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J. Cell Sci. 2, 473-480 (1967) Printed in Great Britain 473 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. LEBLOND Departments of Anatomy, Laval University, Quebec City andMcGill Montreal, Canada University, SUMMARY The interphase nucleolus in root meristematic cells of Allium cepa may be divided into four regions, three of which are always present: the fibrillar, granular and lacunar regions, while the fourth or vacuolar region may be missing. The sites of protein synthesis in nucleoli were investigated by means of light and electron-microscope radioautography after a 5-min immersion of the roots in a solution of [3H]arginine. The radioautographs of interphase nucleoli showed many silver grains over both the fibrillar and the granular regions. Occasional silver grains were also recorded over, or close to, the lacunar regions, but none were over the vacuolar regions. A 15-min chase period did not change the radioautographic pattern. It is concluded that the three permanent regions of the interphase nucleoli, namely the fibrillar, the granular and the DNA-containing lacunar regions, are sites of protein synthesis. INTRODUCTION Light- and electron-microscope observations reveal that the interphase nucleoli in plant cells are made up of several components segregated into regions distinguishable by 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 of labelled amino acids as detected by radioautography in the light microscope (Woodard, Rash & Swift, 1961; De, 1961; Mattingly, 1963) and by biochemical analyses (Birnstiel, Chipchase & Bonner, 1961; Birnstiel & Hyde, 1963; Birnstiel & Flamm, 1964; Flamm & Birnstiel, 1964). Yet it is not known which of the structural components of the nucleolar mass is instrumental in these processes of protein synthesis. The synthesis of protein has also been observed in the nucleoli of animal cells, as shown in the review by Stocker (1963). The uptake of labelled amino acids first observed by Ficq in 1953 was later questioned (Schultze, Oehlert & Maurer, 1958; Carneiro & Leblond, 1959), but with improvements in technique was clearly demonstrated in the light microscope (Leblond & Amano, 1962; Kasten & Strasser, 1966) as well as in the electron 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. Radioautography was used in the light and electron microscopes following pulse-labelling of the proteins with tritiated arginine. MATERIALS AND METHODS Seeds of Allium cepa were germinated on the surface of loosely packed vermiculite kept moist by daily watering. After i week germination at room temperature, the primary 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 water containing 100 /ic/ml of tritiated arginine. The isotope used, L-[G-3H]arginine monohydrochloride (Radiochemical Centre, Amersham, England), has a specific activity of 1830 mc/mM. At the end of the 5-min pulse treatment, the seedlings were rinsed in distilled water and the distal 1-5 mm of some root tips were fixed for 2 h at room temperature in a mixture of formaldehyde and glutaraldehyde (Karnovsky, 1965) in O-IM Sorensen's phosphate buffer, PH7.2; the remainder of the undetached roots were re-immersed for a period of 15 min in a solution of non-radioactive L-arginine monohydrochloride made up to a concentration 100 times that of the radioactive sample. Following this chase period, the seedlings were rinsed with distilled water and the root tips fixed as described above. After fixation, all root tips were placed overnight in O-IM Sorensen's buffer, pH 7-2, at 4 °C; the next day, they were dehydrated over a 2-h period in an ascending series of ethanol concentrations and finally embedded in Epon 812. Half-micron sections of the root meristematic regions were mounted on glass slides with a wire loop and coated by dipping in liquid Ilford L-4 emulsion. After 48 h exposure and development, a drop of filtered 1 % toluidine blue was placed on the slides, allowed to stand at room temperature for 5 min, and rinsed off with distilled water. After dehydration, a coverslip was placed over the sections. The emulsioncoated and exposed sections as well as the uncoated half-micron sections from the same blocks were studied with a Leitz binocular microscope, using a ribbon-filament lamp, Kohler illumination, and a 100 x 1-32 N.A. apochromatic objective. An orange Wratten 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 307 emulsion and, after 2 weeks exposure and proper development, stripped and mounted on 300-mesh copper grids (Salpeter & Bachmann, 1964). Successful double staining of these grids was achieved by floating, emulsion side down, upon drops of a saturated solution 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-microscope examination. The sections were examined in a Siemens Elmiskop I electron microscope 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 chromosomes (Heitz, 1931). Two nucleoli, therefore, are formed at telophase and these, during interphase, may remain distinct (Fig. 1) or adhere to form a dumbbell-shaped structure, or fuse into a large spherical organelle which may reach up to 6/t in diameter (Figs. 3, 4). In 0-5-/4 sections of meristematic cells stained with toluidine blue, the nucleolar material exhibits a purplish blue metachromatic colour (Figs. 1-4). The nucleolus often contains a large, unstained space located centrally or paracentrally, the nucleolar vacuole (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 nucleolus proves to be not homogeneous but made up of two components slightly differing in staining intensity (Figs. 1-4). The less intensely stained component is located along the edge and in the centre of the nucleolus as well as in radial areas extending in 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 in irregular 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 be referred to as lacunae (Figs. 2, 3, arrows). Such lacunae are less readily identified here than in sections of osmium-fixed meristematic cells stained by the Feulgen/methylene blue procedure; they are then seen to be a constant feature of the more intensely stained component of the nucleolus (Chouinard, 1966a, b). Electron microscopy. The nucleoli appear as electron-dense bodies containing light spaces corresponding to the vacuoles and lacunae distinguished in the light microscope. The dense portion shows denser and less dense regions, corresponding respectively to the more and the less intensely stained regions seen in the light microscope (Fig. 9). The denser regions have irregular boundaries with processes projecting into the surrounding less dense regions; small isolated patches of denser material may also be embedded within the less dense regions. The less dense regions of the nucleolus consist mainly of packed granules about 150 A in diameter (Fig. 9). In addition, fibrils are encountered which also are 150 A in diameter and, therefore, would appear as granules when cut across. Occasionally, series of granules are strung like beads; or barely distinguishable granules appear as periodic thickenings along the 150-A fibrils. Finally, a few fine, parallel fibrils approximately 60 A or more in diameter may also be seen among the granules. These less dense regions will be referred to as 'granular'. The denser regions of the nucleolus are composed of an amorphous ground substance containing tightly packed, electron-dense fibrils (Fig. 9). Most of them are thin with a diameter of 60 A. Larger ones may also be observed up to 150 A. The denser regions will be referred to as 'fibrillar'. Of the lighter spaces, some are vacuoles. These contain loosely and uniformly dispersed 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 staining spaces usually smaller than the vacuoles but variable in size and shape and usually exhibiting a loose fibrillar texture (Fig. 9, arrows); in the lacunae it is often possible to 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 obtained indicating 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 lacunae are cross-sections of a channel containing the nucleolar organizing region of the nucleolar chromosome (Chouinard, 1966 c). In summary, the interphase nucleolus in root meristematic cells of A. cepa is made up of 4 structural components segregated into regions distinguishable by differences in staining properties and ultrastructural characteristics: 2 dense ones referred to as granular and fibrillar regions, and 2 light ones, as vacuolar and lacunar regions. In previous electron-microscope observations based on osmium-fixed material, evidence had been presented suggesting that the 2 main structural components of the dense portion of the nucleolus (i.e. the granular and the fibrillar) correspond respectively to what has been referred to in animal nucleoli as nucleolonema (or nucleolonematic network) and pars amorpha (Chouinard, 1966 a, b). Indeed, in several places within the granular regions and more particularly on the fluffy outer surface as well as at the surface of the vacuoles, light irregular narrow spaces may be detected suggesting that the granules and fibrils are assembled into a coarse network, the meshes of which are approximately o-1 ju, in diameter (Fig. 9); this would correspond to the nucleolonema. Sites of incorporation of tritiated arginine into interphase nucleoli Light microscopy. As early as 5 min after immersion of the primary roots of A. cepa into a solution containing [3H] arginine, a radioautographic reaction was observed over all cells of the meristematic region. Representative light-microscope radioautographs of interphase cells (Figs. 5-8) showed many silver grains over the nucleolus, a few over the rest of the nucleus, that is, mainly over chromatin and, finally, a few over cytoplasm. Careful examination of the nucleoli showed grains over both the denser and 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 over the ribosome-rich regions, and, occasionally, over mitochondria. In the nucleus, the silver grains overlay the nucleolus, to a lesser extent the chromatin material, and to a still lesser extent the nuclear sap. Examination of electron-microscope radioautographs of the nucleolus (Figs. 10-13) showed silver grains over both fibrillar (Fig. 11) and granular regions (Fig. 10). Grains were located not only over the main fibrillar regions but also over the small and apparently isolated patches of similar fibrillar material embedded in the granular regions (Fig. 12). Significant numbers of silver grains overlay the 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 vicinity of, the DNA-containing lacunar regions (Fig. 12), whereas there was no significant labelling of the vacuolar regions. Protein synthesis in nucleolus of Allium 477 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 meristematic cells. Except for a 10-20 % reduction in the number of silver grains over the nucleoli, the distribution pattern of the labelling appeared essentially the same as that described in the experiments without chase. DISCUSSION The present observations revealed that, as early as 5 min after immersion of primary roots of A. cepa in a solution of [3H]arginine, a radioautographic reaction was obtained over the cytoplasm and nucleus of meristematic cells. This indicated that the radioactive arginine must have reached even the cells in the centre of the meristem and been taken up into their cytoplasm and nucleus. Because of the high solubility of arginine in water, it was assumed that any free arginine present in the cells would be washed out during fixation and the subsequent 12-h immersion in buffer. However, when the labelled specimens were subjected to large doses of non-labelled arginine in the pulse-chase experiment, the grain count was decreased by 10-20 %. Hence, of the label taken up at 5 min, 10-20% may consist of free arginine which is adsorbed strongly 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 synthesized proteins of the nucleolus have completely turned over during the 15 min period as a result of emigration or breakdown.] But the radioautographic pattern was not visibly modified by the pulse chase. Consequently, the pattern would be caused by the rest of the label, that is, the 80-90 % which was not adsorbed and must therefore have been incorporated into newly synthesized tissue components. Indeed, it was known that, after injection of a labelled amino acid, only that fraction taken up into newly synthesized proteins was retained in the sections (Droz & Warshawsky, 1963; Leblond, 1965). Hence, the distribution pattern of radioactivity after glutaraldehydeformaldehyde fixation indicated the location of protein synthesized during the 5 min of exposure to the labelled amino acid. If it is assumed that this time is too short for significant migration of the new protein, then the radioactive regions are the sites of protein synthesis. It may therefore be concluded that organelles of cytoplasm (ribosomes, 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, it would appear that protein synthesis in that organelle is a continuous process throughout interphase. These observations confirm previous cytological findings that interphase nucleoli in root meristematic cells are sites of continuous protein synthesis (Woodard et al. 1961; De, 1961; Mattingly, 1963). Judging from the relative concentration of silver grains in Figs. 5-8, it also seems as if the rate of protein synthesis in the nucleolus of A. cepa is more rapid than that occurring elsewhere within the cell nucleus, a conclusion confirming previous biochemical observations (Birnstiel et al. 478 L. A. Chouinard and C. P. Leblond 1961; Birnstiel & Hyde, 1963; Birnstiel & Flamm, i964;Flamm&Birnstiel, 1964). The nature of nucleolar proteins which were being synthesized during the 5-min exposure to [3H]arginine is not known. They could be any of the four types of proteins which biochemists claim are present in both plant and animal nucleoli, since all four have been shown to contain arginine and become labelled after administration of [3H]amino acids (Birnstiel, Chipchase & Flamm, 1964; Grogan, Desjardins & Busch, 1966). In the present study, a radioautographic reaction was observed over both the granular and the fibrillar regions of the nucleolus. Hence, it may be concluded that these two regions are sites of protein synthesis. In addition, a few silver grains were located over, or in the immediate vicinity of, the DNA-containing lacunar regions, thus raising the possibility that the intranucleolar DNA also may somehow be involved in protein synthesis. These observations may be interpreted in relation to what is known of RNA metabolism in the nucleolus. A number of authors have observed that RNA synthesis takes place in the fibrillar regions of animal nucleoli and subsequently the newly synthesized RNA migrates to the surrounding granular regions (Granboulan & Granboulan, 1965; Karasaki, 1965; Geuskens & Bernhard, 1966). In root meristematic cells RNA synthesis was also associated with the fibrillar regions of the nucleolus (La Cour & Crawley, 1965). According to Karasaki (1965) and Granboulan & Granboulan (1965) chromatin material present in the fibrillar regions of the nucleolus participates in nucleolar RNA synthesis. It is conceivable that the newly synthesized RNA is in the form of nucleoprotein fibrils (Marinozzi, 1964), which in the course of migration to the granular region become beaded and transform into granules. It is tempting to speculate further that the RNA molecules which are being synthesized by the intranucleolar chromatin and which subsequently invade the rest of the nucleolar mass, are, during their migration, somehow instrumental in the elaboration of 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 nucleolus was observed, a fact indicating that synthesis of proteins does not take place, or is negligible, there. The general conclusion to be drawn from the above observations is that all three of the permanent structural components of the interphase nucleolus in root meristematic cells 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 of the radioautographs. We also thank Mr P. Koen for skilled technical assistance. This investigation was supported by grants from the Medical Research Council of Canada. REFERENCES M., CHIPCHASE, M. & BONNER, J. (1961). Incorporation of leucine-H3 into subnuclear 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 of nucleolar proteins. Biochim. biophys. Ada 87, m-122. BIRNSTIEL, M. & FLAMM, W. G. (1964). Intranuclear site of histone synthesis. Science, N.Y. 145. 1435-1437BIRNSTEIL, Protein synthesis in nucleolus of Allium 479 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, as shown with tritium-labelled amino acids. Science, N.Y. 129, 391-392. CHOUINARD, L. A. (1966a). Nucleolonema and pars amorpha in root meristematic cells of Viciafaba. 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 of Allium cepa. 3. Cell Biol. 31, 139 A. DE, D. N. (1961). Autoradiographic studies of nucleoprotein metabolism during the division cycle. Nucleus, Calcutta 4, 1-24. DROZ, B. (1965). Accumulation de prote'ines nouvellement synthe'tise'es dans l'appareil de Golgi du 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 the detection of newly synthesized proteins. J. Histochem. Cytochem. n , 426-435. 14 FICQ, A. (1953) Incorporation in vitro du glycocolle-i-C dans lesoocytes d'Aste>ies. Experientia 9, 377-379FLAMM, 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. Action de 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 Spirogyra BIRNSTIEL, britannica and Spirogyra ellipsospora. Jl R. microsc. Soc. 84, 347—360. N. & GRANBOULAN, P. (1965). Cytochimie ultrastructurale du nucteole. Etude des 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 Walker tumor. Cancer Res. 26, 775-779. HEITZ, E. (193I). Die Ursache der gestezmassigen Zahl, Lage, Form und Grosse pflanzlicher Nukleolen. Planta iz, 775-792. HYDE, B. B., SANKARANARAYANAN, K. & BIRNSTIEL, M. (1965). Observations on fine structure in pea nucleoli in situ and isolated. J. Ultrastruct. Res. 12, 652-667. KARASAKI, S. (1965). Electron microscopic examination of the sites of nuclear RNA synthesis during amphibian embryogenesis. J. Cell Biol. 26, 937-958. KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol. 27, 137-138. KASTEN, F. H. & STRASSER, F. F. (1966). Amino acid incorporation patterns during the cell cycle 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 in nucleoli. Chromosoma 16, 124-132. LAFONTAINE, J. G. & CHOUINARD, L. A. (1963). A correlated light and electron microscope study 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 for Cell 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 that in the rest of the cell. J. Histochem. Cytochem. 10, 162-174. MARINOZZI, V. (1964). Cytochimie ultrastructurale du nucleole-RNA et prot&nes intranucltolaires. 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. GRANBOULAN, 191, 1325-1327. 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. SALPETER, 480 L. A. Chouinard and C. P. Leblond B., OEHLERT, W. & MAURER, W. (1959). Autoradiographische Untersuchung zum Mechanismus der Eiwissneubildung in Ganglien Zellen. Beitr. pathol. Anat. 120, 58-84. STOCKER, E. (1963). Autoradiographische Untersuchungen zur Aminosaure Inkorporation im Nukleolus. Z. Zellforsch. mikrosk. Anat. 58, 790-797. WOODARD, J., RASH, E. & SWIFT, H. (1961). Nucleic acid and protein metabolism during the mitotic cycle in Vicia faba. J. biophys. biochem. Cytol. 9, 445-462. SCHULTZE, {Received 24 April 1967) Figs. 1-4. Representative light micrographs of interphase nuclei showing the nucleolus in the midst of a network of chromatin. The stainable portion of the nucleolus has 2 components differing slightly in staining intensity. The less intensely stained component is located along the edge and in the centre of the nucleolus as well as in radial areas extending in between (Figs. 1, 2); this component also surrounds the unstained vacuoles (Figs. 3, 4, v). The more intensely stained component is arranged in irregular patches 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) may be recognized in Figs. 2 and 3. x 4000. Figs. 5-8. Representative light microscope radioautographs of interphase nuclei after 5 min immersion of the roots in [3H]arginine solution. Many silver grains are located over the nucleolus, a few over the rest of the nucleus, and, finally, a few over the 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 less intensely stained regions of the rest of the nucleolus. (Ilford L-4 emulsion, 48 h exposure.) x 4000. Journal of Cell Science, Vol. 2, No. 4 t 5 • L. A. CHOUINARD AND C. P. LEBLOND 'IV Fig. 9. Electron micrograph depicting an interphase nucleolus. Two types of structural components, one granular, the other fibrillar, each segregated into regions distinguishable by differences in electron density, are found within the dense portion of the nucleolus. The less-dense granular regions are located along the edge and in the centre of the nucleolus with radial areas extending in between. Light, irregular narrow spaces within these regions suggest that the granular elements are assembled into a coarse 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 lacunae show a core of dense chromatin-like material, which is clearly visible at the double arrows, x 27000. Journal of Cell Science, Vol. 2, No. 4 L. A. CHOUINARD AND C. P. LEBLOND Figs, io, I I . Electron-microscope radioautographs of interphase nucleoli after 5 min immersion of the roots in [3H]arginine solution. The radioautographic reaction occurs over the dense fibrillar regions (Figs, io, 11) and the less dense granular regions, as is well shown in Fig. 10. Some of the lacunae (double arrows) show a core of denser chromatin-like material. (Gevaert 307 emulsion; 2 weeks exposure.) x 22000. Journal of Cell Science, Vol. 2, No. 4 L. A. CHOUINARD AND C. P. LEBLOND Figs. 12, 13. Electron-microscope radioautographs of interphase nucleoli after 5 min immersion of the roots in [3H]arginine solution. The radioautographic reaction occurs over the dense fibrillar and the less-dense granular regions of the nucleolus. Many silver grains also overlie the irregular and in places diffuse boundaries between fibrillar and granular regions. A few silver grains are located over or adjacent to some of the lacunar regions (arrows). Some lacunae (double-arrows) show a core of denser chromatin-likc material. (Gevaert 307 emulsion; 2 weeks exposure.) x 22000. Journal of Cell Science, Vol. 2, No. 4 L. A. CHOUINARD AND C. P. LEBLOND