some properties of ribosome crystals ...1972). sheets of ribosome tetramers have been seen in the...

J. Cell Sci. 14, 301-317 (1974) 301

Printed in Great Britain

SOME PROPERTIES OF RIBOSOME CRYSTALS

ISOLATED FROM HYPOTHERMICALLY

TREATED CHICK EMBRYOS

P. G. DONDI AND D. C. BARKER*

University of Cambridge and Medical Research Council,Dunn Nutritional Laboratory, Milton Road, Cambridge, England

SUMMARY

Ribosome crystals observed in hypothermically treated chick embryos exist as large denseaggregates and, for this reason, could be purified from a post-nuclear supernatant by centri-fugation through a high-density discontinuous sucrose gradient.

A ribosome crystal fraction isolated in this way was observed, by electron microscopy, tocontain intact crystals, resembling those seen in sectioned whole cells, with only minor contami-nation from morphologically indistinct particles.

The ribonucleic acid (RNA) content of this fraction was found to be the same as ribosomalRNA from mature chickens. However, their resistance to mild RNase treatment, lack of transferRNA and endogenous messenger RNA activity indicate that such ribosome crystals are not aspecific type of polysome.

The intertetramer bonds (i.e. bonds between tetramers in a crystal array) are much moresusceptible to mild protease treatment than to treatment with ribonuclease (RNase), indicatinga dependence on protein—protein interactions for their formation.

INTRODUCTION

Since the original electron-microscope observations (Byers, 1966, 1967), thephenomenon of ribosome crystallization in hypothermically treated chick embryos hasbeen of interest for two basic reasons. First, as a possible source of crystals whichcould be used in determining the structure of ribosomes by X-ray diffraction, andsecondly, from a functional viewpoint, to determine the significance of such structures.

Morphological studies remain at the electron-microscope level, concentrating mainlyon sectioned material for crystals (Byers, 1966, 1967; Maraldi & Barbieri, 1969;Barbieri, Simonelli, Simoni & Maraldi, 1970), with a more detailed structural analysisof tetramers achieved using negatively stained preparations (Carey, Hobbs & Cook,1972).

Biochemical investigations have been restricted to the basic unit of the crystals,which is an aggregate of 4 ribosomes. This tetramer may be characterized, in terms ofsucrose density gradients, as a discrete molecular species (Bell, Humphreys, Slayter &Hall, 1965; Carey, 1970) and it is this property which has undoubtedly aided thedetermination of many of its biochemical parameters, obtained from studies carried

• Present address: MRC Unit for Biochemical Parasitology, Molteno Institute, Universityof Cambridge, Downing Street, Cambridge, England.

302 P. G. Dondi and D. C. Barker

out both in vitro (Morimoto, Blobel & Sabatini, 1972a) and in vivo (Morimoto, Blobel& Sabatini, 19726).

Other crystal-like aggregates of ribonucleoprotein particles have been described in anumber of organisms, occurring during special in vivo conditions. Hexagonal arraysof packed ribosome helices have been observed in trophozoites and cysts of Entamoebainvadens(Barker & Deutch, 1958) and these 'chromatoid bodies' have been attributedwith properties thought to be unusual to a differentiating system (Barker & Swales,1972). Sheets of ribosome tetramers have been seen in the oocyte and follicular cellsof the lizard Lacerta sicula Raf., during the animal's winter rest (Taddei, 1972). Itis also of interest to note the report that tetrameric crystals occur in necrotic tissue ofchick embryos which have not been subjected to hypothermia (Mottet & Hammar,1972).

The following paper describes the isolation and partial characterization of intactcrystals from chick embryos in an attempt to elucidate their functional significanceand to determine whether crystal ribosomes exhibited any basic property unusual tothe embryonic system.

MATERIALS

Embryonated eggs incubated for 5 days at 37 °C were obtained from Chivers Farm (ArburyRoad, Cambridge).

Creatine phosphate, creatine kinase, adenosine triphosphate (ATP) and guanosine triphos-phate (GTP) were purchased from the Boehringer Co. (London), Trizma (Tris base) from theSigma Chemical Co. (St Louis, Mo.), Ribonuclease (ex bovine pancrease) and Triton X-100from Koch-Light Laboratories (Colnbrook, Bucks.), Proteinase K from E. Merck (Darmstadt,Germany), and [*H]leucine (469 Ci/mmol) from the Radiochemical Centre (Amersham). Allother reagents were analytical grade, mostly from B.D.H. Chemicals Ltd. (Poole, Dorset,England).

METHODS

Buffer solution: TKMX, 50 mn KC1, 5 mM MgClj, 50 imi Tris [tris(hydroxymethyl)aminomethane]-HCl buffer, pH 75.

Isolation of ribosome crystals and control polysomes

Fertile eggs which had been preincubated at 37 °C for 5 days were incubated for a further24 h at 4 °C. After this time the following procedure was adopted, all operations being carriedout at 4 °C.

Embryos were removed from eggs, carefully excised from the amnion in TKM[ and thenwashed twice in TKMi buffer. Between 100 and 120 embryos, in 3 volumes of TKMj, werehomogenized using 6 strokes of a motor-driven Teflon-glass homogenizer at 500 rev/min. Thehomogenate was centrifuged at 1200 gaV for 10 min in the MSE Mistral 4L (Measuring andScientific Equipment Ltd., London). This treatment sediments a large majority of the celldebris, which unfortunately includes large ribosome crystals. However, since homogenizationtakes place in a hypotonic medium, this pellet was discarded rather than increase the risk ofcontamination by nuclear contents.

The post-nuclear supernatant was then adjusted to 1 % Triton X-100 and layered over ahigh-density discontinuous sucrose gradient, consisting of 10 ml of 25 M sucrose-TKMj and10 ml 20 M sucrose-TKMi, and centrifuged in the 3 x 65 swing-out rotor at 60000 gaV for4-5 h in the MSE Superspeed 65 ultracentrifuge. The supernatant was removed and the

Ribosome crystals isolated from chick embryos 303

centrifuge tubes inverted to allow the viscous sucrose to drain. The resulting pellets weretransparent and almost completely colourless and could be resuspended by gentle shaking in asmall volume of T K M , buffer for storage at — 196 °C.

Assuming a value of 135 for the E.}ij;nm (Tashiro & Siekevitz, 1965) the yield of ribosomes inthe crystal fraction was approximately 0 2 mg/g of embryo, with a ratio of absorbances at260 and 280 nm of 1-65-1-7. Electron micrographs (Fig. 9) of stained sections from pelletsprepared in this way show minor contamination.

To obtain control polysomes, 5-day-chick embryos were isolated as described above withoutundergoing hypothermia. The same quantity of embryos were homogenized in 2-5-3 v°l °f0-25 M sucrose-TKMx and then centrifuged at 20000 (*aV for 15 min in the 8 x 50 rotor of theMSE High-speed 18 centrifuge. The supernatant was adjusted to 1 % Triton X-100 and layeredover 5 ml of 2 0 M sucrose-TKMi in the 8 x 25 rotor of the MSE Superspeed 65 and centri-fuged at 160000 )|av for 3-5 h. The resulting colourless, transparent pellets could be resuspendedin a small volume of T K M , buffer and stored at — 196 °C. Suspensions of these control poly-somes gave 260/280 nm ratios of 1-7-1-9.

Electron microscopy

For sectioned material samples were fixed in 2-5 % glutaraldehyde in 0 1 M sodium cacodylatebuffer (pH 7-2-7-4) for 1 h at 4 °C, then washed in 0 1 M cacodylate buffer for 16 h at 4 °C,postfixed in 1% OsO4 in Zetterquist's buffer (pH 7-2-7-4) at room temperature for 1-5 h,'block stained' in 0-5% uranyl acetate for 1 h and dehydrated in a graded series of ethanols.This was followed by two 30-min treatments with propylene oxide, soaking overnight in50:50 propylene oxide:Araldite and embedding in freshly prepared Araldite. Sections were cuton a Reichert ultramicrotome and further stained in a 5 % aqueous solution of uranyl acetateat 60 °C for 15 min followed by lead citrate at room temperature for 20 min (Reynolds, 1963).

To allow a more rapid qualitative determination of crystal samples, several negative stainingtechniques were employed. The procedure which gave the most repeatable results was asfollows. All operations were carried out at 4 °C using pre-cooled Celloidin/carbon-coatedcopper grids.

Samples were applied to grids, using 1 % bovine serum albumin as a spreading agent. Ashort period of fixation in 8 % neutralized formaldehyde (in a solution having the same saltconcentration as TKM,) was found necessary for stabilizing the crystals on the grid. Thistreatment also facilitated washing of the material to remove excess salts and sucrose, especiallywhen samples were taken straight from sucrose density gradients. After removal of excess liquid,negative staining was achieved using an aqueous 1 % solution of uranyl acetate (Carey et al.1972).

Extraction and analysis of ribonucleic acid (RNA)

RNA was extracted from suspensions of ribosome crystals and control polysomes by completedigestion of all proteins present, using chromatographically pure Proteinase K. Separation ofhigh (18 and 28 s) and low (5 and 4s) molecular weight RNA was achieved using LiCl and thenalcohol precipitation (Wiegers & Hilz, 1972).

Analysis of the high-molecular-weight RNA was carried out using 2 4 % acrylamide gels(Loening, 1969). For molecular-weight determinations, rRNAs from mature chick intestine(kindly supplied by Dr J. S. Emtage) and cultured Xenopus cells (kindly supplied by Dr U. E.Loening) were used as standards with known molecular weights (Loening, 1968).

The gels were loaded with 25-50 /ig RNA and run at 8 V/cm length of gel for 3-5 h at 4 °C.Low-molecular-weight RNA was analysed on 5 % acrylamide gels, electrophoresis at 8 V/cm

length of gel for 2-5 h at 4 CC.In both cases, gels and running buffer contained 0 2 % sodium dodecyl sulphate as a ribo-

nuclease inhibitor.Unstained gels were fixed in 5 % acetic acid and scanned at 265 nm using the Joyce-Loebl

ultraviolet gel scanner.


In vitro amino acid incorporation

The incorporation mixture contained in a volume of I ml: 500 fig RNA (i.e. crystals orpolysomes), ~y O.D.u,, units of Sephadex G25 cell sap from chick embryos, 1 fimol ATP,0-4 fimol GTP, 7 fimol creatine phosphate, 50 fig creatine kinase, 0-5 /tmol dithiothreitol, amixture of 19 amino acids (without leucine) giving a final concentration for each amino acid of1 fimol, 0-5 fid ["HJleucine and TKMj buffer to adjust to the final volume.

The above reagents were mixed at o °C and then incubated in a 37 °C water bath to initiatethe reaction. At certain times 5O-/tl samples were removed from the incubation medium andpipetted on to Whatman 3MM paper disks, which were processed as described by Mans &Novelli (1961). The radioactivity in the disks was measured by liquid scintillation countingin 10 ml of toluene containing PPO and POPOP (Packard Instrument Co. Ltd., Illinois) asprimary and secondary scintillants.

Analytical sucrose gradients

Linear sucrose gradients normally used in the analysis of polysomes were most unsuitablefor studying the crystals and their constituents. Even after short centrifugation of say,100000 gav for 10 min, most of the crystal material had pelleted, whilst tetramer and monomerpeaks were completely unresolved. Although polysomes represent a mixture of macromolecules,which cover a range of molecular weights, they may be resolved on gradients into peaks whichincrease in discrete steps according to a regular series. With crystals however, the range is muchgreater and less discrete, with the molecular weights involved sometimes being an order ofmagnitude greater than polysomes.

The following gradient was found to be the most suitable: 5-20% sucrose-TKMi lineargradients (9-5 ml) were generated over 2-5 M sucrose-TKMj (3 ml) in tubes of the SW 36 rotor(Beckman Instruments Ltd., California). Gradients were loaded with 0-2-0-4 ml of samplecontaining between 50 and 150 fig RNA (crystals). To avoid breakdown of crystals by shearingeffects, crystal solutions were always manipulated with wide bore pipettes. Centrifugation at30000 rev/min and 2-4 °C was usually carried out for 35-45 min in SW 36 rotor.

The optical density at 252 nm throughout the gradient was determined using an LKBUvicord II (0'4 cm flow cell) attached to a Vitatron Chart Recorder UR 401. To determine thedistribution of radioactivity, fractions of ~O'5 ml were collected.

Determination of radioactivity in fractions from sucrose density gradients

Fractions (~o-5 ml) were adjusted to 0-3 N NaOH and incubated at 37 °C for 1 h. Then300 fig of bovine serum albumin was added to each, plus an excess of 8 % trichloroacetic acid(TCA) containing 1 mg leucine/ml and the fractions left to stand for 30 min to allow precipitation.The precipitates were then collected on Whatman GF/C glass fibre filter disks using a Milliporefilter system and the disks dried in an oven for several hours. After cooling, the radioactivityin the disks was determined by liquid scintillation counting in 10 ml of toluene/PPO, POPOP.

RESULTS

RNA content of ribosome crystals

High-molecular-weight RNA extracted from the crystal fraction, analysed on 2-4%polyacrylamide gels, was seen to consist of 2 discrete molecules (Fig. IA). Whencrystal RNA and ribosomal RNA (rRNA) from mature chick intestine were loadedon the same gel (Fig. IB), the molecules migrated coincidentally and thus had thesame electrophoretic mobility. Since electrophoretic mobility is directly proportionalto the logarithm of the molecular weight (Bishop, Claybrook & Spiegelman, 1967;Loening, 1969), this indicated that there was no difference between mature chickrRNA and crystal RNA. To confirm this result, crystal RNA and Xenopus rRNA were


2 4 6Migration distance, cm


0 2 4 6 8Migration distance, cm

Fig. i. 2 4 % polyacrylamide gels. Electrophoresis at 8 V cm"1 length of gel, at 4 °C.A, crystal RNA (~ 50 fig) run for 3 h; B, crystal RNA (~2S fig) and chick intestinerRNA (~ 25 fig) run for 3 h; c, crystal RNA (~ 25 fig) and Xenopus rRNA (~ 25 fig),run for 5-5 h. (X = Xenopus rRNA marker.)

1 3

10

0-5

0

-

"l

B

5s

A1 1 1 1


2 4 6

Migration distance, cm

Fig. 2. s % polyacrylamide gels. Electrophoresis at 8 V cm"1 length of gel, at 4 °C.A, low-molecular-weight RNA from control polysomes run for 2'5 h; B, low-molecular-weight RNA from crystal fraction run for 2-5 h. (4s = transfer RNA.)

run on the same gel (Fig. 1 c). Using published data for the molecular weight ofXenopus rRNA (Loening, 1968) it was possible to determine the molecular weightof crystal RNA molecules as 1-58 x io6 and 0-7 x io6, which are the same as thosedetermined elsewhere for chicken rRNA (Loening, 1968).

The low-molecular-weight RNAfrom crystals, analysed on 5 % polyacrylamide gels,exhibited a similar absorption pattern to that extracted from control polysomes(Fig. 2). The major difference observed was the lower amount (~35%) of transferRNA (tRNA) extracted from crystals. This difference has been observed in isolated


0 5

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Fig. 3. Sucrose gradient analysis of A, control polysomes (~ 100 fig RNA), B, crystalfraction (~8o /ig RNA), and C, polysomes after RNase treatment; D, crystal fractionafter RNase treatment. Gradients loaded with o-3-ml samples and centrifuged at30000 rev/min for 35 min. Shaded area = absorbance at 252 nra due to 2'5 Msucrose 'cushion'.


tetramers, where the presence of tRNA is thought to be caused by 'polysomalcontamination and/or tRNA adsorbed during preparation procedures' (Morimoto,et al. 19720). As will be shown later, this explanation also accounts for the presence ofsmall amounts of tRNA in the crystal fraction.

Analysis on sucrose gradients

Fig. 3 shows typical absorbance profiles obtained when control polysomes (Fig. 3 A)and the crystal fraction (Fig. 3B) were centrifuged in the sucrose gradients previouslydescribed (see Methods).

Fractions corresponding to the peaks of absorbance shown in Fig. 3 B were collectedand negatively stained as described in Methods. Peak T was seen to contain tetramers,whilst the major peak of absorbance, which occurs at the interface between the lineargradient and the high density sucrose cushion, was seen to contain large crystal bodies(Fig. 10).

For reasons which are not clearly understood the analysis of crystal fractions onthis type of sucrose gradient did not give perfectly quantitative results. However, inorder to determine the effect, on crystals, of changing conditions in vitro, the gradientsystem used gave a satisfactory qualitative analysis.

Effect of RNase treatment at o °C

The resistance of tetramers to RNase treatment at o °C has now been demonstratedby several workers (Humphreys, Penman & Bell, 1964; Bell et al. 1965; Morimoto etal. 1972a).

The application of this treatment to the isolated crystal fraction was carried out for2 reasons. First, to determine whether the crystal fraction was contaminated with highmolecular weight polysomes and secondly, to estimate the sensitivity of crystals toRNase.

Fig. 3 A shows the absorbance profile obtained using normal polysomes with the peakof monomers (M) being nearest to the gradient meniscus.

As shown in Fig. 3B, the isolated crystal fraction, when resuspended, consistedmainly of large, rapidly sedimenting crystal bodies, which concentrated at, and to someextent penetrated, the 'cushion' interface. A minor component of free tetramers(peak T), presumably derived from partial breakdown of crystals, was also observed.

After 5 min incubation at o °C with 2-5 /tg RNase the normal polysomes were brokendown to a single monomer peak (Fig. 3 c). With the same treatment the crystal fractiongave rise to 3 peaks, where previously only 2 had been observed (Fig. 3D). This thirdpeak (M) was due to the appearance of monomers and accounted for approximately10% of the total absorbance. Since tetramers are resistant to RNase and the crystalpeak at the interface was again observed, it was assumed that these monomers arosedue to the breakdown of contaminating polysomes.

The slight increase in size of peak T and the narrowing and apparent density shiftof the crystal peak is difficult to interpret but may be due to some non-specific degrada-tion of surface ribosomal RNA. However, prolonged treatment of the crystal fractionat o °C with 5 /ig RNase did not alter, to any great extent, the profile shown in Fig. 3 D.

3o8 P. G. Dondi and D. C. Barker

0Top

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Fig. 4. Crystal fraction suspended in TKMi buffer adjusted to various potassiumconcentrations and analysed on gradients of the same ionic content, A, 50 mM K+;B, 150 mM K+; c, 300 mM K+. Each gradient was loaded with o-3-ml samples contain-ing ~6o/ig RNA and centrifuged at 30 000 rev/min for 35 min. Shaded area = ab-sorbance at 252 nm due to 25 M sucrose 'cushion'.

Ribosome crystals isolated from c/tick embryos 3°9

Effect of increasing potassium (K+) concentration

Above a concentration of 250 mM K+, it has been shown that tetramers dissociateinto small subunits and large subunit tetramers (Carey & Read, 1971; Byers, 1971).In order to determine whether intertetramer bonding depended on the small subunit,crystal suspensions were subjected to increasing concentrations of potassium ions(Fig. 4)-

The normal TKM1 buffer had a concentration of 50 mM K+ and under these con-ditions the crystal suspension contained few free tetramers (Fig. 4A). However, whensubjected to 150 mM K+ and analysed on a gradient of the same K+ concentration,the tetramer peak increased in height, and the crystal peak became concentrated at thecushion interface without penetrating into the high density sucrose (Fig. 4B). Thisindicated a partial breakdown of crystal bodies resulting in smaller, less-dense crystalsand release of free tetramers. When the potassium concentration was further increasedto 300 mM K+, the crystal peak again decreased in height whilst the tetramer peakincreased and a minor monomer peak was produced (Fig. 4c).

It would seem, therefore, that increasing the potassium concentration affects theintertetramer bonds before subunit interactions are disrupted and, that these bonds aremore susceptible than intratetramer bonds to increased potassium concentrations.

Effect ofProteinase K

A suspension of the crystal fraction was treated with 1 /ig of Proteinase K at o °Cfor 5 min. Fig. 5 shows the sucrose gradient profiles of control (A) and treated crystals(B).

c

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0

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Top Volume, ml BottomFig. 5. Crystal fraction A, before, and B, after incubation with i/tg ofProteinase K ato CC for 5 min. Gradients loaded with 03 ml containing ~70/tg RNA and centri-fuged at 30000 rev/min for 35 min. Shaded area = absorbance at 252 nm due to2-5 M sucrose 'cushion'.

P. G. Dondi and D. C. Barker

Volume, ml Bottom

Volume, rnl Co:tom

4 8 Volume, ml

4 8 Volume, ml

A

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Bottom

Fig. 6 . Crystal fraction suspended in TKM, buffer adjusted to the following mag- nesium concentrations, A, control, o OC for 20 min, 5 mhf R1gz+; B, 37 OC for 20 min, 5 mhr MgZ+; c, 37 "C for 20 min, 15 rmf h1gzf; and D, 37 OC for 20 min, 30 mM M$+. Gradients loaded with 0.3 ml containing 70pg RNA and centrifuged at '

30000 rev/min for 35 min. Shaded area absorbance at 252 nm due to 2.5 A* sucrose ' cushion '.


Just mild treatment in this way caused almost complete breakdown of the largecrystal bodies with a concomitant increase in the amount of free tetramers (Fig. 5B).Only a small amount of absorbance remains at the ' cushion' interface, some, if not all,of which may be due to contaminating polysomes.

This clearly shows that the intertetramer bonds, joining tetramers to adjacenttetramers in crystals, are, in some way, dependent mainly on a protein or proteins fortheir formation.

Effect of incubation at 37 °C and the stabilizing effect of increased Mgi+ concentration

Fig. 6 shows the effect of incubation at 37 °C on crystal suspensions in TKM^buffer and TKMi adjusted to different Mg2+ concentrations.

With 5 raM Mg2+ at o °C the crystal suspension exhibited the normal gradientprofile (Fig. 6 A) but on incubation at 37 °C for 20 min the crystals had partiallybroken down giving a substantial number of free tetramers, and monomers (Fig. 6B).

At 15 mM Mg2+ and 37 °C for 20 min the tetramer peak increased whilst the mono-mer peak decreased. However, this apparent stabilization did not apply to the crystalsto the same extent (Fig. 6c). Increasing the concentration of Mg2+ to 30 mM had nofurther effect on the stabilization (Fig. 6D).

In vitro amino acid incorporating activity and its effect on crystal structure

The lack of endogenous activity for in vitro incorporation of radioactively labelledamino acids has been convincingly demonstrated using isolated tetramers (Morimoto

30 r

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Fig. 7. In vitro amino acid L-[4,5-3H]leucine incorporation. 0—%, control polysomes,O—O, crystal fraction.


et al. U)j2a). It was therefore necessary to determine whether tetramers aggregated inthe stable crystal formation exhibited endogenous incorporating ability.

Suspensions of the crystal pellet and control polysomes were used in an incubationmedium as described in Methods. The reaction was started by transferring the mix-tures from ice to a 37 °C water bath, with samples being removed at certain intervals.As can be seen in Fig. 7, the crystal fraction exhibited some incorporating ability, butthis amounted to only 30 % of the activity shown by normal control polysomes.

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Fig. 8. Crystal fraction after in vitro amino acid incorporation, A, 37 °C for 30 min,5 mM Mg*+; B, 37 °C for 30 min, 15 miu Mg*+. Gradients loaded with 0-4 mlcontaining ~ioo/tg RNA and centrifuged at 30000 rev/min for 45 min. O—O,radioactivity in cpm. Shaded area = absorbance at 252 nm due to 2-5 M sucrose'cushion'.

This finding indicated that either the crystals had some incorporating ability orthat the crystal fraction was contaminated by very large polysomes. From the results ofRNase treatment described (p. 307) the latter possibility would seem most likely.

When a sample of crystals, which had been incubated in a protein-synthesizingmedium (see Methods, Amino acid incorporation), was run on the sucrose gradientsystem and fractionated, almost complete breakdown to single ribosomes (M) wasobserved (Fig. 8 A). Since the medium had a concentration of 5 mM Mg2+ breakdownof this type was not expected (cf. Fig. 6B). Hence, some component of the incubationmedium was thought responsible for this efficient disruption of the crystal bodies andtetramers.

Ribosome crystals isolated from cluck embryos 313

A crystal fraction suspended in TKMj was incubated at 37 °C for 20 min in thepresence of the 'energy system' (i.e. ATP, creatine phosphate and creatine kinase) butno cell sap. After gradient analysis a profile similar to that shown in Fig. 6 B was ob-tained, with monomers and tetramers in approximately equal proportions.

In an attempt to eliminate the possible effects of a contaminating RNase, an in vitroincubation was carried out in TKMj with 'energy', chick embryo cell sap and ratliver cell sap, as a source of RNase inhibitor (Roth, 1958). However, after incubationat 37 °C for 20 min, exactly the same breakdown effect was observed as that shown inFig. 8A.

When the incubation medium was adjusted to 15 ITIM Mg2+, both tetramers andcrystals were observed to have been stabilized, but overall protein synthesis of thecrystal fraction had been inhibited by 85 % (Fig. 8B).

Thus, there is some component of the chick cell sap, which under normal in vitroprotein-synthesizing conditions causes efficient breakdown of both crystals andtetramers. Whether this breakdown is due to the mechanisms of protein synthesis orsome undetermined disruptive enzyme is difficult to determine, since increasing theMg2+ concentration was seen to inhibit protein synthesis but may also have affectedother enzyme processes.

Whilst determining the distribution of radioactivity through gradients after invitro protein synthesis with crystal fractions, it was found necessary to process thefractions with mild alkaline conditions as described in Methods, p. 304. If thistreatment was omitted it was found that the fraction corresponding to the residualabsorbance at the 'cushion' interface contained as much radioactivity as the monomerpeak. However, after treatment with sodium hydroxide at 37 °C, which hydrolysesfree aminoacyl-tRNAs, this radioactivity fell to background levels. This finding wouldseem to indicate that crystal bodies have a tendency to adsorb free tRNA.

DISCUSSION

The procedure described gives a simple method for the isolation of a ribosomecrystal fraction which is only partially contaminated by large polysomes (~io%)and morphologically indistinct bodies. Previous isolation procedures (Barbieri et al.1970) have been described as only partially successful (Nanninga, 1973).

The RNA of these ribosome crystal bodies is the same as RNA from normal chickribosomes, in that the large and small subunit RNA molecules from crystals have thesame molecular weights as corresponding molecules from mature chicks. A 5scomponent is also present in crystal ribosomes. These results are compatible with thosedetermined for isolated tetramers (Byers, 1971; Carey & Read, 1971; Morimoto et al.1972 a) but differ markedly from those found in the Entamoeba crystal system (Barker& Swales, 1972).

Ribosome crystals are generally resistant to mild RNase treatment, lack sufficientamounts of tRNA and have little or no endogenous activity for in vitro amino acidincorporation. These findings would seem to indicate that crystals are not equippedto carry out in vivo protein synthesis, and in principle are in agreement with the

314 P- G. Dondi and D. C. Barker

properties already determined for isolated tetramers (Morimoto et al. 1972 a). A lackof in vivo protein synthesis has also been observed in the tetrameric crystal bodies oflizard oocytes (Taddei, 1972).

The instability of intertetramer bonds under increasing concentrations of potassiumand the breakdown of these bonds by mild protease treatment shows their dependenceon protein-protein interactions.

Although crystal formation in chick embryos is induced by artificial cooling,hypothermia is undoubtedly a condition which could be experienced by embryos innatural surroundings. The high viability of embryos, reincubated at 37 °C afterartificial hypothermia, with concomitant breakdown of crystal bodies (Byers, 1966)might, at first sight, attribute crystal formation to some special embryonic defencemechanism against adverse conditions. However, such a complex mechanism isdifficult to envisage and has certainly not been conclusively demonstrated. Indeed,the formation of ribosome crystals may be purely and simply a physical phenomenon.

Nevertheless, the isolation of such crystals provides a unique opportunity to study ahomogeneous population of inactive ribosomes produced in vivo. A detailed and com-parative study of ribosomal proteins from crystals and active polysomes may yielduseful information concerning the protein-dependent intertetramer bonds, and couldalso lead to a better understanding of the control of protein synthesis.

This work was supported by a Medical Research Council Scholarship for training in ResearchMethods. We would like to thank Dr E. Kodicek for his interest and encouragement. We aregrateful to Miss L. S. Swales for her expert technical assistance with the electron microscopyand Mr K. R. Symonds for his skilful technical help.

NOTE

The results reported in a paper (Taddei et al. 1973: Expl Cell Res. 78, 159-167)published after the completion of the above work, attribute the tetrameric crystals ofthe lizard, Lacerta sicula Raf. with in vitro endogenous messenger activity. Taddeiet al., however, state that, due to difficulties encountered in the isolation of theseribosomal bodies, such activity may be real or artifactual.

It is felt that the results represented in the above paper, with particular reference toisolation and sucrose gradient analysis, may help resolve this problem.

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BARKER, D. C. & DEUTCH, K. (1958). The chromatoid body of Entamoeba invadens. Expl CellRes. 15, 604-610.

BARKER, D. C. & SWALES, L. S. (1972). Characteristics of ribosomes during differentiation fromtrophozoite to cyst in axenic Entamoeba sp. Cell Differentiation 1, 297-306.

BELL, E., HUMPHREYS, T., SLAYTER, H. S. & HALL, C. E. (1965). Configuration of inactive andactive polysomes of the developing down feather. Science, N. Y. 148, 1739—1741.

BISHOP, D. H. L., CLAYBROOK, I. R. & SPIEGELMAN, S. (1967). Electrophoretic separation ofviral nucleic acids on polyacrylamide gels. J. molec. Biol. 26, 373-387.


BYERS, B. (1966). Ribosome crystallization induced in chick embryo tissues by hypothermia.J. CellBioL 30, C1-C6.

BYERS, B. ( I 967). Structure and formation of ribosome crystals in hypothermic chick embryo cells.J. molec. Biol. 26, 155-167.

BYERS, B. (1971). Chick embryo ribosome crystals: Analysis of bonding and functional activityin vitro. Proc. natn. Acad. Sci. U.S.A. 68, 440-444.

CAREY, N. H. (1970). Ribosomal aggregates in chick embryo tissues after exposure to lowtemperatures. FEBS Lett. 6, 128-130.

CAREY, N. H., HOBBS, J. R. W. & COOK, E. A. (1972). The structure of nbosomes as indicatedby studies on tetramers from hypothermic chick embryos. Biochem. J. 130, 871-877.

CAREY, N. H. & READ, G. S. (1971). The arrangement of ribosomes in ribosome tetramers fromhypothermic chick embryos. Biochem. J. 121, 511-519.

HUMPHREYS, T., PENMAN, S. & BELL, E. (1964). The appearance of stable polysomes during thedevelopment of chick down feathers. Biochem. biophys. Res. Commun. 17, 618—623.

LOENINC, U. E. (1968). Molecular weights of ribosomal RNA in relation to evolution. J. molec.Biol. 38, 3S5-365-

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FEBS Lett. 23, 77-82.{Received 8 June 1973)

C E L 14

P. G. Dondi and D. C. Barker

0-5 urni f i

Fig. 9. Stained sections from isolated crystal pellets, x 44000.

Ribosome crystals isolated from chick embryos

i m

•5*0-5 //m

10BFig. io. Negatively stained preparations, A, crystal suspension after thawing from— 196 °C, x 45000; D, fraction obtained from ' cushion' interface of a sucrose gradient,x 34000; and c, fraction obtained from peak T of a sucrose gradient, x 68000.

some properties of ribosome crystals ...1972). sheets of ribosome tetramers have been seen in the...

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