sites of protein synthesis in nucleoli of root meristematic cells of

J. Cell Sci. 2, 473-480 (1967) 473Printed in Great Britain

SITES OF PROTEIN SYNTHESIS

IN NUCLEOLI OF ROOT MERISTEMATIC

CELLS OF ALLIUM CEP A AS SHOWN BY

RADIOAUTOGRAPHY WITH [3H]ARGININE

L. A. CHOUINARD AND C. P. LEBLONDDepartments of Anatomy, Laval University, Quebec City andMcGill University,Montreal, Canada

SUMMARY

The interphase nucleolus in root meristematic cells of Allium cepa may be divided into fourregions, three of which are always present: the fibrillar, granular and lacunar regions, while thefourth or vacuolar region may be missing. The sites of protein synthesis in nucleoli were investi-gated by means of light and electron-microscope radioautography after a 5-min immersion ofthe roots in a solution of [3H]arginine.

The radioautographs of interphase nucleoli showed many silver grains over both the fibrillarand the granular regions. Occasional silver grains were also recorded over, or close to, thelacunar regions, but none were over the vacuolar regions. A 15-min chase period did not changethe radioautographic pattern. It is concluded that the three permanent regions of the interphasenucleoli, namely the fibrillar, the granular and the DNA-containing lacunar regions, are sitesof protein synthesis.

INTRODUCTION

Light- and electron-microscope observations reveal that the interphase nucleoli inplant cells are made up of several components segregated into regions distinguishableby differences in staining properties and ultrastructural characteristics (Lafontaine& Chouinard, 1963; Hyde, Sankaranarayanan & Birnstiel, 1965; Chouinard, 1966 a, b).That protein synthesis takes place in plant nucleoli has been shown by the uptake oflabelled amino acids as detected by radioautography in the light microscope (Woodard,Rash & Swift, 1961; De, 1961; Mattingly, 1963) and by biochemical analyses (Birn-stiel, Chipchase & Bonner, 1961; Birnstiel & Hyde, 1963; Birnstiel & Flamm, 1964;Flamm & Birnstiel, 1964). Yet it is not known which of the structural componentsof the nucleolar mass is instrumental in these processes of protein synthesis. Thesynthesis of protein has also been observed in the nucleoli of animal cells, as shownin the review by Stocker (1963). The uptake of labelled amino acids first observed byFicq in 1953 was later questioned (Schultze, Oehlert & Maurer, 1958; Carneiro &Leblond, 1959), but with improvements in technique was clearly demonstrated in thelight microscope (Leblond & Amano, 1962; Kasten & Strasser, 1966) as well as in theelectron microscope (Sandborn, 1963; Droz, 1965).

The present investigation was undertaken to pinpoint the sites of synthesis of

474 •£• A. Chouinard and C. P. Leblond

proteins in interphase nucleoli of root meristematic cells in Allium cepa. Radio-autography was used in the light and electron microscopes following pulse-labellingof the proteins with tritiated arginine.

MATERIALS AND METHODS

Seeds of Allium cepa were germinated on the surface of loosely packed vermiculitekept moist by daily watering. After i week germination at room temperature, theprimary roots of the seedlings had reached approximately i cm in length. The roots,still attached to the seedlings, were then immersed for 5 min in distilled watercontaining 100 /ic/ml of tritiated arginine. The isotope used, L-[G-3H]arginine mono-hydrochloride (Radiochemical Centre, Amersham, England), has a specific activity of1830 mc/mM. At the end of the 5-min pulse treatment, the seedlings were rinsed indistilled water and the distal 1-5 mm of some root tips were fixed for 2 h at roomtemperature in a mixture of formaldehyde and glutaraldehyde (Karnovsky, 1965) inO-IM Sorensen's phosphate buffer, PH7.2; the remainder of the undetached rootswere re-immersed for a period of 15 min in a solution of non-radioactive L-argininemonohydrochloride made up to a concentration 100 times that of the radioactivesample. Following this chase period, the seedlings were rinsed with distilled waterand the root tips fixed as described above. After fixation, all root tips were placedovernight in O-IM Sorensen's buffer, pH 7-2, at 4 °C; the next day, they were dehy-drated over a 2-h period in an ascending series of ethanol concentrations and finallyembedded in Epon 812.

Half-micron sections of the root meristematic regions were mounted on glass slideswith a wire loop and coated by dipping in liquid Ilford L-4 emulsion. After 48 hexposure and development, a drop of filtered 1 % toluidine blue was placed on theslides, allowed to stand at room temperature for 5 min, and rinsed off with distilledwater. After dehydration, a coverslip was placed over the sections. The emulsion-coated and exposed sections as well as the uncoated half-micron sections from thesame blocks were studied with a Leitz binocular microscope, using a ribbon-filamentlamp, Kohler illumination, and a 100 x 1-32 N.A. apochromatic objective. An orangeWratten filter was used in the illumination system.

Ultrathin pale gold sections, cut from the same tissue blocks as the o-5-/t sections,were mounted on celloidin-coated glass slides, coated with a layer of Gevaert 307emulsion and, after 2 weeks exposure and proper development, stripped and mountedon 300-mesh copper grids (Salpeter & Bachmann, 1964). Successful double stainingof these grids was achieved by floating, emulsion side down, upon drops of a saturatedsolution of uranyl acetate in 50% ethanol (30 min) followed by lead citrate (30 min).Uncoated sections were also stained in the same manner for electron-microscopeexamination. The sections were examined in a Siemens Elmiskop I electron micro-scope using the double condenser, 80 kV and 50-/6 objective aperture.

Protein synthesis in nucleolus of Allium 475

OBSERVATIONS

Structural organization of the interphase nucleolus

Light microscopy. A. cepa is a diploid species with a single pair of nucleolar chromo-somes (Heitz, 1931). Two nucleoli, therefore, are formed at telophase and these,during interphase, may remain distinct (Fig. 1) or adhere to form a dumbbell-shapedstructure, or fuse into a large spherical organelle which may reach up to 6/t indiameter (Figs. 3, 4).

In 0-5-/4 sections of meristematic cells stained with toluidine blue, the nucleolarmaterial exhibits a purplish blue metachromatic colour (Figs. 1-4). The nucleolusoften contains a large, unstained space located centrally or paracentrally, the nucleolarvacuole (Figs. 3, 4). The vacuole may be missing, or there may be more than one(Fig. 3). With an orange filter, the metachromatically stained material of the nucleolusproves to be not homogeneous but made up of two components slightly differing instaining intensity (Figs. 1-4). The less intensely stained component is located alongthe edge and in the centre of the nucleolus as well as in radial areas extendingin between (Figs. 1, 2); this component also surrounds the vacuole when present(Figs. 3, 4). The more intensely stained component, on the other hand, is arranged inirregular patches usually located between the edge and the centre of the nucleolus(Figs. 2, 4). Within these patches, there are a few tiny unstained spaces which will bereferred to as lacunae (Figs. 2, 3, arrows). Such lacunae are less readily identified herethan in sections of osmium-fixed meristematic cells stained by the Feulgen/methyleneblue procedure; they are then seen to be a constant feature of the more intenselystained component of the nucleolus (Chouinard, 1966a, b).

Electron microscopy. The nucleoli appear as electron-dense bodies containing lightspaces corresponding to the vacuoles and lacunae distinguished in the light micro-scope. The dense portion shows denser and less dense regions, corresponding respect-ively to the more and the less intensely stained regions seen in the light microscope(Fig. 9). The denser regions have irregular boundaries with processes projectinginto the surrounding less dense regions; small isolated patches of denser materialmay also be embedded within the less dense regions.

The less dense regions of the nucleolus consist mainly of packed granules about150 A in diameter (Fig. 9). In addition, fibrils are encountered which also are 150 Ain diameter and, therefore, would appear as granules when cut across. Occasionally,series of granules are strung like beads; or barely distinguishable granules appear asperiodic thickenings along the 150-A fibrils. Finally, a few fine, parallel fibrils approxi-mately 60 A or more in diameter may also be seen among the granules. These lessdense regions will be referred to as 'granular'.

The denser regions of the nucleolus are composed of an amorphous ground substancecontaining tightly packed, electron-dense fibrils (Fig. 9). Most of them are thin witha diameter of 60 A. Larger ones may also be observed up to 150 A. The denser regionswill be referred to as 'fibrillar'.

Of the lighter spaces, some are vacuoles. These contain loosely and uniformlydispersed granules and fibrils. The vacuoles are enclosed within the granular region.

476 L. A. Chouinard and C. P. Leblond

On the other hand, within the fibrillar region, the lacunae appear as light stainingspaces usually smaller than the vacuoles but variable in size and shape and usuallyexhibiting a loose fibrillar texture (Fig. 9, arrows); in the lacunae it is often possibleto distinguish a core of dense material staining with the same intensity as the chromatin(Figs. 9-13, double arrows). In this connexion, evidence has recently been obtainedindicating that, as in the case of the large Spirogyra nucleoli (O'Donnel, 1961; Godward& Jordan, 1965), the lacunae in question contain DNA. It is likely that these lacunaeare cross-sections of a channel containing the nucleolar organizing region of thenucleolar chromosome (Chouinard, 1966 c).

In summary, the interphase nucleolus in root meristematic cells of A. cepa is madeup of 4 structural components segregated into regions distinguishable by differencesin staining properties and ultrastructural characteristics: 2 dense ones referred to asgranular and fibrillar regions, and 2 light ones, as vacuolar and lacunar regions.In previous electron-microscope observations based on osmium-fixed material, evi-dence had been presented suggesting that the 2 main structural components of thedense portion of the nucleolus (i.e. the granular and the fibrillar) correspond respect-ively to what has been referred to in animal nucleoli as nucleolonema (or nucleolone-matic network) and pars amorpha (Chouinard, 1966 a, b). Indeed, in several placeswithin the granular regions and more particularly on the fluffy outer surface as wellas at the surface of the vacuoles, light irregular narrow spaces may be detected suggest-ing that the granules and fibrils are assembled into a coarse network, the meshes ofwhich are approximately o-1 ju, in diameter (Fig. 9); this would correspond to thenucleolonema.

Sites of incorporation of tritiated arginine into interphase nucleoli

Light microscopy. As early as 5 min after immersion of the primary roots of A. cepainto a solution containing [3H] arginine, a radioautographic reaction was observed overall cells of the meristematic region. Representative light-microscope radioautographsof interphase cells (Figs. 5-8) showed many silver grains over the nucleolus, a fewover the rest of the nucleus, that is, mainly over chromatin and, finally, a few overcytoplasm. Careful examination of the nucleoli showed grains over both the denserand less densely stained regions (Figs. 5, 6), but not over the vacuoles (Figs. 7, 8).

Electron microscopy. In the cytoplasm, a radioautographic reaction was found overthe ribosome-rich regions, and, occasionally, over mitochondria. In the nucleus, thesilver grains overlay the nucleolus, to a lesser extent the chromatin material, and to astill lesser extent the nuclear sap. Examination of electron-microscope radioautographsof the nucleolus (Figs. 10-13) showed silver grains over both fibrillar (Fig. 11) andgranular regions (Fig. 10). Grains were located not only over the main fibrillar regionsbut also over the small and apparently isolated patches of similar fibrillar materialembedded in the granular regions (Fig. 12). Significant numbers of silver grains overlaythe irregular and often diffuse boundaries where fibrillar and granular regions merged(Fig. 13). Finally, a few silver grains were located over, or in the immediate vicinityof, the DNA-containing lacunar regions (Fig. 12), whereas there was no significantlabelling of the vacuolar regions.


Pulse-chase experiment. In this case, the roots were immersed first in a solution of[3H]arginine for 5 min and then in a solution of non-radioactive arginine for 15 min.Again light- and electron-microscope radioautographs showed labelling in all meriste-matic cells. Except for a 10-20 % reduction in the number of silver grains over thenucleoli, the distribution pattern of the labelling appeared essentially the same as thatdescribed in the experiments without chase.

DISCUSSION

The present observations revealed that, as early as 5 min after immersion of primaryroots of A. cepa in a solution of [3H]arginine, a radioautographic reaction was obtainedover the cytoplasm and nucleus of meristematic cells. This indicated that the radio-active arginine must have reached even the cells in the centre of the meristem and beentaken up into their cytoplasm and nucleus. Because of the high solubility of argininein water, it was assumed that any free arginine present in the cells would be washed outduring fixation and the subsequent 12-h immersion in buffer. However, when thelabelled specimens were subjected to large doses of non-labelled arginine in thepulse-chase experiment, the grain count was decreased by 10-20 %. Hence, of thelabel taken up at 5 min, 10-20% may consist of free arginine which is adsorbedstrongly enough not to be washed out during processing. [It is possible that the 10-20 %decrease in radioactivity after the 15-min pulse chase is not due to removal of free,labelled arginine. An alternative explanation would be that some of the newly synthe-sized proteins of the nucleolus have completely turned over during the 15 min periodas a result of emigration or breakdown.] But the radioautographic pattern was notvisibly modified by the pulse chase. Consequently, the pattern would be caused bythe rest of the label, that is, the 80-90 % which was not adsorbed and must thereforehave been incorporated into newly synthesized tissue components. Indeed, it wasknown that, after injection of a labelled amino acid, only that fraction taken up intonewly synthesized proteins was retained in the sections (Droz & Warshawsky, 1963;Leblond, 1965). Hence, the distribution pattern of radioactivity after glutaraldehyde-formaldehyde fixation indicated the location of protein synthesized during the 5 minof exposure to the labelled amino acid. If it is assumed that this time is too short forsignificant migration of the new protein, then the radioactive regions are the sites ofprotein synthesis. It may therefore be concluded that organelles of cytoplasm (ribo-somes, mitochondria) and of nucleus (chromatin material, Figs. 5,1 o, 13, and nucleolus,Figs. 5-8 and 10-13) were concomitantly the seats of protein synthesis.

Incorporation of [3H]arginine was observed in all interphase nucleoli. Hence, itwould appear that protein synthesis in that organelle is a continuous process through-out interphase. These observations confirm previous cytological findings that inter-phase nucleoli in root meristematic cells are sites of continuous protein synthesis(Woodard et al. 1961; De, 1961; Mattingly, 1963). Judging from the relative con-centration of silver grains in Figs. 5-8, it also seems as if the rate of protein synthesisin the nucleolus of A. cepa is more rapid than that occurring elsewhere within the cellnucleus, a conclusion confirming previous biochemical observations (Birnstiel et al.


1961; Birnstiel & Hyde, 1963; Birnstiel & Flamm, i964;Flamm&Birnstiel, 1964). Thenature of nucleolar proteins which were being synthesized during the 5-min exposureto [3H]arginine is not known. They could be any of the four types of proteins whichbiochemists claim are present in both plant and animal nucleoli, since all four havebeen shown to contain arginine and become labelled after administration of [3H]aminoacids (Birnstiel, Chipchase & Flamm, 1964; Grogan, Desjardins & Busch, 1966).

In the present study, a radioautographic reaction was observed over both thegranular and the fibrillar regions of the nucleolus. Hence, it may be concluded thatthese two regions are sites of protein synthesis. In addition, a few silver grains werelocated over, or in the immediate vicinity of, the DNA-containing lacunar regions,thus raising the possibility that the intranucleolar DNA also may somehow be involvedin protein synthesis. These observations may be interpreted in relation to what isknown of RNA metabolism in the nucleolus. A number of authors have observed thatRNA synthesis takes place in the fibrillar regions of animal nucleoli and subsequentlythe newly synthesized RNA migrates to the surrounding granular regions (Granboulan& Granboulan, 1965; Karasaki, 1965; Geuskens & Bernhard, 1966). In root meriste-matic cells RNA synthesis was also associated with the fibrillar regions of the nucleolus(La Cour & Crawley, 1965). According to Karasaki (1965) and Granboulan & Gran-boulan (1965) chromatin material present in the fibrillar regions of the nucleolusparticipates in nucleolar RNA synthesis. It is conceivable that the newly synthesizedRNA is in the form of nucleoprotein fibrils (Marinozzi, 1964), which in the course ofmigration to the granular region become beaded and transform into granules. It istempting to speculate further that the RNA molecules which are being synthesizedby the intranucleolar chromatin and which subsequently invade the rest of thenucleolar mass, are, during their migration, somehow instrumental in the elaborationof protein moieties in both the fibrillar and the granular regions of the nucleolus.

In the present study, no significant labelling of the vacuolar regions of the nucleoluswas observed, a fact indicating that synthesis of proteins does not take place, or isnegligible, there.

The general conclusion to be drawn from the above observations is that all three ofthe permanent structural components of the interphase nucleolus in root meristematiccells of A. cepa, namely, the fibrillar, granular and DNA-containing lacunar regions,are sites of protein synthesis.

The authors gratefully acknowledge the assistance of Dr Beatrix Kopriwa in preparation ofthe radioautographs. We also thank Mr P. Koen for skilled technical assistance. This investiga-tion was supported by grants from the Medical Research Council of Canada.

REFERENCES

BIRNSTEIL, M., CHIPCHASE, M. & BONNER, J. (1961). Incorporation of leucine-H3 into sub-nuclear components of isolated pea nuclei. Biochem. biophys. Res. Commun. 6, 161-166.

BIRNSTIEL, M., CHIPCHASE, H. & FLAMM, W. G. (1964). On the chemistry and organization ofnucleolar proteins. Biochim. biophys. Ada 87, m-122.

BIRNSTIEL, M. & FLAMM, W. G. (1964). Intranuclear site of histone synthesis. Science, N.Y.145. 1435-1437-


BIRNSTIEL, M. & HYDE, B. B. (1963). Protein synthesis by isolated pea nucleoli. J. Cell Biol.18,41-50.

CARNEIRO, J. & LEBLOND, C. P. (1959). Continuous protein synthesis in nuclear chromatin, asshown with tritium-labelled amino acids. Science, N.Y. 129, 391-392.

CHOUINARD, L. A. (1966a). Nucleolonema and pars amorpha in root meristematic cells ofViciafaba. Can. jf. Bot. 44, 403-411.

CHOUINARD, L. A. (19666). Nucleolar architecture in root meristematic cells of Allium cepa.Natn. Cancer Inst. Monogr. 23, 125-143.

CHOUINARD, L. A. (1966c). Localization of intranucleolar DNA in root meristematic cells ofAllium cepa. 3. Cell Biol. 31, 139 A.

DE, D. N. (1961). Autoradiographic studies of nucleoprotein metabolism during the divisioncycle. Nucleus, Calcutta 4, 1-24.

DROZ, B. (1965). Accumulation de prote'ines nouvellement synthe'tise'es dans l'appareil de Golgidu neurone; 6tude radioautographique en microscopie 61ectronique. C. r. hebd. Se'anc. Acad.Sci., Paris 260, 320—322.

DROZ, B. & WARSHAWSKY, H. (1963). Reliability of the radioautographic technique for thedetection of newly synthesized proteins. J. Histochem. Cytochem. n , 426-435.

FICQ, A. (1953) Incorporation in vitro du glycocolle-i-C14 dans lesoocytes d'Aste>ies. Experientia9, 377-379-

FLAMM, W. G. & BIRNSTIEL, M. (1964). Studies on the metabolism of nuclear basic proteins.In The Nucleohistones (ed. J. Bonner & P. Ts'o), pp. 231-241. San Francisco: Holden-Day.

GEUSKENS, M. & BERNHARD, W. (1966). Cytochimie ultrastructurale du nucle'ole. III. Actionde l'actinomycine D sur le me'tabolisme du RNA nucl^olaire. Expl Cell Res. 44, 579-598.

GODWARD, M. B. E. & JORDAN, E. G. (1965). Electron microscopy of the nucleolus of Spirogyrabritannica and Spirogyra ellipsospora. Jl R. microsc. Soc. 84, 347—360.

GRANBOULAN, N. & GRANBOULAN, P. (1965). Cytochimie ultrastructurale du nucteole. Etudedes sites de synthdse du RNA dans le nucl^ole et le noyau. Expl Cell Res. 38, 604-619.

GROGAN, D. E., DESJARDINS, R. & BUSCH, H. (1966). Nucleolar proteins of rat liver and Walkertumor. Cancer Res. 26, 775-779.

HEITZ, E. (193I). Die Ursache der gestezmassigen Zahl, Lage, Form und Grosse pflanzlicherNukleolen. Planta iz, 775-792.

HYDE, B. B., SANKARANARAYANAN, K. & BIRNSTIEL, M. (1965). Observations on fine structurein pea nucleoli in situ and isolated. J. Ultrastruct. Res. 12, 652-667.

KARASAKI, S. (1965). Electron microscopic examination of the sites of nuclear RNA synthesisduring amphibian embryogenesis. J. Cell Biol. 26, 937-958.

KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for usein electron microscopy. J. Cell Biol. 27, 137-138.

KASTEN, F. H. & STRASSER, F. F. (1966). Amino acid incorporation patterns during the cellcycle of synchronized human tumor cells. Natn. Cancer Inst. Monogr. 23, 353-368.

LA COUR, L. F. & CRAWLEY, J. W. C. (1965). The site of rapidly labeled ribonucleic acid innucleoli. Chromosoma 16, 124-132.

LAFONTAINE, J. G. & CHOUINARD, L. A. (1963). A correlated light and electron microscopestudy of the nucleolar material during mitosis in Viciafaba. J. Cell Biol. 17, 167-201.

LEBLOND, C. P. (1965). What radioautography has added to protein lore. In The Use of Radio -autography in Investigating Protein Synthesis, vol 4. Symposia of the International Society forCell Biology (ed. C. P. Leblond & K. B. Warren), pp. 321-339. New York: Academic Press.

LEBLOND, C. P. & AMANO, M. (1962). Synthetic activity in the nucleolus as compared to thatin the rest of the cell. J. Histochem. Cytochem. 10, 162-174.

MARINOZZI, V. (1964). Cytochimie ultrastructurale du nucleole-RNA et prot&nes intra-nucltolaires. J. Ultrastruct. Res. 10, 433-456.

MATTINGLY, A. (1963). Nuclear protein synthesis in Viciafaba. Expl Cell Res. 29, 314-326.O'DONNEL, E. H. J. (1961). Deoxyribonucleic acid structures in the nucleolus. Nature, Lond.

191, 1325-1327.SALPETER, M. M. & BACHMANN, L. (1964). Autoradiography with the electron microscope. A

procedure for improving resolution, sensitivity and contrast. J. Cell Biol. 22, 469-477.SANDBORN, E. (1963). Amino acid incorporation in the neurons of the semilunar ganglion of the

rat. Anat. Rec. 145, 280.


SCHULTZE, B., OEHLERT, W. & MAURER, W. (1959). Autoradiographische Untersuchung zumMechanismus der Eiwissneubildung in Ganglien Zellen. Beitr. pathol. Anat. 120, 58-84.

STOCKER, E. (1963). Autoradiographische Untersuchungen zur Aminosaure Inkorporation imNukleolus. Z. Zellforsch. mikrosk. Anat. 58, 790-797.

WOODARD, J., RASH, E. & SWIFT, H. (1961). Nucleic acid and protein metabolism during themitotic cycle in Vicia faba. J. biophys. biochem. Cytol. 9, 445-462.

{Received 24 April 1967)

Figs. 1-4. Representative light micrographs of interphase nuclei showing the nucleolusin the midst of a network of chromatin. The stainable portion of the nucleolus has 2components differing slightly in staining intensity. The less intensely stained compo-nent is located along the edge and in the centre of the nucleolus as well as in radialareas extending in between (Figs. 1, 2); this component also surrounds the unstainedvacuoles (Figs. 3, 4, v). The more intensely stained component is arranged in irregularpatches usually located between the edge and the centre of the nucleolus (Figs. 2, 4);these patches contain unstained, barely resolvable lacunae, two of which (arrows) maybe recognized in Figs. 2 and 3. x 4000.

Figs. 5-8. Representative light microscope radioautographs of interphase nucleiafter 5 min immersion of the roots in [3H]arginine solution. Many silver grains arelocated over the nucleolus, a few over the rest of the nucleus, and, finally, a few overthe surrounding cytoplasm. In the nucleolus, the unstained vacuoles (seen in Figs. 7, 8)are not labelled, whereas silver grains overlie both the more intensely and the lessintensely stained regions of the rest of the nucleolus. (Ilford L-4 emulsion, 48 h expo-sure.) x 4000.

Journal of Cell Science, Vol. 2, No. 4

t

5

• ' I V

L. A. CHOUINARD AND C. P. LEBLOND

Fig. 9. Electron micrograph depicting an interphase nucleolus. Two types of structuralcomponents, one granular, the other fibrillar, each segregated into regions distinguish-able by differences in electron density, are found within the dense portion of thenucleolus. The less-dense granular regions are located along the edge and in thecentre of the nucleolus with radial areas extending in between. Light, irregular narrowspaces within these regions suggest that the granular elements are assembled into acoarse network. The denser fibrillar regions, circumscribed by a solid black line,appear as irregular patches located between the edge and the centre of the nucleolus.These regions contain several small light-staining lacunae (arrows). These lacunaeshow a core of dense chromatin-like material, which is clearly visible at the doublearrows, x 27000.



Figs, io, I I . Electron-microscope radioautographs of interphase nucleoli after 5 minimmersion of the roots in [3H]arginine solution. The radioautographic reaction occursover the dense fibrillar regions (Figs, io, 11) and the less dense granular regions, as iswell shown in Fig. 10. Some of the lacunae (double arrows) show a core of denserchromatin-like material. (Gevaert 307 emulsion; 2 weeks exposure.) x 22000.



Figs. 12, 13. Electron-microscope radioautographs of interphase nucleoli after 5 minimmersion of the roots in [3H]arginine solution. The radioautographic reaction occursover the dense fibrillar and the less-dense granular regions of the nucleolus. Manysilver grains also overlie the irregular and in places diffuse boundaries between fibrillarand granular regions. A few silver grains are located over or adjacent to some of thelacunar regions (arrows). Some lacunae (double-arrows) show a core of denser chrom-atin-likc material. (Gevaert 307 emulsion; 2 weeks exposure.) x 22000.



Documents

sites of protein synthesis in nucleoli of root meristematic cells of