg) through a 15O/,, surose ushion was resuspended, prepared and banded in CsCl as desribed in Methods (32000 rev./min, 24 h, 2O, Spino SW 56). (C) Cytoplamni partile fration wntaminated with nulear ribonuleoprotein partiles. Cells were labelled for 16 h (100 pc [3H]uridine, 31 x lo4 ells/ml, 300 ml, 0,05 pg/ml atinomyin). Preparation and analysis in a CsCl density gradient aa desribed in (B). 0, absorbane at 260 nm; 0, [*H]uridine ativity onlusions : the presene of partiles due to nulear leakage and the formation of artifiial assoiations of RNA with protein. Preparation of Cytoplasmi xtrats Free of Nulear Material A prerequisite for the ontinuation of our investigation was to exlude, or to redue to a negligible amount, the ontamination of ytoplasmi extrats with nulear frations due to extration and/or breakage of nulei. The ontaminating giant nulear RNA is assoiated with proteins also [5], and possibly with hromatin, whih onfer to it, aording to Penman et al. [12] sedimentation values of up to 3000 s. t may band in CsCl gradients at densities from 1.40 to 1.45 g/m3 [26], together with free ytoplasmi mesenger-like ribonuleoprotein partiles [23]. Thus the analysis of free ytoplasmi messenger-like ribonuleoprotein partiles in CsCl density gradients is possible only if nulear ontaminations are redued to a negligible amount. We ontrol nulear leakage by several methods. As shown by Perry and Kelley [7], the pool of purified polyribosomes does not ontain any free messenger ribonuleoprotein partiles banding at the density of 1.40 to 1.45 g/m3. Thus the absene of label at this density is a, proof of the absene of nulear ontamina- tion. Fig.3A shows the density profile of a polyribosome pool with little or no nulear ontamination. Fig.3B shows the banding pattern in a CsCl gradient of the ytoplasmi material sedimented for 3 h at xg (ytoplasmi partile pellet) ontaining RNA labelled for 6 h. Polyribosome-assoiated messenger ribunoleoprotein bands slightly lighter than single ribosomes at densities between 1.60 and 1.58 &/em3 (the monoribosomes band at e = 1.55 to 1.60) the free messenger-like ribonuleoprotein bands at a density below 1.48 g/m3. vident nulear leakage is shown in Fig.3C (due in this experiment to the fragility of nulei after 16 h, too long an inubation with atinomyin) : not only does a large amount of radioativity appear at densities of 1.42 to 1.46 g/m3 but also a slight amount of material absorbing at 260 nm is visible in the same area as a separate peak. Thus not only the amount of radioativity but also the presene of a peak in absorbane serves as an indiation of nulear leakage. Another argument for the distintion of ytoplasmi and nulear ribonuleoproteins in ytoplasmi extrats is the kineti pattern of labelling and deay itself, the nulear mlrna being haraterized by a partiularly high rate of turnover [28]. The inorporation kinetis of tritiated uridine into ytoplasmi messenger ribonuleoprotein has shown

7 302 Messenger RNA in HeLa Cells ur. J. Biohem. A C z m z m 2 w z Frations Frations Fig. 4. Artifiial assoiation of purified RNA with proteins present in the ytoplasmi partile fration. Polyribosomes, ribosomes and subribosomal partiles were sedimented into one pellet and resuspended in buffer aording to Methods. Purified [W]- uridine-labelled RNA was added and inubated in the old for 15 min, the mixture was fixed with formaldehyde and banded in CsCl density gradients (50000 rev./min, 18 h, 2O, Spino SW 65). (A) 28 S ribosomal RNA added, (B, C) nulear mlrna added that a steady state of synthesis and deay is reahed at approximately 6 h (f. Fig.ll). On the ontrary, the nuleoplasmi heterogeneous RNA approahes steady state already after 2 h [12]. This extensive disussion should point out the importane we give to the ontrol of the ytoplasmi origin of the omplexes subjet to this type of analysis. The method finally adopted for preparing ytoplasmi extrats is desribed in Methods. The restoration of isotoniity before ellular lysis limited the extration of nulei. (Hypotoniity during ell homogenisation led inevitably to nulear leakage.) The limitation of homogenization to a level where about 700/, of nulei only are liberated redued nulear breakage to a negligible level. Control of Artifiial Assoiation of RNA with Protein in the Cytoplasmi xtrats Girard and Baltimore [ll] had shown that labelled poliomyelitis virus RNA added to ytoplasmi extrats of HeLa ells forms artifiial ribonuleoprotein omplexes. However, Perry and Kelley [7] laimed in their demonstration of the presene of mrna - protein omplexes in polyribosomes that labelled ellular RNA did not form suh artifats. Our ontrol experiments (Fig.4A) show that ribosomal type RNA indeed binds to a limited extent only to proteins ontained in the ytoplasmi partile fration. However, mrna and mlrna assoiate with proteins ontained in the ytoplasmi partile fration and band in CsCl at densities between 1.40 and 1.55 g/m3 (Fig. 4 B and 4 C). The nature of these artifiial omplexes is so far not known. Their definition as artifats is ambiguous. ndeed if speifi proteins exist whih bind to RNA it is reasonable to expet similar omplexes to form in vitro as those present in vivo. However experimental riteria allow one to distinguish them. Sedimentation Charateristis of Polyribosome Bound mrna. protein Complexes and Free Cytoplasmi mlrna* protein Partiles A omparison of the sedimentation profile of polyribosome bound mrna protein omplexes and free ytoplasmi mlrna * protein omplexes demonstrates that both types of Ribonuleoproteins are represented with the same spetrum of apparent sizes. Thus both may ontain RNA with the same spetrum of moleular weights. The ytoplasmi extrat of ells labelled for 6 h with [3H]uridine was frationated on a surose gradient into polyribosomes, monoribosomes (70 to 100s) and the subribosomal zone (30 to 70s) as shown in Fig.5A and B. The partiles ontained in the respetive zones were olleted by a 3 h entrifugation at x g and the resuspended larified material was analysed by sedimentation after the addition of 2 pmoles DTA per absorbane unit at 260 nm. The sedimentation pattern of the free labelled ribonuleoprotein frations is shown in Fig.6D-. The mrna - protein partiles extrated from polyribosomes by addition of DTA sediment between 20 and 70 S (Fig.5C). A lighter peak may represent

8 0 W 8 2.o N.m a, s m 1.0.D a 2 A Polyribosomes 1 B JJ C bottom Frations top bottom r 1.0 Jo Frations top 0.50 J lo bottom Frations top bottom Frations top bottom Frations top Fig. 5. Sedimentation profile of messenger ribonuleoprotein ontained in polyribosomes, monoribosomes and the subribosomal fration. The ells were labelled for 6 h (800 pc C3H]uridine, 34 x lo4 ellslml, 1.2 1, 0.05 pg/ml atinomyin). The postmitohondrial extrat prepared aording to Methods was frationated on surose gradients (10 to 40 /,, rev./min, 2 h (A) or 6 h (B), Spino SW 27). Polyribosomes, monoribosomes (70 to 100 S) and the subribosomal fration (30 to 70 S) were pooled aording to (A) and (B) olleted by entrifugation (3 h, Xg). The pellets were resuspended in 0.25 M surose in suspension buffer, treated with DTA (20 pmoles/mg of ribosomes plus ompensation of Mgff in buffer) and analysed on a surose gradient 5-20 /, in Mg+ free suspension buffer (40000 rev./min, 3 h, 2", Spino SW 40). (C) Polyribosomes; (D) monoribosomal zone; () Subribosomal zone. -, absorbane at 260 nm; O-----O, rh]uridine ativity

9 304 Messenger RNA in HeLa Cells ur. J. Biohem. trna bound to ativating enzymes as well as small mrna - protein partiles. The mlrna protein partiles of the 70 to 100 S zone (Fig.5D) whih, as demonstrated in Fig. 7 B are essentially not bound to ribosomal material, sediment also between 10 and 70 S. The free mlrna. protein partiles from the subribosomal zone show a predominane of partiles in the sedimentation zone below 50 S (Fig.5). The gradient analysis of free mlrna - protein partiles shown in Fig. 5 D and was arried out in the presene of DTA in analogy to Fig. 5C. Control experiments showed that DTA does not alter the sedimentation veloity of labelled RNA in the free messenger-like ribonuleoprotein fration. Thus the partiles of the entire 30 to 100 S area (Fig.BD+ ) whih ontains the free mlrna * protein show the same size distribution as the mrna * protein dissoiated from polyribosomes by DTA treatment. We onlude that free mlrna.protein partiles and polysome-bound mrna - protein-ontaining partiles, or in other terms inative messengerlike ribonuleoprotein and ative messenger ribonuleoprotein, show the same sedimentation behaviour. Thus they should ontain the same size lasses of RNA. Sedimentation Charateristis of Labelled RNA xtrated from Free and Polyribosome-Bound mrna *protein Partiles The sedimentation analysis of phenol extrated RNA from the various ytoplasmi frations demonstrates that mrna - protein partiles ontain RNA speies with a spetrum of moleular weights analogous to that we found earlier by pulse labelling in HeLa ell polyribosomes [20]. HeLa ells were labelled to steady state (6 h) with midine as desribed in Methods and the postmitohondrial ytoplasmi partiles were frationated on surose gradients into polyribosomes, monoribosomes and a subribosomal fration. The RNA of the free mlrna-protein partiles and polysome-bound mrna * protein ontained in these frations was extrated by dodeylsulfate dissoiation (and diretly sedimented [29]) or by the hot phenol method. Both methods gave omparable results. Fig. 6 demonstrates that RNA extrated from the polyribosome-bound messenger ribonuleoprotein and from free messenger-like ribonuleoprotein gives rise to the same overall sedimentation profile spreading from 6 S to about 30 S. As expeted, the separately extrated heavy free messenger-like ribonuleoprotein partiles osedimenting with monoribosomes ontain RNA of higher moleular weight then those in the subribosomal zone (Fig.6B and C resp.). Polyarylamide gel analysis of the phenol extrated RNA leads to essentially the same results (Fig.6D-F). The sedimentation of mrna and mlrna relative to rrna an be ompared to that of the orresponding messenger ribonuleoprotein partiles relative to ribosomal subunits (Fig.5). The overall pattern of the RNA or ribonuleoprotein spetra are the same : polyribosomes ontain the full spetrum of sizes whereas the free messenger-like ribonuleoprotein partiles or their RNA have higher sedimentation values in the monoribosomes area ompared to those in the subribosomal zone. The fat that in this experiment the monoribosome pool ontains not only mlrna with high sedimentation values orresponding to large mlrna partiles but also smaller types may be attributed either to the presene of some messenger ribonuleoprotein bound to small polyribosomes ( mono-or di-polyribosomes ), sine the entire 70 to 100 S area was pooled, or to the fat that in the absene of DTA or of high salt some assoiation between messenger-like ribonuleoprotein and ribosomal material may possibly our. Our general onlusion is that in the ytoplasm of HeLa ells (free of mitohondria) mrna and mlrna types exist, polyribosome-bound or in free ribonuleoprotein partiles, with moleular weights ranging from to about 1 million daltons. Heavier RNA was found in negligible amounts in experiments where no nulear leakage had ourred. Density Profiles of ndividual Messenger and Messenger-Like Ribonuleoprotein Frations The shallow CsCl density gradients obtained at lower speed in the SW-56 Rotor allow analysis of the partiulate fration of ytoplasmi extrats in more detail (Fig. 7). Postmitohondrial ytoplasmi extrats from ells labelled for 3 or 6 h were frationated on surose gradients into pure polyribosomes (more than 150 S), monoribosomes (70 to 100 S) and subribosomal partiles (30-70 s). The pooled frations were analysed on CsCl gradients after fixation. The polyribosomal material bands in an area at a density of g/m3, the free messenger-like ribonuleoprotein present in the 70 S to 100 S pool bands at a buyoant density of approximately g/m3 and that of the 30 S to 70 S sedimentation zone bands at a density lower than 1.45 g/m3. The S zone ontains little labelled messenger ribonuleoprotein bound to monoribosomes. The fat that the free ribonuleoprotein partiles ontaining mlrna from the heavier sedimentation zone band at a slightly higher density than those of the subribosomal area may be attributed to intrinsi differenes in omposition of different lasses of these partiles or perhaps to the attahment of small messenger ribonuleoprotein partiles to ribosomal subunits. However, as shown in Fig.5D the S zone ontains mainly large, messenger ribonuleo-

10 ? W 0 01 N 0 A bottom Frations top bottom Frations tw bottom Frations top R Slie number Slie number Fig.6. Analysis of RNA extrated from polyribosomes, monoribosomes and the subribosomal fration. Cells were labelled for 6 h aording to Fig.5 and frationated into the polyribosome, monoribosome and subribosomal partile zone (f. Fig.5A and B). (A-C) The pooled frations were sedimented (3 h, xg) resuspended in 0.25 M surose in gradient buffer, adjusted to 1 O/, sodium dodeylsulfate, 0.5O/, deoxyholate and 0.5O/, urea, diluted with buffer to a surose onentration of 3.5O/, and analysed on a surose gradient (0.01 M triethanolamine, ph 7.4, 0.01 M NaC, 0.01 M DTA) ontaining 0.2O/, sodium deoxyholate and 0.20/, sodium dodeylsulfate (5 to 20 /,, rev./min, 6 h, 2", Spino SW 40). (D-F) The pooled frations were extrated with hot phenol and analysed aording to Mirault et al. [30] on polyarylamide exponential gels (2--15O/, gel in -RNA buffer, 9 h, 2", 10 volts/m, 3 ma/tube). (A, D) RNA of the polyribosome zone; (B, ) RNA of the monoribosome zone (70 to 100 S); (C, F) RNA of the subribosomal partiles zone (30 to 70 S). -, absorbane at 260 nm; and , [SHuridine ativity

11 306 Messenger RNA in HeLa Cells ur. J. Biohem. A Polyribosms B Maor ibo s o m e s 70-03s zme C Subribosml partiles 30-70s m 0.4, 8 N * 8? 0.3 z s Density (g/ml) Density (g/ml) Denw'ty (gm3 ~ D F Polyribosomes + DTA Mono r i bo s o m e s + DTA 0.10 Sukibosomal partiles DTA - w * Density (giml) Fig. 7. Buoyant density profiles of messenger ribonuleoprotein partiles ontained in polyribosomes, monoribosomes and the subribosomal fration before and after DTA treatment. Cells were labelied for 3 (C and F) or 6 (A, D, and B, ) h (800 $2, [9H]- uridine, 33 x lo4 ells/ml, 1.5 1, 0.05 pg/ml atinomyin), the postmitohondrial extrat was prepared aording to Methods and frationated on a surose gradient into polyribosomes, monoribosomes and the subribosomal partile zone (f. Fig. 5). The pooled frations were sedimented (3 h, x g) resuspended in 0.25 M surose in suspension buffer, treated or not with DTA (20 pmoles/mg ribosome plus ompensation for Mg in buffer). CsCl density analysis aording to Methods (32000 rev./min, 24 h, 2", Spino SW 56). (A, D) polyribosomes; (B, ) monoribosomes; (C-F) subribosomal partiles. (A-C) ontrols; (D-F) DTA treated. 0-0, absorbane at [3H]uridine ativity protein partiles. This favours the first interpretation rather than the seond. t is interesting to note that the ribosomal material of the polyribosome zone bands at densities slightly lighter than the monoribosomes (Fig. 7 A and B). This partial separation an be onfirmed by eletron mirosopy, investigating the material ontained in eah zone as shown below. The binding of messenger ribonuleoprotein onfers a slight derease in density to ribosomes, as would be expeted of a omplex formed by assoiation of a omponent ontaining 75O/, protein with one ontaining BOO/, protein. This also explains why in the ytoplasmi partile pellet the radioativity assoiated with polyribosomes has a lower mean buoyant density than the absorbany whih orresponds to inative monoribosomes. The fat that the monoribosome zone bands heavier than polyribosomes and arries little radioativity under steady state onditions demonstrates that monoribosomes are not engaged in protein synthesis. Furthermore, this serves as a ontrol that during the isolation proedure polyribosomes have not been degraded to monoribosomes assoiated with messenger ribonuleoprotein.

12 Vol.17, x0.2, 1970 G. SPOHR, N. GRANBOULAN, C. MORL. and <. SCHRRR N m 5: a < Frations Frations ar.n a N 2.o d m i? 5: Frations Fig. 8. Labelling pattern in density profiles of free messenger-like ribonuleoprotein and polyrihosome bound messenger ribonuleoprotein. Cells were labelled for the indiated time periods (150 pc [3H]uridine diluted to 0.1 pv, 66 x lo4 ells/ml, 600 ml, 0.05 pg/ml atinomgin) as indiated in Methods. Aliquots of 125 ml were hilled, the ytoplasmi partile pellet was prepared and analysed in CsCl density gradients as indiated in Nethods (50000 rev./min, 18 h, 2", Spino SW 65). 0, absorbane at 260 nm; 0, [3H]uridine ativity Frations Kinetis of Synthesis and Deay of Ribonuleoprotein Complexes of mlrna and Polyribosorne Bound mrna The inorporation of labelled uridine into polyribosomal mrna * protein and free mlrna * protein was determined for up to 2 h under onditions of bloked rrka synthesis. After 6h a steady state of labelling and deay had been reahed and a high onentration of atinornyin > (5 &ml) was added to follow the deay of the ribonuleoprotein partiles ontaining mrna or mlrna, respetively. LO+ The labelling pattern after various times of synthesis and deay of mrna. and mlrna. protein was analysed in steep (Fig.8) and in the better resolving shallow CsCl gradients (Fig.9). For every time point the entire partiulate fration was sedimentd for 3 h at xg from a postmitohondrial ptoplasmi extrat. The resuspended and larified pellets were diretly analysed in CsCl gradients after fixation with formaldehyde. After a 30min exposure to [3H]uridine free mlrna * protein is labelled almost exlusively

13 D A C h i t y (glvn3) F G 0.e Density (g/un3) Fig. 9. Pattern in density profiles of labelling and deay of messenger ribonueleoprotein partiles. Cells were labelled for 6 h (500 pc [3H]uridine, 33 x lo'ells/ml, 1300 ml, 0.05 pg/ml atinomyin), aording to Methods; after 6 h the ulture was adjusted to 5 pg/ml atinomyin in order to indue the hase. After the indiated times 150 ml aliquots were hilled and the ytoplasmi partile pellet prepared and analysed in CsCl density gradients (32000 rev./min 24 h, 2", Spino SW 56) as in& ated in Methods. (A) 1 h; (B) 2 h; (C) 4 h; (D) 6 h; () 6 h and 1 h hase; (F) 6 h and 3 h hase; (G) 6 h and 6 h hase. 0-0, absorbane at 260 [3H]uridine ativity U M -' f 0 B

14 Vo1.17, No.2, 1970 G. SPOHR, N. GRANBOULAN, C. MORL, and K. SCHRRR s 12 Time (h) Time (h) Pig.10. Kinetis of RNA labelliny in whole ells and in the ytoplasmi partile fration. Cells were labelled as indiated in Fig. 9. To determine the radioativity in whole ells 1 ml aliquots of the ulture were sedimented at 280 x g, washed twie with old arle s saline and lysed in isotoni saline with 0.5 sodium dodeylsulfate. 10 trihloroaeti preipitable radioativity was determined as desribed in Methods. The speifi ativity of the ytoplasmi partile fration prepared as indiated in Fig.9 was determined aording to Methods. (A): whole ells; (B): ytoplasmi partile pellet. (-) ontrol; (----) 5 pg/ml atinomyin added at 6 h, as indiated by the arrow (Fig. 8A). Subsequently more enters the ytoplasm and some polyribosome-bound label appears (Fig. 8B and C). After 4 h of labelling (Fig. 8D) approximatively the same amount of radioativity is present in mrna * protein and free mlrna * protein. A similar pattern may be observed in more shallow gradients (Fig.9A to D). norporation of label into free mlrna * protein preedes always inorporation into polyribosomes. n the presene of the low atinomyin onentration used a steady state of synthesis and deay is reahed after approximately 6 h. The synthesis of mrna. and mlrnaprotein was followed for up to 12 h. This represents the pratial time limit for these experiments due to the inreased fragility of the nulei after prolongued exposure to atinomyin. f after 6 h of labelling a high dose of atinomyin (5 pg/ml) was added to inhibit ompletely further RNA synthesis, the deay of both polysome-bound mrna * protein and free mlrna * protein ould be observed. n these shallow gradients a different pattern of synthesis and deay was evident in the various density zones. n several experiments it was observed that the light mlrna * protein of the.40 g/m3 density zone disappear more rapidly than others (Fig.9-G). f in the first hour of atinomyin hase the disappearane of free mlrna * protein seems to be more rapid than in polyribosome-bound mlrna. protein, a ontinued hase reveals approximately the same rate of deay in both frations. n order to express quantitatively the pattern of mrna * and mlrna * protein synthesis and deay observed, the radioativities orresponding to the free and polyribosome bound ribonuleoprotein zones were integrated. Speifi ativities were alulated by normalisation relative to the total ribosomal material present in a gradient. For omparison, Fig. 10A shows the radioativity pattern during labelling and hase in whole ells, and Fig. 10B in the total ytoplasmi partile fration. n both ases a steady state is reahed after 6 h. t is established slightly faster in the total ell sine, aording to Penman et al. [Z], nulear label is already maximal after 3 h. The radioativity in the entire ytoplasmi partile fration enters steady state after 6 h. The half life of the labelled RNA is also about 6 h. However it has to be onsidered that this analysis inludes the metabolially stable soluble RNA whih is ontinuously labelled in the presene of a low atinomyin dose [31]. The kinetis of mrna. protein labelling in several typial experiments are shown in Fig. 11. These patterns demonstrate the general finding that the labelling of free mlrna-protein always preedes that of the mrna protein bound in polyribosomes to a more or less pronouned degree. However, the speifi ativities of both frations finally reah approximately the same value. Under the hosen experimental onditions in steady state only 50 /, of the newly synthesized ribonuleoprotein partiles are engaged in ative polyribosomes and thus arry funtional mrna. The label remaining in free partiles raises the problem if these ontain inativated mrna speies not able to bind ribosomal subunits, or if they represent preursors to the polyribosome-bound mrna - protein ontaining the same messenger whih aumulate as a onsequene of a limitation in ribosomes available. n order to answer this question hase experiments were arried out (Fig.9-G and Fig. A, B). They demonstrated learly that on the average the free mlrna * protein partiles deay as rapidly as the mrna * protein partiles engaged in polyribosomes, although speifi mlrna - protein partiles of one density lass may disappear more rapidly than those of an other lass. Thus on the average inative mlrna - protein speies have the same stability as mrna * protein-ontaining messenger partiles and annot be hased into polyribosomes. This pattern of deay indiates that at steady state either a majority of mlrna in free partiles is inativated and does not represent preursors of the polyribosome-bound mrna, or that the equilibrium

15 -! ' A [ ',,~, &~,.\, b ~ ~ Time (h) 4 8 Time (h) D 4 8 Time [h) Time (h) Fig. 11. Kinetis of uridine inorporation into polyribosome bound messenger ribonuleoprotein and free ytoplasmi messenger-like ribonuleoprotein. Cells were labelled in the presene of atinomyin (0.05 pg/ml) and ytoplasmi partile pellets were prepared analysed and as reported in Methods and in the legends of Fig.8 and 9. The radioativity of frations with densities from 1.36 to 1.46 g/m3 was attributed to free messenger-like ribonuleoprotein (0) and between to polyribosome bound messenger ribonuleoprotein (o), the values were integrated and normalized relative to the ribosomal material in the profile. Fig. (A) to (D) refer to four different experiments. Note the different time sale in D. The arrow indiates the addition of atinomyin D (5 pg/ml) to indue the hase (----) between ative and inative forms of mrna is disturbed by the high dose of atinomyin added for the hase. n the attempt to answer this question pulsehase experiments were arried out and will be published in detail elsewhere. They tend to show that a fration of mlrna had been shifted during the hase from the free mlrna * protein partile pool to the polyribosomes, being thus a preursor of the funtional mrna. However, the major part of ytoplasmi mlrna labelled in 40min annot be hased into polyribosomes and may be inativated. The possibility has to be taken into aount that limiting fators may be neessary to ativate free mlrna. protein omplexes and enable them to beome bound to ribosomes. t was evident in these experiments that the stability of mrna or mlrna is an intrinsi property of the ribonuleoprotein partile itself, quite independent of its eventual assoiation with ribosomes and its funtion in the ative omplex during protein synthesis. The time limitation in this type of experiment whih is the onsequene of the presene of atinomyin at a very low dose, made it impossible to follow the fate of the messenger ribonuleoprotein beyond 6 h of hase. The rate of deay during this period of both types of partiles orresponds to an

16 100 A 100 i 100 A A Fig. 12. The visualisation of non-ribosomal small partiles in the ytoplasmi partile frations. (a, b,, d, e, g). Negatively stained small partiles found in CsCl gradients of the ytoplasmi partile fration. From (a) to (e) the size and the more or less ompat aspet of these partiles seem to depend on the length of the strand 35 fi in diameter, forming them by oiling. n (g) their morphologial feature an be ompared to that of 305 and 50s ribosomal subunits. (Magnifiation: ~.) (0) 30S, (+) 505 ribosomal subunits. (f) Three partiles in positive staining. (Magnifiation: ) (h) Negatively stained sample of the polyribosomal zone in a CsCl gradient of the ytoplasmi partile fration. (Density: 1.55g/m3; tube: 12; Fig. 13B.) (+) Small partiles loated lose to polyribosomes. (Magnifiation: x.) The large amorphous area of 300 fi diameter and larger are staining artefats. (i) Shadowed preparation of polyribosoma1 fration of a surose gradients. (+) Small partiles whih seem to be linked to polyribosomes. Note the fine strand linking the ribosomes in the upper right orner. (Magnifiation: x )

17 312 Messenger RNA in HeLa Cells ur. J. Bioehem. average half life of 3 h. n an earlier investigation we had shown that the half life of polyribosomes and thus of messenger RNA funtion is also of the order of 3 h in HeLa ells [20]. n view of these experiments the disappearane of ative polyribosomes and thus the arrest of messenger RNA funtion is the onsequene of the deay of the messenger or of the mrna * protein omplex itself. The Visualisation of Ribonuleoprotein Partiles Containing mrna and mlrna in the letron Mirosope Besides easily identified ribosomal material suh as polyribosomes, monoribosomes and ribosomal subunits, eletron mirographs of the ytoplasmi partile fration (15 to 400 S) from HeLa ells show a distint type of partile. We observed small, generally round partiles, with a diameter varying from 110 to 200 d. They seem to be formed by the oiling of a thin ontinuous strand presenting regularly loated small knobs along its length (Fig. 12a, b,, d, e, g). This strand measures about 35 d in thikness. The size of the partiles and their more or less ompat aspets seem to vary with the length of the oiled strand. Thus, some of them appear with a small entral hole (Fig. 12a, ) and, most frequently, they are ompat and eletron dense (Fig. 12d, e, g). The presene of these partiles an be demonstrated as well by negative staining, uranyl aetate giving best results, as by shadowing. They are also detetable by positive staining but not so easily as ribosomal material (Fig. 12f). Observations on CsCl Gradient Frations The material orresponding to a density of 1.58 g/m3 (Fig. 13B, fration 9) is omposed of monoribosomes. Very few small partiles were found attahed to monoribosomes : exeptionally, free partiles may be seen. n the density zone from 1.49 to 1.56 g/m3 (Fig.3A, frations: 8, 12; Fig.l3B, frations 12, 13, 14, 16) the material onsists prinipally of polyribosomes inluding from two to more than ten ribosomes. Some partiles an be distinguished attahed to polyribosomes. t was espeially diffiult to view the partiles attahed to polyribosomes due to their small size and the possibility of a superposition effet (Fig. 12h). They are better demonstrated by shadowing (Fig. 12i). These polyribosomal frations also ontain monoribosomes and very rare free small partiles. At densities from 1.44 to 1.48g/m3 (Fig.13A: frations 18, 19; Fig.13B: frations 18, 20) the observed material onsists of ribosomal subunits, monoribosomes and free small partiles. These latter partiles are more frequent than in the preeding frations. n the density frations of 1.39 to 1.44 g/ om3 (Fig. 13A; frations 22, 23; Fig. 13B, frations 22, to 24) whih orrespond to the region of mlrna. A 1.7 T (v 3 g ' R m f _. Lo 3-00._.- v * bottom Frations top bottom Frations top Fig. 13. Frequeny in buoyant density gradients of the small partiles deteted by eletron mirosopy. Cells were labelled for 6 h aording to Fig. 9 and the ytoplasmi partile fration was analysed on CsCl density gradient as desribed in Methods. (32000rev./min, 24 h, 2", Spino SW 56.) (A) and (B) refer to two different experiments. (0-o), absorbane at 260nm; ), number of small partiles deteted by eletron mirosopy (o---a), radioativity; (+ --+

18 Vol. 17, No. 2, 1970 G. SPOHR, N. GRANBOULAN, C. MORL, and K. SCHRRR 313 protein omplexes as defined in the biohemial experiments, the free small partiles are found more frequently than in frations of higher density or those of lower density. These frations ontain also some more or less degraded ribosomal subunits. The frequeny of the free small partiles dereases in the following frations orresponding to Q = 1.33 to 1.38g/m3 (Fig. 13A: fration27; Fig. 13B, fration 26) where the zone of pure proteins free of RNA begins. Sine the small partiles did not appear as the only material in the mlrna * protein region of the gradients, an attempt was made to ount them on the mirographs and to ompare their mean number per eletron mirosopial area to the pattern of radioativity. These data are given with some reservation due to the problem of regular distribution on eletron mirosope grids. Unfortunately, the material was not abundant enough to use the filtration tehnique whih is known to give a better distribution [32]. t an be seen (Fig.13) that the frequeny distribution of the small partiles roughly follows the urve of radioativity. The rihest tubes are found in the zone of the labelled mlrna. and mrna protein omplexes. t has to be taken into onsideration that many small partiles attahed to polyribosomes may not be seen due to the above mentioned superposition effet or their unwinding into the 35 d wide strand. Observation of Pure Polyribosomes n a pure fration of polyribosomes isolated on a surose gradient some small partiles attahed to polyribosomes are visible. Free partiles are exeptional (Fig. 12i and 14a). When this fration is treated with DTA (0.5 or 0.1 pmoles per A,,, unit, 30 min, O0) before formaldehyde fixation and preparation for eletron mirosopy, some free small partiles an be deteted either by negative staining or by shadowing (Fig. 14b and ). The ribosomal material is prinipally in the form of 30 S and 50 S subunits. Some of them present a tail (Fig.14~) whih may be interpreted as an elongated piee of the 35 d diameter strand whih would, after omplete ondensation, form the small partiles by oiling. The knobs are learly visible in the developed 35 d strand. After exposure of the polyribosomal fration to 3 M urea (30 min, 20 ), free small partiles are detetable (Fig. 14d). The ribosomal strutures are profoundly altered by this treatment presenting extended strutures distintly larger than the tails mentioned above. Thus the small partiles seem to be more resistant than ribosomes although their struture has beome looser, as an be deteted in negatively stained preparations. DSCUSSON This investigation of ytoplasmi messenger RNA in animal ells was started in order to find new lines of evidene for the physiohemial reality in vivo of messenger-ribonuleoprotein omplexes, whih have been desribed by several laboratories [6-10, 231. Their biohemial haraterisation is ruial in view of the role suh ribonuleoprotein assoiations may play in messenger RNA stability, transport, funtion, and possibly regulation. n the following we will disuss some of the experimental evidene and present our onlusions. Control of Artifiial RNA-Protein Assoiation The artifiial assoiation of proteins with RNA observed first by Girard and Baltimore [ll and reently by Baltimore and Huang [34] onstitutes a major argument whih ould ast doubt on the reality and signifiane of the mrna * protein omplexes observed in vivo. Perry and Kelly [7] demonstrated that purified mlrna did not bind ytoplasmi proteins in L ells. However artifiial assoiations were observed under our onditions in HeLa ells (f. Fig. 4) in agreement with Baltimore and Huang [34]. We did not observe the formation of a speifi band at Q = 1.45 g/m3 whih Spirin [23] demonstrated to form in partile free ytoplasmi extrats mixed with purified RNA, but found rather a non-speifi absorption of RNA to preexisting strutures aross the zone of ative polyribosomes and mlrna * protein partiles. Two onsiderations should be taken into aount : a) f mrna-protein omplexes play a physiologial role in the ell, then the formation of ribonuleoprotein omplexes upon mixing of their onstituents must be expeted a priori. The extent of their formation on addition of purified RNA to a lysate or partile fration will depend on the availability of the orresponding proteins and their binding equilibrium, i. e. the physiohemial stability of the omplex. b) f some ytoplasmi proteins may bind purified RNA it does not follow neessarily that ribonuleoprotein partiles already present will bind RNA to the same extent and with equal stability. The two phenomena may be of very different nature. The resistane of native mrna * protein partiles to DTA or to 3M urea (a treatment whih destroys ribosomes) speaks in favour of a relatively tight binding of the protein in the omplex, a situation that may not neessarily apply to the absorption of externally added RNA. Thus the problem is to develop a biohemial distintion between the native partile and a nonspeifi absorption of RNA. Suh an experimental distintion may be derived from the speifi time dependent pattern of syn-

19 314 Messenger RNA in HeLa Cells ur. J. Biohem A T ~ ~ l_r l Fig. 14a-d

20 Val. 17, No.2, 1970 G. SPOHR, N. GRANBOULAN, C. MORL, and K. SCHRRR 315 thesis of two distint types of mrnaeprotein partiles. Furthermore the demonstration of Orhinnikov et al. [35] and Henshaw and Loebenstein [36] that unlabelled purified RNA added to the lysis buffer will not redue the speifi ativity of the omplex labelled in vivo shows that a differentiation is possible. All these arguments are in favour of the real existene of mrna. protein omplexes in the ell. Messenger Charateristis of the RNA in Ribonuleoprotein Partiles One of the ritial questions in the interpretation of our experimental results is the attribution of messenger quality to the labelled RNA ontained in ribonuleoprotein omplexes. n the absene of a diret test in vitro produing a speifi protein in a ell free ribosomal system after addition of the speifi mrna, the only RNA to whih messenger quality an be attributed is by definition the funtional mrna in the polyribosome. However, a labelled RNA in the polyribosome an only be identified with the messenger if it an be demonstrated that the ontinuation of protein synthesis depends on formation and attahment to ribosomes of mrna newly synthesized during the labelling period. This ondition is fulfilled in tissue ulture-ells, even in a ondition of arrested growth, as ould be shown in this investigation. Furthermore the size of mrna is a ruial feature sine, in a given type of ells and assuming a prevalene of monoistroni translation, the mrna moleules should orrespond in size to the types of polypeptide hains being synthesized in this ell. The observed sedimentation spetrum of mrna in HeLa ells is in agreement with the expetation: the vast majority of polyribosomal labelled mrna moleules orresponds to moleular weights of 0.1 to 1.0~10~ daltons and thus to polypeptide hains of to daltons, prevalent in animal ells. t may be pointed out that essentially no mrna or mlrna sedimenting more rapidly than 35 S ould be found in polyribosomes. The presene of RNA Fig. 14. Polyribosomal fration isolated from a surose gradient and treated with DTA or urea. (a) Negatively stained sample of a polyribosomal surose gradient fration (-+) small partiles attahed to polyribosomes (Magnifiation: x ). (b, ) Same preparation of polyribosomes treated with DTA (20 pmoles/mg, 30 min, 0 ) before formaldehyde fixation. The polyribosomal struture is destroyed ; only monoribosomes and ribosomal subunits are present. On shadow-asted preparations some of them present a tail (3);(+) free small partiles. (Magnifiation: (b) ~ ; () x ). (d) Same preparation of polyribosomes as in (a) treated with 3 M urea (30 min 20 ) before formaldehyde fixation : Destrution of the polyribosomal struture. Alter- ations in the morphologial appearane of ribosomal material. (+) Free small partiles; tails (3)(Magnifiation: x ) with higher moleular weights was orrelated with the ourrene of nulear ontamination. The attribution of messenger quality to the RNA ontained in the free mlrna 1 protein partiles is muh more ambigous than the identifiation of the mrna in polyribosomes. Therefore we lassify this RNA as messenger-like in order to distinguish it from true mrna. However several arguments an be brought forward favouring the presene of true messenger in free mlrna protein partiles. (a) As will be shown elsewhere [24] biologial ativity an be deteted in subribosomal frations, free of polyribosomes and ribosome bound mrna * protein, by inubation in vivo under the onditions for ell-free protein synthesis. (b) By ompetitive hybridization we ould show that ommon sequenes must exist in labelled RNA extrated from polyribosomes and from free mlrna * protein partiles [25]. () The spetrum of mrna sizes is essentially the same in polyribosomes and in free mlrna. protein omplexes (f. Fig. 6). (d) n pulse hase experiments a fration of the RNA in free mlrna protein an be hased into polyribosomes. We may thus onlude that true mrna is present both in polyribosome bound mrna * protein and to some extent in free mlrna * protein partiles. The Ribonuleoprotein Complex and Messenger RNA Transport The results presented in this paper also permit some onlusions relative to the mehanism of messenger RNA transport. t was suggested that mrna is transported assoiated with 45 S native ribosomal subunits [21,37,38] or with inomplete ribosomal partiles [39]. Sidebottom and Harris [22] proposed reently that nuleolar ribosome formation was essential for mrna transport. Spirin and oworkers [40] showed on the ontrary that newly synthesized vainia virus-speifi mrna has all the harateristis of a free mlrna *protein omplex and is not assoiated with polyribosomes. Our experimental evidene supports Spirin s model by ontraditing the existene of a linkage between messenger transport and the transfer of newly synthesized ribosomal material into the ytoplasm : a) Protein synthesis and transfer of mrna to the ytoplasm and into polyribosomes proeeds in the absene of rrna synthesis; this was arrested in our experiment by a low dose of atinomyin D as visible in Fig. 2 C and D. b) The labelled mlrna unattahed to polyribosomes sediments in the form of ribonuleoprotein omplexes whih spread over the entire 10 S to 80 S zone of a surose gradient (f. Fig. 5D, ). DTA does not influene this pattern. The presene of labelled material in the 50 S or in the 30 S sedimentation

21 316 Messenger RNA in HeLa Cells Bur. J. Biohem. zone is due to a fortuitous superposition effet. Furthermore, DTA treatment, whih breaks up the mrna-ribosome assoiation (f. Fig. 7A and D) does not alter the density distribution of free mlrna * protein partiles (el. Fig.7C and F). Thus no assoiation of free mlrna. protein with ribosomal material ould be observed. The presene of newly synthesized ribosomal 28 S and 18 S RNA in a CsCl gradient at densities below 1.50 g/m3 shown by Lissitzky et al. [39] to be assoiated mainly with the larifiation pellet of redissolved ytoplasmi partiles may be due to denatured and agglomerated ribosomal material. We ould observe suh material in the eletron mirosope along with the mlrna * protein partiles in the density zone of g/m3. t may be onluded that ytoplasmi mlrna is transported in the form of a ribonuleoprotein omplex as proposed by Spirin et al. [14]. Ribosome synthesis is not neessary for mrna transport. However, there is a limitation to this onlusion. We annot exlude experimentally whether preexisting ribosomal subunits of a nulear pool may play a role in a very short lived, transient phase [41], nor that ribosome formation may be essential for mrna transfer during a phase of regulational adaptation. The Visualisation of mrna Protein Partiles The eletron mirosopial investigation presented here in a preliminary form and whih will be extended in a forthoming paper [42], reveals the presene of small rounded partiles in ytoplasmi frations ontaining ribosomal material, like polyribosomes, monoribosomes or ribosomal subunits. The detailed morphologial appearane and their size is variable. The eletron mirosopial pitures suggest a spetrum of morphologial variations from the smallest type (110 d diameter) with a entral hole or exentri left, to the largest (more that 200 d in diameter) whih seems to be full and more eletron dense; this form is the most frequent found in HeLa ells. These variations an be attributed to differenes in the length of the 35if wide strand whih forms the rounded partiles by oiling. A possible interpretation of the struture seen is that the messenger ribonuleoprotein strand seen in polyribosomes (f. Fig. Zi), upon omplete liberation from (or in between) ribosomes, would oil up into a random oil and be fixed by formaldehyde as the spherial partiles we observe. However, we annot exlude at this stage of the investigation that some different biologial materials may give similar images. n this respet the ultrastrutural resemblane of the smallest partile having a entral hole with RNA polymerase [43,44] or a ontaminant of RNA polymerase preparations [45] should be noted. These enzymes, whih however, should not our in quantity in ytoplasmi extrats, are more homogenous in size and rod shaped rather than spherial. This morphologial feature generally leads to the staking of individual moleules, an effet whih we ould never observe in our preparations. The possibility of a viral ontamination an be exluded by onsiderations of morphology, size and ellular loalisation. Ferritin moleules resembles losely the smallest of the strutures we observed and it annot fully be exluded that some of the small partiles shown (Fig.2a, b) may be ferritin indeed. However ferritin moleules are more homogenous in size (about 00 d in diameter) whereas the full partiles we desribe have diameters of up to more than 200 if. They are also less regular in shape (ferritin appears often retangular) and we never observed the typial tetrad struture. A further onsideration is that, although in HeLa ells ferritin exists, it ould not be demonstrated in our preparations. Similar small partiles an be observed in eletron mirographs illustrating earlier papers on ribosomal subunits [46] or polyribosomes from mammalian ells [47] but no desription of these partiles was given. Slayter et al. [48] mention the presene of partiles 100 d in diameter in rabbit retiuloyte polyribosomes without giving any identifiation. Considering the detailed desription of polyribosomes, monoribosomes and ribosomal subunits given by Shelton and Kuff [49] the morphologial appearane of the small partiles with diameters ranging from 11Od to 200d is easy to distinguish from ribosomal material : the 50 S ribosomal subunit has a diameter of 270 d and the 30 S subunit is ellipsoidal with axes 270 if and 150 d in length (Fig. 12g). The diffiulties enountered in seeing mrna - protein partiles assoiated with polyribosomal strutures may have two explanation. (a) Many may be masked beause of their small size by a superposition effet with ribosomes in negative staining as well as after shadowing. (b) The partial or omplete unoiling of the 35 if wide strand when engaged in polyribosomes is another interesting and suggestive possibility for the diffiulty of their identifiation in the ative omplex of translation. n this respet it should be realled that they ould be identified again as free partiles if the polyribosomal fration was treated with DTA or 3 M urea. n summary we onsider the eletron mirosopial evidene as preliminary only and suggestive but in no way onlusive. The morphologial identity of the free mlrna-protein omplex remains to be proven. The Role of mrna - and mlrna. Protein Complexes The essential result of this investigation is the finding that messenger RNA in the ytoplasm of

22 Vol. 17, No. 2,1970 G. SPOHR, N. GRANBOULAN, C. MORL, and K. SCHRRR 317 HeLa ells is present in the form of ribonuleoprotein omplexes. This observation is in agreement with those of Perry and Kelly [7] in L ells, of Henshaw [8] in liver, of Burny et al. [9] in rabbit retiuloytes and of Cartouzou et al. [lo] in thyroid tissue, that mrna in polyribosomes of animal ells is assoiated with protein. Furthermore, our results are in agreement with those of Spirin and oworkers [6, 14,231 and of Kafatos [50] onerning the existene of free ribonuleoprotein partiles ontaining mlrna in the ytoplasm of developing embryos, of tissue ulture ells and of insets. Our findings support the nformosome model [14,23] of ytoplasmi mrna transport and fit into the general sheme of asade regulation in animal ells [3,4,51]. Suh a mehanism alls for modulating agents whih ould onfer to mrna speifiity in respet to stability, transport and the apability to attah to ribosomal subnits in the proess of initiation of protein synthesis. t is evident that protein moleules would be ideal for suh a mehanism in view of their apability to interat with maromoleules like DNA, RNA and the onstituents of ell membranes, as well as with small moleular weight effetors induing allosteri transitions. Samarina et al. [5,26] and others [53,54], have shown that nasent mlrna in the nuleus is already assoiated with proteins. Muh of these ribonuleoprotein omplexes, whih exist also in HeLa ells [12,55], remain in the nuleus [52, 561. However, some of them are transferred to the ytoplasm onstituting the pool of free mlrna * protein partiles desribed in this investigation. This transfer from the nuleus to the ytoplasm is paralleled by an apparent redution in size of the giant mlrna moleules: although we an detet in the nuleus of HeLa ells, under the labelling onditions used during this investigation, a majority of mlrna moleules in a broad sedimentation zone from 28 S up to an estimated 150 S (as will be shown elsewhere [52]) we never ould detet in the ytoplasm appreiable amounts of RNA with sedimentation onstants of more than 35 S. Sine the appearane of heavier moleules in the ytoplasm ould be orrelated always with nulear leakage, we have to onsider that, at least in HeLa ells, the non-translatable, pre-ribosomal informosomes desribed by Spirin [23] as o-sedimenting with polyribosomes might be of nulear origin. However we found during this investigation that non-translatable, messenger-like ribonuleoprotein omplexes indeed do exist in the ytoplasm of HeLa ells and ontain mlrna spread over sedimentation zones from 6 to 30 S, omparable thus in size to the mrna in polyribosomes. 40 to 60 /, of the ytoplasmi free mlrna does not assoiate with ribosomes in steady state: only a small fration of this free pool an be hased into polyribosomes. The majority of free mlrna * protein omplexes deays as free partiles at the same rate as the polyribosomal mrna-protein omplexes. This average rate of deay is not exponential in time and shows a kineti heterogeneity for the mlrna - protein partiles as well as the polyribosomal mrna protein partiles. We an onlude that they disappear with half-lives of about 1.5 h for the rapidly deaying and of up to about 6h for the more stable frations. We propose that the stability of the translatable messenger RNA and of the non-translated messengerlike RNA of the free pool is an intrinsi property of the ribonuleoprotein omplex. The stability of an mrna may thus be a funtion of the equilibrium onstant of an RNA-protein assoiation whih is in dynami equilibrium with its onstituents. Aording to suh a model, every proteting protein moleule would dissoiate from the omplex with a given statistial frequeny. The size of the non-proteted area and the duration of the periods of non-protetion against hydrolyti or enzymati attaks would determine the life span of an RNA moleule of speifi sequene. Loal nuleotide sequenes are the basis of regions with speifi seondary struture and would determine per se or by a proess of indued fit the binding of the protein moleules. The same kind of onsideration may be justified in regard to the ativation or inativation of an mrna: in fat, the binding onstant of the protein moiety in the mrna. protein omplex might be allosterially influened by ytoplasmi agents. Translational regulation ould be understood assuming that the binding energy of this protein would be in ompetition with the energy provided by the translational system for transloation sine ribosomes moving along the mrna would have to displae temporarily the protein from the omplex. n fat it is a surprising finding that about 50 /, of mlrna in the ytoplasm does not enter the translation proess. This amount may possibly be explained by the low atinomyin dose used for this investigation. However, sine the rate of protein synthesis is affeted less, suh an explanation annot aount for the amount of free mrna. Thus a more physiologial interpretation of the equilibrium between ribonuleoprotein omplexes of mrna and mlrna may be justified. Further work will have to be done in order to determine the atual equilibrium between these omplexes under normal onditions. n embryoni tissue large amounts of maternal messenger RNA are present whih had been ompletely masked ) in the ooyte prior to fertilisation [57]. They may aount for the large amount of nontranslatable mrna in early development. The large fration of inativated messenger ribonuleoprotein in tissue ulture ells points to the possibility that a more general system of regulation not limited to developing systems may be involved. t would ex-

23 ,, 318 G. SPORR et al.: Messenger RNA in HeLa Cells ur. J. Biohem. plain many of the experimental fats brought forward by Tomkins et al. [58] and others in favour of posttransriptional ontrols operating in animal protein synthesis, possibly within the frame of a multilevel regulational system [51]. We gratefully aknowledge the exellent assistane of our tehnial ollaborators: Raymonde Cornuz, who provided us with ells, Pierre-AndrB Briand, RBmy Moret and Claudine Coet. We thank Mr. Alain Gautier, diretor of the Centre de Mirosopie letronique de l Universit6 de Lausanne, for providing us the failities of his laboratory and for helpful disussions. The assistane during the preparation of this manusript of RBmy Moret, Otto Jenny, Annette Muhlbauer and Claude MBrel, and the administrative assistane of Mr.. Marovith (Virology Department, SRC) and of the Central Servies of our nstitute (Mr. M. Zagnoli) are gratefully aknowledged. Our speial gratitude goes to our olleagues Ronald Hanok and Niholas Aheson for ritial reading of this manusript. This work was supported by the Swiss National Foundation (grants , and ). One of us (C. M.) is indebted to the World Health Organization for a Researh Training Grant. (M8/181/4/M. 77). RFRNCS 1. Sherrer, K., n xperimental Biology and Mediine (edited by. Hagen, W. Weshler, and P. Zilliken), Vol., Morphologial and Biohemial Aspets of Cytodifferent;ation, S. Karger, Base1 and New York 1967, pp Sherrer, K., and Maraud, L., Bull. SOC. Chim. Biol. 47 (1965) Sherrer. K.. Maraud. L.. Zaide1a.F.. London.. M.. and Gros,F., Pro. Nat. A&?. ji. U.S.A. 56 (1966a) i Sherrer, K., Maraud, L., Zajdela, F., Brekenridge, B., and Gros, F., Bull. SOC. Chim. Biol. 48 (1966b) Samarina, 0. P., Krihevskaya, A. A., and Georgiev, G. P., Nature (London), 210 (1966) Spirin, A. S., and Nemer, M., Siene, 150 (1965) Perry, R.P., and Kelley, D.., J. Mol. Biol. 35 (1968a) Henshaw,. C., J. Mol. 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A. 69 (1968) Sherrer, K., and Maraud, L., J. Cell. Physiol. 72, Sup. 1 (1968) Sherrer, K., Spohr, G., Granboulan, N., Morel, C., Gros- Claude, J., and Chezzi, C., Cold Spring Harbor Symp. Quant. Biol. 35 (1970), in press. 53. Parsons, J. T., and MCarthy, K. S., J. Biol. Chem. 243 (1968) Kohler, K., and Arends, S., ur. J. Biohem. 5 (1968) Morel, C., Spohr, G., and Sherrer, K., unpublished observations. 56. Shearer, R. W., and MCarthy, B. J., Biohemistry, 6 (1967) Spirin, A. S., Curr. Top. Dev. Biol. 1 (1966) Tomkins, G. M., Gelehrter, T. D., Grannen, R. D., Martin, D., Samuels, H. H., and Thompson,. B., Siene, 166 (1969) G. Spohr and K. Sherrer nstitut Suisse de Reherhe8 xperimentales sur le Caner Rue du Bugnon 21, CH-1005 Lausanne, Switzerland C. Morel s permanent address : Fauldade de Cihias M6dias Universidade de Brasilia, Brasilia, D. F., Brasil