J. CellSci. 19, 85-101 (1975) 85

Printed m Great Britain

AN ELECTRON-MICROSCOPE STUDY OF THE

INTRANUCLEOLAR CHROMATIN DURING

NUCLEOLOGENESIS IN ROOT MERISTEMATIC

CELLS OF ALLIUM CEPA

L. A. CHOUINARD

Department of Anatomy, Laval University, Quebec City, Canada

SUMMARY

The various states of condensation of the chromatin material, contained inside the lacunarregions of the reforming nucleolus in Allium cepa, have been investigated by means of con-ventional electron-microscope techniques. The observations reveal that, in the emerging earlyto late telophase nucleoli, the intralacunar chromatin material in question appears both in anextended and a condensed condition; from late telophase to the mid G1 period of interphase,the intralacunar chromatin material of the rapidly growing and developing nucleoli is presentin an extended state only.

An attempt is made to interpret these morphological findings in the light of current knowledgeconcerning the structural relationship of the nucleolar organizing region of the nucleolarchromosome with the interphase nucleolus in plant cells. The relevant observational evidencewould be consistent with the view that the chromatin-containing lacunar regions of the reformingnucleolus in Allium cepa correspond, in fact, to cross- or oblique sections of a meanderingchannel through which the nucleolar organizing segment of the nucleolar chromosome passes.Assuming the applicability to intranucleolar chromatin of the general concept of condensed-inactive versus extended-active chromatin, it is concluded that gradual uncoiling and subsequentdecondensation of the chromatin of the nucleolar organizing region in the form of a convolutedloop structure are key morphological and functional events associated with the process ofnucleologenesis in the species investigated.

INTRODUCTION

Previous ultrastructural observations have revealed that the chromatin material,located inside the small lacunar spaces confined to the dense fibrillar regions of thenucleolar mass in root meristematic cells of Allium cepa, occurs in various states ofcondensation and configuration (Chouinard, 1974). In a number of lacunar profiles,the chromatin material in question indeed appeared in an extended form only; inother lacunar profiles of the same or different nucleoli, the chromatin material waspresent both in an extended and a condensed condition. Moreover, in some lacunarprofiles, a single mass of chromatin in a condensed state was observed; in others,several discrete and often seemingly interconnected masses of condensed chromatinwere visualized.

Since the above investigations have been limited to the interphase nucleolus, itwas thought of interest to extend our observations to the organizational features ofthe intralacunar chromatin material during the entire process of nucleolar recon-

86 L. A. Chouinard

stitution (nucleologenesis) as seen in telophase and early interphase nuclei of rootmeristematic cells also in Allium cepa.

MATERIALS AND METHODS

Seeds of Allium cepa were germinated on the surface of loosely packed Vermiculite keptmoist by daily watering. After i week's germination at room temperature, the primary rootsof the seedlings had reached approximately I cm in length. Root tips, 0-5-1 mm long, werethen excised from these primary roots and fixed for 30 min in an ice-cold 1 % solution of osmiumtetroxide in 0-5 M Sorensen's phosphate buffer, pH 7-2. After decanting the fixative, the roottips were rapidly dehydrated in a series of increasing concentrations of cold ethanol beginningwith 70 % and brought slowly to room temperature in absolute ethanol. Dehydration was com-pleted with 2 additional 15-min changes of absolute ethanol and 2 15-min changes of propyleneoxide. Subsequent embedding of the specimens was carried out in Epon 812 according tocurrent procedures. Ultrathin silver-to-pale gold sections cut from the root meristematicregions were picked up on uncoated 300-mesh copper grids and double-stained with uranylacetate and lead citrate. The preparations were examined in a Siemens Elmiskop iA electronmicroscope using the double condenser, 60 kV and 5o-/tm objective aperture.

OBSERVATIONS

As originally established by Heitz (1931) in the species investigated, the nucleolusarises at telophase in intimate association with a specific subterminally located region(the nucleolar organizing region: NOR) of the so-called nucleolar chromosome. Inthe present study, the youngest observed or recognized early to mid-telophasenucleoli are seen to be only slightly larger than the width of the nucleolar organizingregion on to which they develop. Under electron microscopy, these emerging nucleoliare already seen to exhibit a heterogeneous structure consisting of fine fibrillar material,staining less intensely than the chromatin of the adjoining chromosome segments,and of a few elongated or irregularly shaped more electron-transparent lacunar regionscontaining chromatin material in various states of condensation ranging from theextended to the condensed (Figs. 1-4). In some nucleolar profiles, favourable sectionsindeed reveal that the lacunar regions in question contain, in addition to chromatinin an extended state, a number of small, ill-defined clumps or strands made up of aslightly more condensed form of chromatin material (Figs. 1, 2). In other nucleolarprofiles, the intralacunar chromatin material is represented by a few relativelyvoluminous masses of chromatin, structurally and tinctorially indistinguishable fromextranucleolar chromatin in its condensed form, embedded in a loosely and uniformlydispersed microfibrillar material that can be equated with chromatin in an extendedstate (Figs. 3, 4). These intralacunar microfibrils of extended chromatin are seen tomerge more or less imperceptibly with the compact meshwork of fibrils making upthe bulk of the nucleolar mass. Within any given lacunar region, there also appearsto be continuity of microfibrils between the condensed and extended forms ofchromatin. In favourably transected lacunar regions, one often gains the impressionthat the strands or masses of condensed chromatin, often with intricate contours, aresomehow interconnected and thus structurally continuous with one another. Inappropriate preparations also, contiguity and even continuity between the chromatin

Intranucleolar chromatin in Allium cepa 87

content (whether in extended or condensed state) of lacunar regions and strands ofcondensed chromatin originating from the adjoining condensed segment of thenucleolar chromosome can be readily visualized (Figs. 1, 3).

From mid to late telophase, the nucleolar mass undergoes a marked increase in sizeand is seen to consist predominantly of a compact moderately electron-dense finefibrillar material in which are enclosed a number of more electron-transparent lacunarregions of varying sizes and shapes (Figs. 5-8). At mid telophase, the lacunar regionsin question contain, in addition to loosely and uniformly dispersed fibrillar materialallegedly corresponding to chromatin in an extended state, a few small clumps orstrands made up of a slightly more condensed form of chromatin material (Figs. 5-7).At late telophase, the intralacunar chromatin material appears to be present in anextended form only (Fig. 8). During the transition from mid to late telophase, it isof interest to note the gradual appearance of a number of rather uniformly distributedmoderately electron-dense 20- to 35-nm granular elements closely associated withthis extended form of intralacunar chromatin material (Figs. 7, 8).

At very late telophase or in early Gx nuclei, the bulk of the still enlarging nucleolarmass is seen to be occupied by compact moderately electron-dense fibrillar material(Fig. 9). Here and there, a number of relatively small widely scattered more electron-lucent areas corresponding to profiles of the lacunar regions is clearly recognizablewithin the nucleolar body. Favourable sections reveal that these lacunar regionscontinue to exhibit within their confines chromatin material in an extended stateassociated with moderately electron-dense granular elements similar to those describedpreviously. During that period, the typical preribosomal granular component of thenucleolar mass makes its appearance at the edge and in the centre of the nucleolarbody as well as in narrow radial strips extending in between; at that time, however,this component made up of 15-20-nm elements still represents only a relatively minorportion of the total nucleolar mass.

From the early to the mid Gx period of interphase, the still growing nucleolar bodygradually acquires the ultrastructural features typical of the interphase nucleolus inthe species investigated. Growth and development of the nucleolar mass during thatperiod is brought about partly by an increase in the amount of the dense fibrillarmaterial and partly by the acquisition of a substantial quantity of the granular com-ponent. At the end of our observation period, the nucleolus is seen to consist of 4structural components segregated into regions distinguishable by differences instaining properties and ultrastructural characteristics: 2 dense ones referred to as thegranular and the fibrillar regions and 2 light ones, as the vacuolar and the lacunarregions (Fig. 10). Of these 4 regions, only the vacuolar may be missing from thenucleolar body. The dense granular regions, consisting predominantly of particles15-20 nm in diameter, are located along the edge and in the centre of the nucleolusas well as in radial areas extending in between. The dense fibrillar regions, made up oftightly convoluted fibrils 6-15 nm in width, are arranged in irregular patches betweenthe edge and the centre of the nucleolar mass. Of the light-staining regions, some arevacuoles. Such vacuoles, which contain loosely and uniformly dispersed granules andfibrils, are always seen to be enclosed within the granular regions of the nucleolus.

88 L. A. Chouinard

Other light-staining areas, variable in size and shape, correspond to the lacunarregions; such lacunar regions, which are seen to be restricted to the dense fibrillarareas of the nucleolar mass, are characterized by the presence within their confinesof a loosely and uniformly distributed fibrillar material that can be equated withchromatin in an extended state. The previously described 20- to 35-nm moderatelyelectron-dense granular elements associated with the intralacunar chromatin materialare no longer observed in the fully reconstructed nucleolus.

DISCUSSION

The exact structural relationship of the uncoiled nucleolar organizing region (NOR)of the nucleolar chromosome with the plant interphase nucleolus has been successivelydocumented by Godward (1950), Godward & Jordan (1965) and Jordan & Godward(1969) in Spirogyra, by O'Donnell (1961, 1965) in Euglena, by La Cour (1966), LaCour & Wells (1967) in Ipheion and by Hyde (1.967) in Plantago. On the basis of theirobservations, these authors conclusively showed that the chromatin of the nucleolarorganizing region exists in the form of a continuous contorted loop structure visiblymeandering inside a channel of variable width running within the nucleolar body.Subsequent electron-microscope and cytochemical observations on the architecturalorganization of the interphase nucleolus in Altium cepa revealed that the chromatin-containing lacunar spaces confined to the fibrillar regions of the nucleolar mass corre-spond, in fact, to cross- or oblique sections of this meandering channel through whichthe chromatin of the nucleolar organizing region passes (Chouinard, 1970, 1971).

Our present observations clearly reveal the presence of chromatin-containinglacunar regions within the nucleolus at all stages of its reconstitution from earlytelophase to the mid G1 period of interphase. Assuming that the chromatin-containinglacunar regions in question and those observed within the interphase nucleolus arehomologous structures, there can be little doubt that gradual uncoiling or unfoldingof the chromatin of the nucleolar organizing region in the form of a convoluted loopstructure is a key morphological event in the process of nucleologenesis in the speciesinvestigated. From early to mid telophase, the young emerging nucleoli are onlyslightly larger than the width of the nucleolar organizing region on to which theydevelop, and their lacunar regions are seen to contain chromatin material in variousstates of condensation ranging from the extended to the condensed. We believe thatsuch a variability in the state of condensation of this intralacunar chromatin materialat that stage merely reflects the variability in the degree of compaction observed inthe chromatin contained in the nucleolar organizing region in formed metaphase andanaphase chromosomes. Previous light- and electron-microscope observations haveindeed shown that the slightly narrower chromosomal segment, that (judging from itslocation and size) corresponds most likely to the nucleolar organizing region, displaysvariations in its overall length as well as in the degree of condensation of its containedchromatin material (Chouinard, 1971). In a number of cases, the nucleolar organizingregion could barely be recognized as such and the chromatin material at that site wasshown to exhibit a texture and density almost matching those of the condensed

Intranucleolar chromatin in Allium cepa 89

chromatin in the remainder of the chromosome. In other cases, the nucleolar organizingregion could easily be visualized and the moderately dense chromatin material at thatlocus was shown to consist of a lower-density fibrillar material associated with whatappeared to be laterally or obliquely sectioned chromonematic gyres. Variability in themorphological expression of the chromosomal region associated with the formationof the nucleolus has usually been considered as a consequence of the more or lesstardy detachment of the nucleolar material during the preceding prophase such as tointerfere with normal chromatin condensation at that site (Moses, 1964; Callan, 1966;Hsu, Brinkley & Arrighi, 1967; Ohnuki, 1968; Miller & Brown, 1969; Brinkley &Stubblefield, 1970).

Our observations show that the intralacunar chromatin material occurs in anextended state throughout the period of rapid growth and development of thenucleolus, i.e. from late telophase onwards. Assuming the applicability to intra-nucleolar chromatin of the general concept of condensed-inactive versus extended-active chromatin (Hay & Revel, 1963; Littau, Allfrey&Frenster, 1964; Frenster, 1965;Milner, 1969; Brasch & Setterfield, 1974) it is reasonable to hypothesize that theextended state of the intralacunar chromatin material is largely related to its functionalactivity during the complex process of nucleologenesis. In other words, when fullyactive in the synthesis and accumulation of nucleolar constituents, the intranucleolarchromatin material in question would indeed be expected to be mostly unwound andtherefore in an extended condition. In this connexion, it should be recalled that, inthe case of the rapidly developing nucleoli in oocytes (lampbrush stage) of variousamphibians as well as in those of the giant primary nucleus of Acetabularia, the trans-criptionally active chromatin material associated with the fibrillar regions of thenucleolar mass has been shown to exist in a highly extended state (Miller & Beatty,1969; Miller & Hamkalo, 1971; Trendelenburg, Spring, Scheer & Franke, 1974;Spring et al. 1974). In relation to the problem at hand, it is of interest to note that,in the reforming nucleolus of Allium cepa, the microfibrils of extended chromatinpresent in the peripheral portions of the lacunar regions appear to merge more or lessimperceptibly with the reticulum of fibrils making up the dense fibrillar regions of thenucleolar body. As previously suggested for the interphase nucleolus, such micro-fibrils of extended chromatin in close association with the dense fibrillar region of thenucleolar mass are probably best considered as corresponding to the transcriptionallyactive chromatin material during the process of nucleologenesis (Chouinard, 1974).

The present study points to the extensive and intimate association of the uncoilednucleolar organizing region (in the form of a convoluted chromatin loop) with thefibrillar regions of the developing telophase and early interphase nucleolus. On thesegrounds, it is difficult to avoid the conclusion that the genes located within thischromatin loop are somehow instrumental in the elaboration of at least some of thenucleolar components. One of these components is certainly ribosomal RNA, sinceall of the DNA base sequences complementary to that molecular species have indeedbeen shown to be clustered in the nucleolar organizing region of the nucleolar chromo-some (McConkey & Hopkins, 1964; Ritossa & Spiegelman, 1965; Birnstiel, Wallace,Sirlin & Fischberg, 1966; Birnstiel et al. 1968; Avanzi, Durante, Ciomini & d'Amato,

o,o L. A. Chouinard

1972; Brady & Clutter, 1972; Pardue&Hsu, 1975). Autoradiographic and biochemicalstudies also reveal that the reconstituting nucleolus is an active site of RNA andpossibly also of protein biosynthesis (Das, 1963; Mittermayer, Braun & Rusch, 1964;Schiff, 1965; Das & Alfert, 1966; Schowacre, Cooper & Prescott, 1967; Gaffney &Nardone, 1968; Jain, Raut & Nerwal, 1969). The present study being essentiallymorphological in character, no definite conclusion can be drawn concerning thesignificance of the 20- to 35-nm granules that have been observed in association withthe extended intralacunar chromatin during nucleologenesis in the species investi-gated. Similar granular elements, which are larger than the usual preribosomalgranules, have previously been described in both plant and animal interphase nucleoliand in association with the dense fibrillar regions of the nucleolar mass (Sankaranarayan& Busch, 1965; Hyde, 1967; Verbin, Goldblatt, Saez & Farber, 1969; Shinozuka,1970; Goldblatt, Verbin & Sullivan, 1970; Smetana & Busch, 1974).

Our findings on the sequential appearance of the fibrillar and granular componentsof the nucleolus during the process of nucleologenesis in Allium cepa closely parallelthose reported by a number of workers who have previously studied the develop-mental stages of the nucleolus at the electron-microscope level (Jacob & Sirlin, 1963;Lafontaine & Chouinard, 1963; Karasaki, 1965; Stevens, 1965; Chouinard, 1966;Hay & Gurdon, 1967; Jacob, 1969; Noel, Dewey, Abel & Thompson, 1971; Rose,Setterfield & Fowke, 1972; Lafontaine & Lord, 1974; Goessens & Lepoint, 1974).

In summary, the relevant observational evidence presented in this paper would beconsistent with the view that nucleolar reconstitution at telophase and early interphaseis the morphological expression - like the puffs of the polytene and the loops andspheroids of the lampbrush chromosomes - of differential gene activity on the partof a chromatin loop derived through unfolding or uncoiling from the nucleolar organ-izing region of the nucleolar chromosome. The nucleolus would thus correspond toa specialized structural device allowing for the formation, stabilization and packagingof essential genes products derived from its intrinsic chromatin material.

REFERENCES

AVANZI, S., DURANTE, M., CIONLNI, P. G. & D'AMATO, F. (1972). Cytological localization ofribosomal cistrons in polytene chromosomes of Phaseolus coccineus. Chromosoma 39, 191-203.

BIRNSTIEL, M. L., SPIERS, J., PURDOM, J., JONES, K. & LOENING, U. E. (1968). Properties andcomposition of the isolated ribosomal DNA satellite of Xenopus laevis. Nature, Lond. 219,454-473-

BIRNSTIEL, M. L., WALLACE, H., SIRLIN, J. L. & FISCHBERG, M. (1966). Localization of the

ribosomal DNA complements in the nucleolar organizer region of Xenopus laevis. Natn.Cancer Inst. Monogr. 23, 431-447.

BRADY, T. & CLUTTER, M. E. (1972). Cytolocalization of ribosomal cistrons in plant polytenechromosomes. J. Cell Biol. 53, 827-832.

BRASCH, K. & SETTERFIELD, G. (1974). Structural organization of chromosomes in interphasenuclei. Expl Cell Res. 83, 175-185.

BRINKLEY, B. R. & STUBBLEFIELD, E. (1970). infrastructure and interaction of the kinetochoreand centriole in mitosis and meiosis. In Advances in Cell Biology, vol. 1 (ed. D. M. Prescott,L. Goldstein & E. McConkey), pp. 119-185. New York: Appleton-Century-Crofts.

CALLAN, H. G. (1966). Chromosomes and nucleoli of the axolotl, Ambystoma mexicanum.J. CellSet. 1, 85-108.

Intranucleolar chromatin in Allium cepa 91

CHOUINARD, L. A. (1966). Nucleolar architecture in root meristematic cells of Allium cepa.Natn. Cancer Inst. Monogr. 23, 125-143.

CHOUINARD, L. A. (1970). Localization of intranucleolar DNA in root meristematic cells ofAllium cepa.J. Cell Set. 6, 73-85.

CHOUINARD, L. A. (1971). Behavior of the structural components of the nucleolus duringmitosis in Allium cepa. In Advances in Cytopharmacology, vol. 1 (ed. F. Clementi & B.Ceccarelli), pp. 69-87. New York: Raven Press.

CHOUINARD, L. A. (1974). An electron-microscope study of the intranucleolar chromatin inroot meristematic cells of Allium cepa.J. Cell Set. 15, 645-657.

DAS, N. K. (1963). Chromosomal and nucleolar RNA synthesis in root tips during mitosis.Science, N.Y. 140, 1231-1233.

DAS, N. K. & ALFERT, M. (1966). Nucleolar RNA synthesis during mitotic and meioticprophase. Natn. Cancer Inst. Monogr. 23, 337-351.

FRENSTER, J. H. (1965). Ultrastructural continuity between active and repressed chromatin.Nature, Lond. 205, 1341-1342.

GAFFNEY, E. V. & NARDONE, R. M. (1968). Nucleolar RNA synthesis in synchronous culturesof strain L-929. Expl Cell Res. 53, 410-416.

GODWARD, M. B. E. (1950). On the nucleolus and nucleolar-organizing chromosomes of Spiro-gyra. Ann. Bot. 14, 39-53.

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

GOESSENS, G. & LEPOINT, A. (1974). The fine structure of the nucleolus during interphase andmitosis in Ehrlich tumour cells cultivated in vitro. Expl Cell Res. 87, 63-72.

GOLDBLATT, P. J., VERBIN, R. S. & SULLIVAN, R. J. (1970). Induction of nucleolar segregationby actinomycin D following inhibition of protein synthesis with cycloheximide. Expl CellRes. 63, 117-123.

HAY, E. D. & GURDON, J. B. (1967). Fine structure of the nucleolus in normal and mutantXenopus embryos..?. Cell Sci. 2, 151-162.

HAY, E. D. & REVEL, J. P. (1963). The fine structure of the DNP component of the nucleus:an electron microscopic study utilizing autoradiography to localize DNA synthesis. J. CellBiol. 16, 29-51.

HEITZ, E. (1931). Die Ursache der gesetzmassigen Zahl Lage, Form und GrOsse pflanzlicherNukleolen. Planta 12, 775-815.

Hsu, T. C , BRINKELY, B. R. & ARRIGHI, F. E. (1967). The structure and behaviour of thenucleolus organizers in mammalian cells. Chromosoma 23, 137-153.

HYDE, B. B. (1967). Changes in nucleolar ultrastructure associated with differentiation in theroot tip. jf. Ultrastruct. Res. 18, 25-54.

JACOB, J. (1969). Changes in nucleolar ultrastructure during amphibian development. ExplCell Res. 54, 281-284.

JACOB, J. & SIRLIN, J. L. (1963). Electron microscope studies on salivary gland cells. I. Thenucleus of Bradysia mycorum Frey (Sciaridae), with special reference to the nucleolus. J. CellBiol. 17, 153-165.

JAIN, H. K., RAUT, R. N. & NERWAL, S. K. (1969). Nucleolar organizer as a hyperactive locusfor RNA synthesis. Heredity 24, 59-67.

JORDAN, E. G. & GODWARD, M. B. E. (1969). Some observations on the nucleolus of Spirogyra.J. Cell Sci. 4, 3-15.

KARASAKI, S. (1965). Electron microscopic examination of the sites of nuclear RNA synthesisduring amphibian embryogenesis.,7. Cell Biol. 26, 937-958.

LA COUR, L. F. (1966). The internal structure of nucleoli. In Chromosomes Today, vol. 1 (ed.C. D. Darlington & K. P. Lewis), pp. 150-160. Edinburgh and London: Oliver and Boyd.

LA COUR, L. F. & WELLS, B. (1967). The loops and ultrastructure of the nucleolus of Ipheionuniflorum. Z. Zellforsch. mikrosk. Anat. 82, 25-45.

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

LAFONTAINE, J. G. & LORD, A. (1964). A correlated light- and electron-microscope investiga-tion of the structural evolution of the nucleolus during the cell cycle in plant meristematiccells (Alliumporrum). J. Cell Sci. 16, 63-93.

92 L. A. Chouinard

LITTAU, V. C , ALLFREY, V. G. & FRENSTER, J. H. (1964). Active regions of nuclear chromatinas revealed by electron microscopy autoradiography. Proc. natn. Acad. Sri. U.S.A. 52,93-100.

MCCONKEY, E. H. & HOPKLNS, J. W. (1964). The relationship of the nucleolus to the synthesisof ribosomal RNA in HeLa cells. Proc. natn. Acad. Sri. U.S.A. 51, 1197-1204.

MILLER, L. & BROWN, D. D. (1969). Variation in the activity of nucleolar organizers and theirribosomal gene content. Chromosoma 28, 430-/1^.

MILLER, O. L., JR. & BEATTY, B. R. (1969). Visualization of nucleolar genes. Srience, N.Y.164, 955-957-

MILLER, O. L., JR. & HAMKALO, B. A. (1971). Visualization of genetic transcription. In Mole-cular Genetics and Developmental Biology (ed. M. Sussman), pp. 183-199. New Jersey:Prentice Hall.

MILNER, G. R. (1969). Nuclear morphology and the ultrastrucrural localization of deoxyribo-nucleic acid synthesis during interphase. J. Cell Sci. 4, 569-582.

MITTERMAYER, C, BRAUN, R. & RUSCH, H. P. (1964). RNA synthesis in the mitotic cycle ofPhysarum polycephalum. Biochim. biophys. Acta 91, 399-405.

MOSES, M. J. (1964). The nucleus and chromosomes: a cytological perspective. In Cytology andCell Physiology (ed. G. H. Bourne), pp. 423-558. New York and London: Academic Press.

NOEL, J. S., DEWEY, W. C, ABEL, J. H., JR. & THOMPSON, R. P. (1971). Ultrastructure of thenucleolus during the Chinese hamster cell cycle. J. Cell Biol. 49, 830-847.

O'DONNELL, E. H. J. (1961). Deoxyribonucleic acid structures in the nucleolus. Nature, Lond.191, 1325-1326.

O'DONNELL, E. H. J. (1965). Nucleolus and chromosomes in Euglena gracilis. Cytologia 30,118-154.

OHNUKI, Y. (1968). Structure of chromosomes. I. Morphological studies of the spiral structureof human somatic chromosomes. Chromosoma 25, 402-428.

PARDUE, M. L. & Hsu, T. C. (1975). Locations of 18s and 28s ribosomal genes on the chromo-somes of the Indian muntjac. J. Cell Biol. 64, 251-254.

RITOSSA, F. M. & SPIEGELMAN, S. (1965). Localization of DNA complementary to ribosomalRNA in the nucleolus organizer region of Drosophila melanogaster. Proc. natn. Acad. Sci.U.S.A. 53, 737-745-

ROSE, R. J., SETTERFIELD, G. & FOWKE, L. C. (1972). Activation of nucleoli in tuber slices andthe function of the nucleolar vacuoles. Expl Cell Res. 71, 1-16.

SANKARANARAYAN, K. & BUSCH, H. (1965). Dense granules in nucleoli of Walker 256 carcino-sarcoma cells of the rat. Expl Cell Res. 38, 434-437.

SCHIFF, S. O. (1965). Ribonucleic acid synthesis in neuroblasts of Chortophaga viridifasciata(de Geer), as determined by observations in individual cells in the mitotic cycle. Expl CellRes. 40, 264-276.

SHINOZUKA, H. (1970). Intranucleolar dense particles in rat hepatic cell nucleoli..7. Ultrastruct.Res. 32, 430-442.

SHOWACRE, J. L., COOPER, W. G. & PRESCOTT, D. M. (1967). Nucleolar and nuclear RNAsynthesis during the cell life cycle in monkey and pig kidney cells in vitro. J. Cell Biol. 33,273-279.

SMETANA, K. & BUSCH, H. (1974). The nucleolus and nucleolar DNA. In The Cell Nucleus (ed.H. Busch), pp. 73-147. New York and London: Academic Press.

SPRING, H., TRENDELENBURG, M. F., SCHEER, U., FRANKE, W. W. & HERTH, W. (1974). Struc-tural and biochemical studies of the primary nucleus of two green algal species, Acetabtdariamediterranea and Acetabularia major. Cytobiologie 10, 1-65.

STEVENS, B. (1965). The fine structure of the nucleolus during mitosis in the grasshopperneuroblast cell. J. Cell Biol. 24, 349-368.

TRENDELENBURG, M. F., SPRING, H.( SCHEER, U. & FRANKE, W. W. (1974). Morphology ofnucleolar cistrons in a plant cell, Acetabularia mediterranea. Proc. natn. Acad. Sri. U.S.A.71, 3626-3630.

VERBIN, R. S., GOLDBLATT, P. J., SAEZ, L. & FARBER, E. (1969). A dense particulate com-ponent of nucleoli of rat intestinal crypt cells. Expl Cell Res. 56, 167-169.

(Received 18 March 1975)

Intranucleolar chromatin in Allium cepa 93

Figs. 1-4. For legend see p. 94.

94 L. A. Chouinard

Figs. I - IO. Representative electron micrographs depicting the various states ofcondensation assumed by the chromatin material contained within the lacunar regionsof the nucleolar mass during the process of nucleologenesis.

Figs. 1-4. Electron micrographs depicting emerging early to mid telophasenucleoli. The light-staining lacunar regions (large arrowheads) are seen to contain,in addition to chromatin in an extended state, a number of small ill-defined clumps orstrands (Figs, i, 2) or more discrete masses (Figs. 3, 4) of chromatin material (smallarrowheads) structurally and tinctorially indistinguishable from extranucleolarchromatin in its condensed form. In Figs. 1 and 3, contiguity and even continuitybetween the more condensed chromatin masses contained within favourably transectedlacunar regions and strands of condensed chromatin originating from the adjoiningcondensed segment of the nucleolar chromosome can be readily visualized, x 38000.

Figs. 5-7. Electron micrographs depicting young mid to late telophase nucleoli.Favourably transected lacunar regions (large arrowheads) of these enlarging mid-telophase nucleoli display a few small clumps or strands (small arrowheads) of con-densed chromatin material staining with the same intensity as that of the extra nucleolarmasses of condensed chromatin. These clumps or strands of intralacunar condensedchromatin are embedded in a loosely and uniformly dispersed microfibrillar materialthat can be equated with chromatin in an extended state. In Fig. 7, a number of ratheruniformly distributed moderately electron-dense 20- to 35-nm granular elements arealso seen to be closely associated with thie extended form of intralacunar chromatinmaterial, x 38000.

Intranucleolar chromatin in Allium cepa

/

96 L. A. Chouinard

Fig. 8. Electron micrograph depicting a late telophase nucleolus. The more lightlystained lacunar regions (large arrowheads) are seen to contain chromatin material inan extended state only. The constituent microfibrils of extended chromatin appearquite uniformly distributed within each of the lacunar regions where they form aloosely arranged meshwork of moderate electron opacity; interspersed among themicrofibrils of extended chromatin are a number of widely scattered 20- to 35-nmgranular elements, x 38000.

iIntranucleolar chromatin in Allium cepa

* » . •

a

C E L 1 9

L. A. Chouinard

Fig. 9. Electron micrograph depicting a very late telophase or early Gt nucleus.Favourable sections reveal that the more lightly stained lacunar regions (large arrow-heads) continue to exhibit within their confines chromatin material in an extendedstate associated with a number of moderately electron-dense 20- to 35-nm granularelements. The typical preribosomal granular component of the nucleolar mass(granular regions of the nucleolus: gr) has made its appearance at the edge and in thecentre of the nucleolar body as well as in narrow radial strips extending in between,x 30000.

Intranucleolar ckromatin in Allium cepa 99

7-2

ioo L. A. Chouinard

Fig. 10. Electron micrograph depicting a fully reconstituted nucleolus at about themid Gi period of interphase. The nucleolar mass is then seen to consist of 4 structuralcomponents segregated into regions distinguishable by differences in staining proper-ties and ultrastrucrural characteristics: 2 dense ones referred to as the granular (gr)and the fibrillar (Jr) regions and 2 light ones, as the vacuolar (vr) and the lacunarregions (large arrowheads). Such lacunar regions, variable in size and shape and seento be restricted to the dense fibrillar areas of the nucleolar mass, are characterized bythe presence within their confines of a loosely and uniformly distributed fibrillarmaterial that can be equated with chromatin in an extended condition. The 20- to35-nm moderately electron-dense granular elements previously described in closedassociation with the intralacunar chromatin material are no longer observed in thefully reconstructed nucleolus. x 28000.

Intranucleolar chromatin in Allium cepa IOI