118 Cytologia 30

Nucleolus and Chromosomes in Euglena gracilis1

E. H. J. O'Donnell, D. SC.

The New York Botanical Garden, Research Laboratory, Bronx Park, Bronx 58, New York, U. S. A.2

Received June 23, 1964

Introduction

Feulgen positive structures have been described inside the nucleolus of a variety of cell types (7, 15, 16, 17, 18, 36, 43, 84, 87, 88, 105, 108, 109, 110, 139, 141); and a "nu cleolar" DNA fraction has been reported in chemical analysis of isolated nucleoli (19, 29, 30, 44, 104, 106, 130, 136, 149). This chemically identified nucleolar DNA has been regarded as contamination by chromatin; however, nucleolar incorporation of DNA precursors has been shown to occur (65, 68, 69, 121) and a nucleolar constituent has been demonstrated to be hydrolyzable by DNAse (15, 108, 109, 110, 139, 141). This evidence validates the ex istence of a nucleolar structure containing DNA. The author's observations on the cells of Spirogyra (Chlorophyceae) (108, 109), confirmed early interpretations of the chromosomal nature of the Feulgen positive nucleolar structures (61, 87, 88, 89), but the light microscopy used did not resolve details of the nucleolar struc tures containing DNA and failed to disclose the expected association of intranucleolar DNA with extranucleolar DNA (chromosomes) (Figs. 1-8). The experimental approach used to examine the nucleolar organization of Spirogyra was adapted here for the investigation, at the fine structure level, of the nucleolar organization of another algal representative: Euglena gracilis var. bacillaris. This study was designed to discern the patterns of enzymic degradation of nuclear components by nucleases and proteinases, and to derive information about the chemical nature and spatial aggregation of nucleolar constituents. This in turn might give insights on the association between nucleolus and nucleolar chromosomes, and on the mode of formation of nucleoli.

Materials and methods

Euglena gracilis was cultured in liquid medium and sampled in the logarithmic phase.

Nucleases and/or proteinases used were added to living cells in cultures of standardized population density. Cell suspensions with the enzymes were incubated at 37•Ž and samples were taken at 1, 2, 3, 4 or 6hr and prepared for light and electron microscopy. Controls were run in parallel for each set of experiments. Since the in vivo enzymatic hydrolysis demanded incubation at 37•Ž, control samples were also incubated at 37•‹. Additional controls were prepared with untreated, "non-incubated" cells to investigate possible changes in the living material by the incubation temperature as compared with normal culture conditions

(20•Ž). The criteria for extent of nuclease activity were the diminished basophilia follow ing RNAse treatment and the Feulgen reaction after DNAse treatment. The enzyme solutions were used as follows: RNAse (specific activity 2500U/mg) 0.4 mg/ml in distilled water, pH 6.8 (veronal buffer); RNAse A3 (specific activity 3500U/mg) 1 This research has been supported by a grant from the Damon Runyon Memorial Fund for Cancer Research (DRG-535 B). 2 Present address: Cornell University Medical College, 1300 York Avenue , New York, U. S. A. 3 Prepared free from proteinase activity. 1956 Nucleolus and Chromosomes in Euglena gracilis 119

0.4mg/ml in distilled water, pH 6.8 ("tris" buffer); DNAse 1 (minimum activity 2000U/mg)

0.5mg/ml in a 0.003M solution of MgSO4 pH 6.9 (veronal buffer); trypsin (minimum ac tivity 10,000U/mg) 3mg/ml in distilled water, pH 7.8 (adjusted with 0.2M NaOH), and ƒ¿- chymotrypsin4 (minimum activity 11,000U/mg) 0.5mg/ml in distilled water, pH 8.2 (adjusted with 0.2M NaOH). Experiments were performed with nuclease or proteinase alone or with either nuclease followed by a proteinase. DNAse and RNAse were routinely checked for proteinase activity before experiments and the hydrolysis of nuclear components upon nu clease treatments was specific for DNA-or RNA-depolymerization. To 0.5ml of concentrated cell suspension, 5ml of enzyme solution was added and gently agitated periodically to better contact with the enzyme. Nuclease treatments were extended until definite patterns of enzymatic action emerged, rather than going to extreme de polymerization, else extreme treatment destroyed the photosynthetic apparatus: RNAse hydrolyzed the interlamellar matrix and the lamellae themselves; DNAse, on the other hand, interrupted the continuity of certain lamellae in the vicinity of the pyrenoid. The fixatives used for electron microscopy were 10% neutral formalin alone or followed by 1% OsO4, 1% OsO4, and 2% KMnO4 alone or followed by 1% OsO4. One per cent OsO4 gave best preservation of nuclear material at pH 7.4. Different buffers were tested: phos phate buffer was found to degrade a chromosomal constituent rendering regions of the chromonemata hardly visible; similar modification of chromatin was observed on lowering the fixation pH below 7.4; disruption of the chromonemata here gave the chromosomes the appearance of being constituted by numerous fibrils rather than by coiled and continuous filaments extended throughout the chromosomes' width and length. Acetate-veronal buffer gave good preservation and was selected for the routine. After enzyme treatment, the material sampled for electron microscopy was fixed at 0•Ž in 1% OSO4, buffered at pH 7.4 with acetate-veronal buffer, changed to fresh fixative after

the first 15min and left overnight in it; optimal preservation of nuclear material was at

tained after prolonged fixations (12-16hr). Material was embedded in methacrylate (60% n-butyl, 40% ethyl-methacrylate) with addition of Luperco CDB (1.5%) for oven polymeri zation (60•Ž) and without the catalyst for U. V. polymerization (151). U. V. polymerization

minimized polymerization damage and gave the blocks desirable cutting qualities. Both

polymerization procedures were extended for 48hr. Sections were cut with a LKB Ultratome at 200A and at 300A and stained in uranium acetate (saturated aqueous solution). Unstained sections also showed good contrast under the electron beam. Electronmicrographs were taken

with RCA EMU-2D and RCA EMU-3B microscopes.

Results

The nuclear organization in control cells of Euglena gracilis. The

fine organization of control cells was compared before and after incubation

at 37•Ž. The incubated cells showed a looser aggregation of the constituents

of the nuclear envelope and of the chromosomal matrix caused by the in

cubation temperature, 17•‹higher than the temperature at which the cultures

were normally grown. In general, when proteins are heated even at moderate temperatures, unfolding of the peptide chain is known to occur. Such unfolding and even uncoiling of peptide chains does not require actual hydrolysis of peptide linkages. In the control and enzyme-treated cells, structures containing considerable amounts of protein have a blurred ap pearance attributable to this temperature-induced uncoiling and unfolding of

4 RNAse, RNAse A, DNAse 1, trypsin and ƒ¿-chymotrypsin from Worthington Biochemical

Corp.

Cytologia 30, 1965 9 120 E. H. J. O'Donnell Cytologia 30 proteinaceous fibrils (Figs. 10, 11, 15, 25, 26). The topography of cellular organelles was not modified and there was not impairment of enzyme activity.

The round to ovoid nucleus of Euglena gracilis-5 to 7ƒÊ long by 4 to

5ƒÊ wide-is enclosed by a double-layered nuclear envelope, each membrane

50 to 60A thick and separated by a 60A space. The nuclear membrane pores are 30-55mƒÊ in diameter. The centrally located nucleolus is rather large-1.5 to 3ƒÊ long by 0.6 to 1.5ƒÊwide-often showing 1 or 2 indenta

tions5 crossed by coiling fibrils about 100A in diameter (Fig 9). At interphase the chromatin is uniformly distributed throughout the nuclear space, the moderately coiled chromonemata being about 100A in diameter. Thinner fibrils-less than 75A in diameter-were observed amidst the chromonemata. The nucleolus consists of two morphologically dis tinguishable components, both fibrillar; where they aggregated compactly, the nucleolar area showed high electron density. One component had long, electron-dense filaments, 100A in diameter, coiling and folding throughout the nucleolar mass (Fig. 9). Short, coiled fibrils less than 50A in diameter are the second morphologically distinguishable nucleolar component. Within the resolution of our microscopy, they seemed to be made up of elements coiling almost at right angles with the main axis of the long, electron-dense filaments (Fig 10). In a variety of cell types a third nucleolar component with granular organization has been described by several authors (2, 13, 16, 25, 27, 32, 75, 80, 81, 82, 84, 93, 133, 137). During the course of this investigation, neither in the control nor in the enzyme-treated samples were granules ob served in the nucleolus of Euglena. Cross-sections of the coiled filaments and fibrils, however, resembled very closely the description of the nucleolar granules by Lafontaine (81) with a core of less electron-dense material. The so-called granules, 150A in diameter, were actually a pair of coiling filaments describing a gyre. Less electron-dense material was attached to the filaments towards the center of the enclosed space and peripherally (Fig. 14). At the onset of prophase, chromatids were thicker and shorter than at interphase. Localized compactness became noticeable in certain regions of the chromosomes and later progressed to the entire chromosome (Figs. 10-15). The chromonemata of the mitotic chromosomes were represented by two closely apposed long filaments, coiled in diagonal across the chromosome body as reported by Davis (42) (Fig. 11). Longitudinal sections of chromo somes suggest the chromonemata are continuous, rather than made up of several fibrils or a network, as has been described for other cell types (20, 69, 76, 124, 125, 126, 156). Within the confines of the condensed chro- 5 Dutta et al . (46) have mistakenly identified the nucleolar indentations as an in tranucleolar body for which they proposed the name of "nucleololus". Cytologia 30, 1965 Plate I

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis

9* 122 E. H. J. O'Donnell Cytologia 30 mosome the chromonemata were highly coiled. Short fibrils accumulated in close association with the chromonemal threads as described by Yasuzumi (157) and in a similar way the short nucleolar fibrils related to the main nucleolar component (Fig. 10). During prophase, the nucleolus expanded and the indentations enlarged, giving the nucleolus a doughnut or ring shape (Figs. 10, 13-17). Late at prophase, the ring-shaped nucleolus opened up in bizarre aggregates of nu cleolar material in which the extended electron-dense filaments could be seen (Fig. 11). Some of the nucleolar electron-dense filaments are continuous with the chromonemata of the nucleolar chromosomes condensed nearby (Figs. 9-15). At metaphase, the chromosomes arranged their long axis almost parallel to the longitudinal axis of the cell rather than organizing in a metaphasic plate (85, 86, 146). During this investigation, 4 chromosomes were counted in the haploid set; the nucleolar chromosome was easily identified at all times of mitosis through its characteristic morphology (Figs. 11, 25-26). The nuclear membranes pesisted until metaphase. No signs of mitotic ap paratus were found after different fixations. Very early at anaphase, some nucleolar material remained attached to the nucleolar chromosome. This remnant nucleolar material was drawn by each nucleolar chromosome, moving towards the pole. Remnant nucleolar material has been observed with light microscopes in various cell types, including the Euglena cell, but it has been erroneously described as "per sistent nucleolus" (28, 52, 85, 159). The findings during this investigation supported an early interpretation of "persistent" nucleolar material (88) and confirmed that the nucleolar body is no longer organized after prophase. The telophase chromosomes arrayed closely and the nucleolus became promptly organized at a particular chromosome (Figs. 25, 26). While the nucleolus enlarged, the chromonemata uncoiled and extended until the interphase con dition was attained. Nuclear organization after RNAse treatment. RNAse-treated cells prepared for light microscopy showed a diminished basophilia as enzymatic treatment continued. Organelles containing RNA showed after RNAse treat ment a disorganized appearance caused by the specific removal of RNA. Following treatment with either of both ribonucleases investigated, the RNA in the interlamellar matrix of the chloroplasts (Fig. 12), the cytoplasmic RNA and the chromosomal RNA (Fig. 14) were removed to a greater degree than the nucleolar RNA. It should be mentioned that degradation of cell organelles by RNAse acting in vivo, coincided with the depolymerization patterns observed in materials exposed to RNAse after fixation. The re sistance of nucleolar RNA to RNAse action has also been observed in samples treated with the enzyme after mild fixation (15, 62, 84). In the living material, certain nucleolar constituents were modified only Cytologia 30, 1965 Plate II

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis 124 E. H. J. O'Donnell Cytologia 30 after incubation in RNAse for periods extending up to 4 and 6hr. Where the enzyme acted, degraded nucleolar material had a blurred appearance (Figs. 12, 14, 15); a filamentous structure RNAse-resistant then became visible. At the stages observed, nucleolar RNA seems to be associated with the short fibrils of the second nucleolar component. The delicate fibrils crossing the lumen of the nucleolar indentations at interphase, thickened during early prophase up to 500A-700A. Not only do they resemble the chromonemata, but they present a similar pattern of degradation after RNAse (Fig. 14). RNAse modified the organization of certain regions of the early prophase chromosome (Fig. 14). After condensation-when the chromonemata be came surrounded by matrical proteins-RNAse partially degraded the matrix without affecting the chromonemata (Figs. 12, 13); this would indicate that at early prophase chromosomal RNA is intimately associated with chromosomal DNA(DNA-RNA complex?) (67), while after condensation it is associated with chromosomal matrical proteins as previously reported (44, 101, 102). After prophase condensation only RNAse-resistant fibrils of less than 100A in diameter were seen crossing the lumen of the nucleolus (Fig. 15). Ueda (146) identified these as nucleoplasmic fibrils; in these author's opinion they are chromonemal. Nucleolar material was continuous with the chromatin at points in the periphery (Figs. 12-15).

RNAse A, having about 1.4•~the specific activity of RNAse, degraded

RNA-containing structures apparently in similar fashion to RNAse, but attained equivalent disorganization levels in shorter periods of contact. Nuclear organization after DNAse treatment. DNAse effectively de polymerized DNA-containing nuclear structures even after short incubation a digestion successfully attained in vivo without the pepsin pretreatment reported by Leduc and Bernhard (84) as necessary for fixed material. Hy drolysis of the chromonemata was observed from late interphase until very late telophase. The chromonemata in the condensed regions of the early prophase chromosomes were more affected by DNAse than expanded regions which were surrounded by an electron-dense material (Figs. 16, 17). At equal contact times with the enzyme, the prophase and early metaphase chromonemata were more susceptible to DNAse activity than during late metaphase, anaphase, and early telophase. During the same mitotic stages, the chromosomes reached maximum condensation; this condition perhaps made the chromonemata more resistant to enzyme action in the living state. In the nucleolus, DNAse depolymerized the long electron-dense filaments of the first nucleolar component described (Fig. 16). The nucleolar areas hydrolyzed by DNAse at late prophase were wider than those hydrolyzed at interphase or early prophase which indicates that the DNA-containing structures widened during prophase (Figs. 17, 18). The structures crossing the lumen of the nucleolus were also degraded by this nuclease (Figs. 17, 18). Nuclear organization after proteinase treatment and nuclease followed Cytologia 30, 1965 Plate III

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis 126 E. H. J. O'Donnell Cytologia 30

by proteinase. Of the two proteinases employed, trypsin and ƒ¿-chymotrypsin, the former has been reported as not penetrating the living cell (107) and as degrading natural proteins very slowly. Extent of proteinase activity on

Euglena cells was also controlled by light microscopy. Samples were drawn of the proteinase-incubated cultures at the end of 15, 30, 45, 60, 90, 120,

150 and 180min of exposure. The viability of the Euglena cells was judged from locomotion and metaboly. At the end of 30min contact with trypsin,

Euglena cells moved more slowly than the control cells: chloroplast degrada tion was noticed after 1-hr contact; after 3-hr, locomotion was repressed, metaboly persisted as a very slow motion in 60% of the cells sampled. ƒ¿- chymotrysin showed little effect on the vital movements of Euglena after short exposures; locomotion slowed down only after 2-hr contact, but meta boly remained unchanged; chloroplasts began to disaggregate and decolorize after 1hr. In cells prepared for electron microscopy, both proteases ex tensively hydrolyzed cell organelles even after 1-hr contact. This indicates

that ƒ¿-chymotrypsin, as well as trypsin, penetrated living cells.

Trypsin or ƒ¿-chymotrypsin attenuated the interphase and the very early prophase chromonemata until the chromonemal threads became faint. In the condensed regions of the mid-prophase chromosomes, the proteinase treatment intensely degraded short fibrils associated with the chromonemata

(Fig. 21). The short fibrils constituted a proteinaceous matrix around the chromonemata. High electron-dense regions of the chromosomes, lacking

this matrix, resisted proteolytic degradation (Fig. 21).

Trypsin and ƒ¿-chymotrypsin modified the nucleolar appearance de

polymerizing the short fibrils of the second nucleolar component. Proteinase induced modifications were not discerned in the long, electron-dense filaments of the first nucleolar component (Fig. 20).

Euglena cells were also treated by nuclease followed by a poteinase.

Following RNAse and either trypsin or ƒ¿-chymotrypsin, long RNAse-resistant

nucleolar filaments became conspicuous, apparently unchanged by the combined

treatment; they were coiled tightly and ran along the whole nucleolar body

(Fig. 19) to continue into the chromonemata outside the nucleolar boundaries. In the combined treatment of DNAse followed by either proteinase, the chromonemata and the electron-dense nucleolar filaments were degraded by the nuclease and the matrical chromosomal fibrils and the short nucleolar fibrils by the proteinases (Figs. 22, 23).

Discussion According to Leduc and Bernhard (84) DNAse does not hydrolyze nuclei of tissues embedded in Durcupan6 unless the sections are pretreated by pepsin. They also described an interchromatinic material of unknown nature as a residual nuclear material after extended DNAse treatment. Durcupan is an

6 Registered trademark of Fluka AG ., Buchs SG, Switzerland. Cytologia 30, 1965 Plate IV

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis 128 E. H. J. O'Donnell Cytologia 30 effective dehydrating agent of tissue and also causes intermolecular cross linkages in proteins and nucleic acids (16, 84)7. This may account for the failure of DNAse when used alone, likewise for the DNAse-resistance of the so-called interchromatinic material. Interchromatinic material was never observed in Euglena after condensation of the chromosomes and before breakdown of the nuclear membrane. The nuclear organization of Euglena gracilis v. bacilaris conforms with the nuclear organization described for many cell types (4, 42, 126, 128, 156, 157). The late interphase chromonema is about 100A and is constituted by a pair of filaments about 43A each, closely apposed in certain regions, relationally coiled in others (Figs. 9, 22, 23). Matrical fibers are laid down later around each filament. The matrical fibrils of Euglena arrange in space as the dendritic process described by Yasuzumi (157) in ascites tumor cells and as the matrix material described by Lafontaine and Chouinard (82) in Vicia faba. A pair of relational coiled filaments becomes associated with another pair to constitute the early prophase chromosome-200-300A wide, which progressively widens up to 700A. During prophase condensation, the con figuration of the chromonemata changed with the addition of another order of coil, along which they fold across the width of the chromosome body (Figs. 10, 11, 13). The differences in the spatial configuration of the chro monemata between interphase and prophase may denote differences in syn thetic processes: synthesis of DNA at interphase; synthesis of matrical proteins at prophase. Uncoiling as well as condensing of chromonemata are asynchronous, as are DNA replication and matrical synthesis. Since each DNA molecule replicates independently, this process can be accomplished when the chromosomal region to be replicated is loosely arranged and extended. Synthesis of matrical proteins, on the other hand, may involve the cooperation of different chromosomal sites. If the production of nonstructural proteins proceeds independently in each chromosome, favorable conditions for close, fast loci interaction are brought about, changing the spatial configuration of the chromonemata from the expanded to the condensed. Localization of chromonemata within the compartments of the condensing prophasic chro mosomes is of twofold significance; it facilitates the displacement of chro mosomes during metaphase and anaphase, and it mediates the closeness for the synthetic activities demanding gene interaction or cooperation. It can be said that the configuration of the chromonemata is the framework that the mitotic chromosome (39, 40). RNA seems to form a complex with DNA in the filamentous organiza

7 In our experiments, Durcupan damaged Euglena gracilis . The cells so embedded had the interlamellar matrix of chloroplast completely degraded, the lamellae disarrayed and partially digested, and disorganized cristae in the mitochondria, and the filamentous organization of chromatin constituents was much modified. Cytologia 30, 1965 Plate V

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis 130 E. H. J. O'Donnell Cytologia 30 tion of interphase chromosomes (67). In Euglena numerous regions of the early prophase chromosomes were degraded by RNAse (Fig. 14). The removal of the RNA complexed with DNA in the chromosome brings about the dis organization of the region and even disruption of the chromosome (67). The chromonemata in the condensed regions, instead, were not modified by incubation with RNAse. It becomes evident that RNA is intimately associated with chromosomes at stages in which a nucleolus is fully organized, i.e., the presence of chromosomal RNA is compatible with the presence of nucleolar RNA. A highly pycnotic region of the nucleolar chromosome, the so-called nucleolar organizer body, is currently believed to control nucleolus formation (59, 70, 97, 131, 132). According to Rattenbury and Serra (119), in organisms lacking the heteropycnotic body the nucleolar organizer may be unresolvable by light microscopy. For Krivshenko (78), not only the nucleolar organizer body takes a direct part in the formation of a nucleolus but "... the unre solvable branches of the nucleolar organizer" as well, considering that very often chromosome strands penetrate into the nucleolus (35, 43, 56, 59, 61, 80, 81, 89, 91, 105, 108, 109, 110, 111, 120, 156). Hybridization experiments, however, indicate that only the intranucleolar regions of the nucleolar chro mosome have a direct role in the formation of a nucleolus (10, 11, 91), as postulated among others by Dearing (43), Lewis (89), Godward (61), Ohno et al. (111), Yasuzumi et al. (156) and O'Donnell (108). Along with this line of evidence, it has been found that the synthesis of intranucleolar RNA depends on intranucleolar DNA (116). The nucleolus indeed possesses fila mentous structures containing DNA structures which are continuous with the chromonemata they resemble very closely (16, 17, 87, 88, 108, 109, 110, 139, 141). A nucleolar DNA fraction has been reported in the chemical analyses of isolated nucleoli (29, 44, 104, 106, 136) and there is nucleolar incorporation of DNA precursors (37, 65, 68, 69, 121) which indicates DNA synthesis and, consequently, replication of the structure bearing the DNA. The nucleolar synthesis of RNA has been proved (a) to occur independently of extranucleolar RNA synthesis (3, 5, 6, 8, 12, 21, 33, 48, 49, 53, 65, 71, 83, 98, 99, 113, 114, 115, 123, 131, 143, 153, 154) and (b) to depend on intra nucleolar DNA (116). The DNA chemically isolated from nucleoli is presently described as a contaminant-i.e., extranucleolar DNA. Although the rest of the nucleolar chromosome, or part of it, may remain attached to the nucleolus and other chromosomal fragments adhere to the nucleoli during isolation procedures, a percentage of the so-called contaminant DNA could well be intrinsic nu cleolar DNA corresponding to the DNA-containing nucleolar structures . Detached nucleoli of the oocyte of the starfish have been reported to lack DNA (100, 148, 149). A similar system, however , detached nucleoli of the Triturus oocyte, contains DNA (74). It is interesting to note that also in Cytologia 30, 1965 Pl ate VI

O'Donnell: Nucleolus and Chromosomes in Englena gracilis 132 E. H. J. O'Donnell Cytologia 30

T. viridescens Duryee (45) observed that to free nucleoli attached to the k-thread, the chromosome should be fractured, which indicates an intimate association. Since all nucleolar RNA fractions have been found to be dependent on nucleolar DNA (55, 57, 72, 73, 116, 118, 129, 130), the interpretation by earlier workers of a nucleolar RNA synthesis being primed by extrinsic RNA synthesis would be untenable (132). In addition to newly synthesized RNA (9, 26, 34) the nucleolus possesses the DNA required in the RNA polymerase (RNA nucleotidyl transferase) reactions for DNA-primed synthesis of RNA (72, 135) and very likely it also contains the RNA polymerase (55, 130, 152). The intranucleolar structures containing DNA are indeed the site of synthesis of nucleolar RNA. In terms of cellular coordination it would seem wasteful to reserve so many close chromosomal loci of the intranucleolar DNA just to collect material presumably synthesized elsewhere. Recent experiments indicate that the RNA polymerase may copy preferentially certain regions of DNA (73); this may explain the localization of RNA synthesis to certain chromosomal regions-the nucleolar organizer in particular. Franklin and Baltimore (55) proposed that structural ribosomal RNA is synthesized in the nucleolus and recent data by Perry confirmed this assumption (116). In Escherichia coli, the DNA with a sequence complementary to homologous ribosomal RNA shows the units contiguous in the DNA structure and not scattered throughout the genome (155). If ribosomal RNA in nucleolated cells is synthesized by intranucleolar DNA, as described, the contiguous subunits in the DNA structure of E. coli may represent in bacterial organization the nucleolar organizer of higher organisms. The DNA-containing structures found in the nucleolus of Euglena have the morphology and organization of the chromonemata with which they are continuous. The intranucleolar filaments are here interpreted as the nucleolar organizer proper and will be so referred to. The organizer concept vs. the nucleolonema concept. Currently, and in the past, a nucleolar filamentous structure has been described under the name of nucleolonema (13, 16, 32, 42, 50, 51, 62, 68, 77, 121, 133, 134, 141, 156). The reasons behind the interpretation of the nucleolar structures of Euglena as the organizer will be better understood if the organizer attributes are discussed in parallel with those credited to the nucleolonema. According to Estable and Sotelo (50, 51), the nucleolonema is a permanent structure of normal and cancerous cells; the nucleolar organizer, being a chro mosomal region, is also a permanent structure of all nucleolated cells, unless a deletion in the nucleolar chromosome includes the organizing region. The nucleolonema is believed to become organized independent of any chromosomal locus (50, 51, 133); the nucleolar organizer is a chromosomal region very likely with multiple genetic loci (10, 78, 112, 127, 131, 148, 150). The nu cleolonema shows diversified aggregation-from simple coiled filaments up Cytologia 30, 1965 Plate VII

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis 134 E. H. J. O'Donnell Cytologia 30

to complex networks of filaments (13, 14, 15, 16, 23, 24, 25, 27, 32, 42, 50, 51, 62, 63, 84, 93, 117, 133); the nucleolar organizer can also be observed in configurations matching those described for the nucleolonema (10, 43, 61, 78, 87, 88, 108, 109, 110) (Figs. 28, 29). The nucleolonema is believed to

contain RNA; this assumption, however, is based on the overall Feulgen negative reaction of nucleoli rather than on structure-specific chemical study (50, 51, 158); according to Gonzalez-Ramirez-(63), however, the argentophil nucleolonema resists RNAse. The intranucleolar organizer is Feulgen-positive (87, 89, 105, 108, 109, 110), is hydrolyzable by DNAse (108, 109, 110, 139), and is RNAse-resistant (15, 84, 108, 109, 110). At mitosis the nucleolonema would fragment during prophase, after which each nucleolonemal segment would join a chromatid by its side; nucleolonemal segments would coalesce to reconstitute the nucleolus at telophase (50, 51, 64, 158). The mitotic behavior of the nucleolonema has been primarily detailed under light micro scopes; electron-microscopical observations failed to confirm it. The nucleolated condition shows Mendelian segregation (54). Further more, the semiconservative segregation of chromatids is favored as the most likely mechanism in the distribution of genetic information (22, 73, 142, 144); the nucleolar organizer being a chromosomal region will, therefore, segregate in a semiconservative pattern if its behavior conforms with that of non nucleolar regions. It is interesting to note that Harris (66) demonstrated that a certain nucleolar proteinaceous constituent of premitotic nucleolus is distributed between both post-mitotic, derivative nucleoli. In line with this finding, Martin (96) reported that certain nucleolar constituents show con tinuity with the next cell generation; one of them is a basic-type protein which segregates semiconservatively. The structures described as the nucleolonema usually average from 600A. up to 1200A in diameter; this width is of the order described for paired chromonemata at prophase. The nucleolar organizer proper is constituted Cytologia 30, 1965 Plate VIII

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis

Cytologia 30, 1965 10 136 E. H. J. O'Donnell Cytologia 30 instead by filaments about 100A, the same order of magnitude observed in the morphological unit of the interphase chromonema and up to 700A after somatic pairing. The information along the nucleolar organizer shows linear arrangement similar to the arrangement of the information storaged in non-nucleolar re gions of chromosomes. It has been argued that the information storaged in the organizer does not involve the actual synthesis of the nucleolar material, but only the collection of the material produced at different loci. The evidence against this interpretation of nucleolar formation has been presented in pp. 16-18. A few considerations are made here about what is considered strong evidence supporting the theory that nucleolar material is contributed by all chromosomes (1, 79, 119, 120): when a nucleolar organizer is lacking, "blobs" of nucleolar-like material are seen in different chromosomes (54, 66). The chromosomal sites believed to synthesize such material are known as the "latent organizers" . The "prenucleolar bodies"-originating in the chro mosomal matrix-described in the early work by Lafontaine (80, 81) were thought to substantiate at the fine structure level this theory of nucleolar formation postulated after observations with light microscopes (1, 119). Lafontaine and Chouinard (82) reevaluated their conclusion: they presently regard the nucleolar material as being synthesized by all telophase chromo somes and not derived from matrical material. Hybridization experiments (10, 91, 92) have shown, however, that only the nucleolar chromosome affects nucleolar formation and nucleolar mass (41). If the nucleolar organizer represses the expression of the "latent organizers", as theorized, deletion of the nucleolar organizer would eliminate the repressor action and chromosomal sites capable of RNA-synthesis, normally present, (140, 145) may show hyperactivity. Anucleolated cells-by deletion of the nucleolar organizer-fail to attain normal levels of cell division and development (10, 54) in spite of the nucleolar-like material produced by the "latent organizers" . Since the quantity of the material produced by the latent organizers combined, often reaches the levels of the material produced by the nucleolar organizer in normal conditions, the failure to reach normal growth may perhaps be attributed to differences of kind, rather than of degree. Kopac and Mateyko (77) suggested that "... The relationship of the nucleolus to the chromosome and to its 'cycle' during mitosis would be more intelligible if the nucleolonema were continuous with the nucleolar organizer or were part of it. Thus, the nucleolonema, the nucleolar organizer, and the nucleolar chromosomes would be the self-replicating structures". If we would assume that the nucleolonema contains RNA and no DNA at all, as postulated by Estable and Sotelo (50, 51), a self-replicating nucleolonema would imply self-replicating abilities for the nucleolonemal RNA. This possibility is ruled out since the biochemical evidence shows that the synthesis Cytologia, 30 1965 Plate IX

Q'Donnell: Nucleolus and Chromosomes in Euglena gracilig

10* 138 E. H. J. O'Donnell Cytologia 30 of all nucleolar RNAs is DNA-dependent (57). In the light of our present information about nucleolar organization and the mechanisms for nucleic acids syntheses, the nucleolonema concept as defined by Estable and Sotelo (50) is no longer tenable. On the other hand, the nucleolar filamentous structures containing DNA which had been described and interpreted in this paper as the true nucleolar organizer region of the nucleolar chromosome are endowed as chromosomal regions, with self-replication. The words nucleolar organizer suggested "in lieu" of nucleolonema not only have priority in the literature but, what is more significant, they inform about the function of this particular chromosomal region. The nucleolus can, therefore, be regarded as a particular structure of the nucleolar chromosome(s) and the nucleolar material as the manifestation of the functional state and synthetic abilities of the loci in the nucleolar organizer region (138). The nucleolar organizer proper-intranucleolar-can be pictured as an extended chromosomal loop highly coiled and folded in space. Nucleolar proteins are laid down as short, coiled fibrils almost at right angles with the organizer and intimately associated with it. These short nucleolar fibrils, which are depolymerizable RNAse and proteinases, may correspond to the ground nucleolar material described by light microscopy under the name of "pars amorpha" and known to be Feulgen-negative and pyroninophilic (50, 51, 61, 62, 64, 87, 88, 103, 108, 109, 110, 133). During prophase the organizers thicken and shorten as non-nucleolar chromosomal regions do. Depending on the configuration of the folds, and on the organizer metabolic conditions, indentations in the nucleolar mass will be noticeable (13, 14, 15, 25, 38, 46, 62, 81, 109, 110, 117, 122). In embryonic cells nucleoli are organized after exhaustion of the maternal proteins (131); this would indicate that the nucleolar organizer is non-functional at the very early stages of development. Dr . Lyser8 (Cornell University Medical College) has observed differential organization in nucleoli during the development of the neural tube of chick embryo: early in embryonic life the nucleolar organization suggests a loose arrangement of the folds in the organizer (Fig. 28) while at later stages the configuration of the overlapping loops gives the nucleolus the meshwork look described so often (Fig. 29). This suggests two possibilities: either all subunits along the organizer are active but with a moderate metabolism at early stages, or different subunits are activated at different stages depending upon the complexity and demand of specific proteins to grow and attain differentiation. The second possibility also seems to hold for the nucleoli of the starfish oocyte in which Edstrom (47) found changes in the base composition of nucleolar RNA during the growth of the oocyte. This finding implies that at different stages of growth, nucleolar RNA is primed by different complementary DNAs-inhomogeneities

8 Personal communication. Cytologia 30, 1965 Plate X

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis 140 E. H. J. O'Donnell Cytologia 30 in base distribution along the chain of DNA molecules have already been demonstrated (95). In actively dividing cells the nucleolar organizer proper should be metabolically active-consequently, in the expanded condition-earlier than other chromosomal regions if nucleolar RNA mediates extranucleolar synthesis from interphase on. In Euglena, both chromatids of the nucleolar chromo some participate in the organization of the nucleolus (Figs. 13, 27). Judging by the number of chromatids entering and leaving the nucleolar body at different stages, the nucleolar organizer replicates early at anaphase, im mediately after the sister chromatids separated (Fig. 24). Somatic pairing must occur between the organizers, as described by Lettre and Siebs (88) and Ohno et al. (111, 112) since the two organizers concur to organize a single nucleolar body. Nucleolar material accumulates first at the medial region of the intranucleolar organizer, later progressing towards the proximal region of the satellite, and the long arm of the nucleolar chromosome (Fig. 25). If the sequence of synthetic periods in the organizer is similar to the sequences observed in other chromosomal regions, this would indicate that the medial region of the organizer is the first to replicate. The organizer, then similar to non-nucleolar regions, shows asynchronous DNA synthesis (5, 12, 90, 118, 144) and, as a consequence, asynchronous RNA and protein syntheses. An electron-dense region was sometimes observed in the nucleolar body of Euglena from telophase until early prophase (Figs. 12, 14, 16, 17, 27). After condensation was completed by all chromosomal regions, the electron dense region was no longer seen (Figs. 10, 11, 13, 15). This intranucleolar region may be the last to engage in synthesis of nucleolar material. The central body in the Euglena nucleus has been described, sometimes, as lacking the attributes of true nucleoli. The word endosome has been used either as a substitute (85, 86) or as a synonym (60) for nucleolus. According to Leedale (85) the main differences between true nucleolus and endosome are: a) the endosome is not associated with particular chromosomes, as nucleoli are; b) the endosome persists during mitosis; and c) the endo some retains RNA during mitosis. Furthermore, the existence of a nucleolar chromosome in the Euglena complement has also been questioned (85). The electron-micrographs in this paper (Figs. 10-14, 16, 17, 24-27) distinctly show the association between the nucleolus and the nucleolar chromosome; such was not indicated by the light microscopy used by previous investigators (85, 86). The validity of the interpretation of the persistence of nucleoli or endosome, for this matter, has been discussed on p. 126. Finally, the remnant nucleolar material still attached to the nucleolar organizer after the 9 Parenthetically , it may be remarked that Bollum (22) suggested that from the stand point of the mechanics of DNA replication, the first event in DNA synthesis-the nucleation involving interaction of DNA and protein-would probably occur at the middle of a DNA chain. Cytologia 30, 1965 Plate XI

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis 142 E. H. J. O'Donnell Cytologia 30 disorganization of the nucleolus accounts for the persistence of RNA denoted in the so-called endosome. In conclusion: the distinction between nucleoli and endosomes is not valid, and the central body of the Euglena cell is a true nucleolus, its organization, metabolism and origin comparable to those of nucleoli of higher organisms, as some cytologists correctly interpreted it in the past.

Synopsis

Nuclear constituents were degraded by nucleases and proteinases. Pat

terns of depolymerization are described in interphasic and in mitotic stages

of Euglena gracilis var. bacillaris treated "in vivo" with DNAse, RNAse,

trypsin and ƒ¿-chymotrypsin for exposure extended from 2 to 6 hours. DNAse

degraded chromatin at all stages, and it also hydrolyzed a nucleolar con

stituent. RNAse moderately degraded one nucleolar component and certain

regions in the prophase chromosomes. Observations on control cells and RNAse

treated cells showed that the nucleolus possesses highly coiled filamentous

structures which resemble chromonemata and extend into the nucleolar chro

mosomes. Treatment by nuclease followed by proteinase demonstrated that

the nucleolar filamentous structure resembling chromonemata was degraded

by DNAse and by either proteinase but was RNAse-resistant. This nucleolar

structure is not regarded as the nucleolonema but as the nucleolar organizer

proper, and as such chromosomal in nature.

Acknowledgments

The author is indebted to the Cornell University Medical College and Dr. George B. Chapman for her training in electron microscopy and for the use of electron microscopes during the course of this research; to Dr. A. L. Mirsky for stimulating discussions; to Dr. Katherine M. Lyser for allowing the inclusion of her unpublished electronmicrographs of chick embryo.

References 1) Albuquerque, R. M. and Serra, J. A. and Serra, J. A. 1951. Nucleolar composition and the nucleolar zones of the chromosomes. Portug. Act. Biol. 3A: 187-93. 2) Allfrey, V. G. 1963. Nuclear ribosomes, messenger-RNA and protein synthesis. Exptl. Cell Res. Suppl. 9: 183-212. 3) Amano, M. and Leblond, C. P. 1960. Comparison of the specific activity time curves of ribonucleic acid in chromatin, nucleolus and cytoplasm. Exptl. Cell Res. 20: 250 - 3. 4) Amano, S., Dohl, S., Tanaka, H., Uchino, F. and Hanaoka, M. 1956. The structure of the nucleus studied by electron microsocopy in ultrathin sections with special reference to the chromonema-an advocation of "subchromonema" and "protochromonema". Cytologia 21: 241-51. 5) Bader, S., Miller, O. J. and Mukherjee, B. B. 1963. Observations on chromosome Cytologia 30, 1965 Plate XII

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis 144 E. H. J. O'Donnell Cytologia 30

duplication in cultured human leucocytes. Exptl. Cell Res. 31: 100-12. 6) Baltus, E. 1954. Observations sur le role biochimique du nucleole. Arch. Int. Physiol. 62: 281-2. 7) Bauer, H. 1933. Die wachsenden Oocytenkerne einiger Inshkten in ihrem Verhalten zur Nuklealfarbung. Zeit. f. Zellfors. 18: 254-98. 8) - and Caspersson, T. 1948. Cytochemical observations on nucleolus formation in Chironomus. Proc. VIII Int. Congr. Genetics. 9) Beerman, W. 1959. Chromosomal differentiation in insects, in Rudnick, D. Develop mental Cytology, The Ronald Press Co., N. Y.: 83-103. 10) - 1960. Der Nukleolus als lebenswichtiger Bestandteil des Zellkernes. Chromosoma 11: 263-96. 11) - 1962. Reisenchromosomen. Protoplasmatologia 6, 1-161. 12) Bender, M. A. and Prescott, D. M. 1962. DNA synthesis and mitosis in cultures of human peripheral leukocytes. Exptl. Cell Res. 27: 221-9. 13) Bernhard, W., Bauer, A., Gropp, A., Haguenau, F. and Oberling, C. 1955. L'ultrastructure du nucleole de cellules normales et cancereuses: etude au microscope electronique. Exptl. Cell Res. 9: 88-100. 14) - 1959. Ultrastructural aspects of nucleo-cytoplasmic relationship. Exptl. Cell. Res. Suppl. 6: 17-50. 15) - and Leduc, E. 1962. L'avenir de l'utilisation de matieres plastiques hydrosolubles en cytochimie ultrastructurale. Proc. 5th Int. Congr. Elect. Microsc., 2: L-4, -5. 16) - and Tournier, P. 1962. Ultrastructural cytochemistry applied to the study of virus infection. Cold Spring Harbor Symp. Quant. Biol. 27: 67-82. 17) - and Granboulan, N. 1963. The fine structure of the cancer cell nucleus. Exptl. Cell Res. Suppl. 9: 19-53. 18) Bhattacharyya, T. K. and Ghosh, A. 1961. On the occurrence of Feulgen positive nucleoli in adrenocortical cells of the pigeon. Naturwissenschaften 48: 389-90. 19) Birnstiel, M. L. and Hyde, B. B. 1963. Protein synthesis by isolated pea nucleoli. J. Cell Biol. 18: 41-50. 20) Bloom, W. and Leider, R. J. 1962. Optical and electron microscopic changes in ultra violet-irradiated chromosome segments. J. Cell Biol. 13: 269-301. 21) Bogoroch, R. and Siegel, B. V. 1961. Some metabolic properties of the nucleolus as demonstrated by recent radioisotope experiments. Acta. Anat. 45: 265-87. 22) Bollum, F. J. 1963. "Primer" in DNA polymerase reactions. Prog. Nucleic Ac. Res. 1: 1-26. 23) Bolognari, A. 1959. Esperienze con enzimi sui nucleoli degli ovociti in accrescimento di Mytilus galloprovincialis Lam. e di Patella coerulea L. Boll. Soc. It. Biol. Speriment. 35: 37-9. 24) - 1959. Aspetti strutturali e citochimici dell'apparato nucleolare degli ovociti in accrescimiento di Patella coerulea L. Acta Histochem. 8: 504-523. 25) - 1960. Precisazioni sul nucleolonema. Experientia 16: 307-9. 26) Bonner, J., Huang, R. C. and Maheshwari, N. 1961. The physical state of newly syn thesized RNA. Proc. Natl. Acad. Sci. (U. S.) 47: 1548-54. 27) Borysko, E. and Bang, F. B. 1951. Structure of the nucleolus as revealed by the electron microscope. Bull. Johns Hopkins Hosp. 89: 468-73. 28) Brown, W. F. and Emery, W. H. R. 1957. Persistent nucleoli and grass systematics. Amer. J. Bot. 4: 585-95. 29) Busch, H., Muramatsu, M., Chien, S. C. and Somers, C. 1962. Isolation and metabolic activity of nucleoli of the Walker tumor. Proc. Am. Assoc. Cancer Res. 3: 308. 30) Busch, H., Muiamatsu, M., Adams, H., Steele, W. J., Liau, M. C. and Smetana, K. 1963. Isolation of nucleoli. Exptl. Cell Res. Suppl. 9: 150-63. 31) -, Byvoet, P. and Smetana, K. 1963. The nucleolus of the cancer cell; a review, Cancer Res. 23: 313-339, Cytologia 30, 1965 Plate XIII

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis 146 E. H. J. O'Donnell Cytologia 30

32) Buvat, R. 1963. Electron microscopy of plant protoplasm. Int. Rev. Cyt. 14: 41-155. 33) Caspersson, T. and Schultz, J. 1940. Ribonucleic acids in both nucleus and cytoplasm, and the function of the nucleolus. Proc. Natl. Acad. Sci. (U. S.) 26: 507-15. 34) - 1962. Nucleo-cytoplasmic relations in normal and malignant cells. In Allen, J. M. The Molecular Control of Cellular Activity, McGraw-Hill, New York: 127 -141. 35) Chayen, J., Davies, H. G. and Miles, U. J. 1953. Observations on some plant inter phase nuclei. Proc. Roy. Soc. London, S. B. 141: 190-9. 36) Chen, T. T. 1936. Observations on mitosis in opalinids (Protozoa, Ciliata). II. The association of chromosomes and nucleoli. Proc. Nat. Acad. Sci. (U. S.) 22: 602-607. 37) Chevremont, M., Bassleer, R. and Baeckeland, E. 1961. Nouvelles recherches sur les acides desoxyribonucleiques dans des cultures des fibroblastes refroidie puis rechauffees. Etudes cytophotometrique et histoautoradiographique. Archives du Biol. 72: 501-23. 38) Crawley, J. C. W. 1963. The fine structure of Acetabularia mediterranea. Exptl. Cell Res. 32: 368-78. 39) Darlington, C. D. 1947. Nucleic acid and the chromosomes. Sym. Soc. Exp. Biol. 1: 252-69. 40) - 1955. The chromosome as a physico-chemical entity. Nature 176: 1139-44. 41) Das, N. K. 1962. Synthetic capacities of chromosome fragments correlated with their ability to maintain nucleolar material. J. Cell Biol. 15: 121-30. 42) Davis, J. M. G. 1963. The ultrastructure of nucleoli and chromosomes during the early stages of liver regeneration and the changes produced in these structures by X radiation. Phil. Trans. Roy. Soc. London 246: 291-303. 43) Dearing, W. H. 1934. The material continuity and individuality of the somatic chro- mosomes of Ambyostoma tigrinum, with special reference to the nucleolus as a chro - mosomal component. J. Morphol. 56: 157-79. 44) Dounce, A. L. 1955. The isolation and composition of cell nuclei and nucleoli. In Chargaff, E. and Davidson, J. N., The Nucleic Acids, Academic Press, Inc., New York, 2: 93-153. 45) Duryee, W. R. 1950. Chromosomal physiology in relation to nuclear structure. Ann. N. Y. Acad. Sci. 50: 921-53. 46) Dutta, C. R., Siegesmund, K. A. and Fox, C. 1963. Light and electron microscopic observations of an intranucleolar body in nerve cells. J. Ultrastructure Res. 8: 542 - 51. 47) Edstrom, J. E. and Beermann, W. 1962. The base composition of nucleic acids in chromosomes, puffs, nucleoli and cytoplasm of Chironomus salivary gland cells. J. Cell Biol. 14: 371-9. 48) Edstrom, J. E. and Gall, J. G. 1963. The base composition of ribonucleic acid in lampbrush chromosomes, nucleoli, nuclear sap, and cytoplasm of Triturus oocytes. J. Cell Biol. 19: 279-84. 49) Errera, M., Hell, A. and Perry, R. P. 1961. The role of the nucleolus in ribonucleic acid and protein synthesis. II. Amino acid incorporation into normal and nucleolar inactivated HeLa cells. Biochem. Biophys. Acta 49: 58-63. 50) Estable, C. and Sotelo, J. R. 1951. Una nueva estructura celular: el nucleolonema. Inst. Invest. Ciencias Biol. 1: 105-26 51) - and - 1954. The behaviour of the nucleolonema during mitoses. In Noordhoff, P., Symp. on Fine Structure of the Cells, 8th Int. Cong. Cell Biol. Leiden: 170-90. 52) Fabbri, F. 1960. Contributo per l'interpretazione della persistenza nucleolare durante la mitosi in Psilotum nudism (L.) Beauv. Caryologia 13: 297-337. 53) Feinendegen, L. E., Bond, V. P., Shreeve, W. W. and Painter, R. B. 1960. RNA and DNA metabolism in human tissue culture cells studied with tritiated cytidine. Exptl. Cell Res. 19: 443-59. 54) Fischberg, M. and Wallace, H. 1960. A mutation which reduced nucleolar number Cytologia 36, 1965 Plate XIV

O'Donnell: Nucleolus and Chromosomes in Euglena gracilis 148 E. H. J. O'Donnell Cytologia 30

in Xenopus laevis. In Butterworth & Co., Ltd., London, The Cell Nucleus: 30-34.

55) Franklin, R. M. and Baltimore, D. 1962. Patterns of macromolecular synthesis in

normal and virus-infected mammalian cells. Cold Spring Harbor Symp. Quant. Biol.

27: 175-95.

56) Furieri, P. 1957. Chromatin association with the nucleolus of various mammals. Boll. Dell. Soc. Ital. Biol. Sper. 33: 260-1.

57) Furth, J. J., Hurwitz, J. and Goldmann, M. 1961. The directing role of DNA in RNA

synthesis. Bioch. and Biophys. Res. Comm. 4: 362-7.

58) -, - and Anders, M. 1962. The role of deoxyribonucleic acid in ribonucleic acid

synthesis. I. Purification and properties of ribonucleic acid polymerase J. Biol. Chem.

237: 2611-19. 59) Gates, R. R. 1942. Nucleoli and related nuclear structures. Sot. Rev. 8: 337-409.

60) Gibbs, S. P. 1960. The fine structure of Euglena gracilis with special reference to the chloroplasts and pyrenoids. J. Ultrastructure Res. 4: 127-48.

61) Godward, M. B. E. 1950. On the nucleolus and nucleolar organizing chromosomes of

Spirogyra. Ann. Bot. 14: 39-53. 62) Gonzalez-Ramirez, J. 1959. Etudes sur 1'ultrastructure du nucleole. Rev. d'Hemat.

14: 333-43.

63) - 1961. Nucleolar physiology. III. Considerations about the meaning of nucleolar

silver impregnations in cultured HeLa cells. Bot. Inst. Est. Med. y Biol. 19: 195-205.

64) Guttes, E. and Guttes, S. 1961. On the filamentous elements in the nucleoli of chick

embryo fibroblasts. Q. J. M. Sc. 102: 23-6 65) Harris, H. 1959. The initiation of deoxyribonucleic acid synthesis in the connective

tissue cell, with some observations on the function of the nucleolus. Bioch. Jour.

72: 54-60. 66) - 1961. The nucleolus. Nature 190: 1077-8.

67) - 1963. Nuclear ribonucleic acid. Progr. Nucleic Ac. Res. 2: 19-59.

68) Hay, E. D. and Revel, J. P. 1962. The DNA component of the nucleolus, studied in

autoradiographs viewed with the electron microscope. Proc. 5th Int. Cong. Elect. Micros. 2: 0-7, -8.

69) Hay, E. D. and Revel, J. P. 1963. The fine structure of the DNP component of the

nucleus. An electron microscopic study utilizing autoradiography to localize DNA syn

thesis. J. Cell. Biol. 16: 29-51. 70) Heitz, E. 1931. Die Ursache der gesetzmaƒÀigen Zahl, Lage, Form and GrosƒÀe pflan

- zenlicher Nukleolen. Planta 12: 775-844.

71) Hsu, T. C. 1962. Differential rate in RNA synthesis between euchromatin and hetero

chromatin. Exptl. Cell Res. 27: 332-4.

72) Hurwitz, J., Furth, J. J., Malamy, M. and Alexander, M. 1962. The role of deoxy ribonucleic acid in ribonucleic acid synthesis. III. The inhibition of the enzymatic

synthesis of ribonucleic acid and deoxyribonucleic acid by actinomycin D and pro

flavin. Proc. Natl. Acad. Sci. (U. S.) 48: 1222-30. 73) - and August, J. T. 1963. The role of DNA in RNA synthesis. Progr. Nucleic Ac.

Res. 1: 59-92.

74) Izawa, M., Allfrey, V. G. and Mirsky, A. E. 1963. Composition of the nucleus and

chromosomes in the lampbrush stage of the newt oocyte. Proc. Natl. Acad. Sci. (U. S.)

50: 811-17. 75) Jacob, J. and Sirlin, J. L. 1963. Electron microscope studies on salivary gland cells.

J. Cell. Biol. 17: 153-65. 76) Kaufmann, B. P. and De, D. N. 1956. Fine structure of chromosomes. J. Biophys.

Bioch. Cyt. 2 (suppl.): 419-25.

77) Kopac, M. J. and Mateyko, G. 1958. Malignant nucleoli: cytological studies and

perspectives. Ann. N. Y. Acad. Sci 73: 237-82.

78) Krivshenko, J. 1959. The divisibility of the nucleolus organizer and the structure of 1965 Nucleolus and Chromosomes in Euglena gracilis 149

the nucleolus in Drosophila buscki. Genetics 44: 520-1. 79) LaCour, L. F. 1963. Ribose nucleic acid and the metaphase chromosome. Exptl. Cell Res. 29: 112-18. 80) Lafontaine, J. G. 1958. Structure and mode of formation of the nucleolus in meri -stematic cells of Vicia faba and Allium cepa. J. Biophy. and Bioch. Cyt. 4: 777-84 . 81) - 1953. A particulate component found in nucleoli of Allium cepa and Vicia faba. J. Biophy. and Bioch. Cyt. 4: 229-30. 82) - and Chouinard, L. A. 1963. A correlated light and electron microscope study of the nucleolar material during mitosis in Vicia faba. J. Cell. Biol. 17: 167-201. 83) Leblond, C. P. and Amano, M. 1962. Synthetic processes in the cell nucleus. IV. Synthetic activity in the nucleolus as compared to that in the rest of the cell. J. Histochem. and Cytochem. 10: 162-74. 84) Leduc, E. H. and Bernhard, W. 1961. Ultrastructural cytochemistry. Enzyme and acid hydrolysis of nucleic acids and protein. J. Biophy. and Bioch. Cyt. 10: 437-55. 85) Leedale, G. F. 1958. Nuclear structure and mitosis in the Euglenineae. Arch. Mikro biol. 32: 32-64. 86) Leedale, G. F. 1962. The evidence for a meiotic process in the Eugleninae. Arch. Mikrobiol. 42: 237-51. 87) Lettre, R. and Siebs, W. 1955. Beobachtungen zur Struktur des Nucleolus in normalen Zellen sowie in Tumorzellen. Zeit. f. Krebsforsch. 60: 564-80. 88) - 1961. Some studies of the nucleolus of cells cultivated "in vitro". Path. Biol. 9: 819-23. 89) Lewis, W. H. 1940. On the chromosomal nature of nucleoli. Bull. Johns Hopkins Hosp. 66: 60-4. 90) Lima de Faria, A. 1959. Differential uptake of tritiated thymidine into hetero and euchromatin Melanoplus and Secale. J. Biophy. and Bioch. Cyt. 6: 457-66. 91) Lin, M. 1955. Chromosomal control of nucleolar composition in maize. Chromosoma 7: 340-70. 92) Longwell, A. C. and Svihla, G. 1959. Specific chromosomal control of the nucleolus and of the cytoplasm in wheat. Exptl. Cell Res. 20: 294-312. 93) Marinozzi, V. 1962. Premieres observations sur la structure fine du nucleole revelees par des methodes de cytochimie ultrastructurale. Proc. 5th Int. Cong. Elect. Microsc. 2: NN-5, -6. 94) - 1962. The role of fixation in electron staining. J. Roy. Microsc. Soc. Ser. B. 95) Marmur, J., Rownd, R. and Schildkraut, C. L. 1963. Denaturation and renaturation of deoxyribonucleic acid. Prog. Nucleic Ac. Res. 1: 231-300. 96) Martin, P. G. 1961. Evidence for the continuity of nucleolar material in mitosis. Nature 190: 1078-9. 97) McClintock, B. 1934. The relation of a particular chromosomal element to the de velopment of the nucleoli in Zea mays. Zeits, f. Zellforsch. 21: 294-328. 98) McMaster, Kaye R. and Taylor, J. H. 1958. Evidence for two metabolically distinct types of ribonucleic acid in chromatin and nucleoli. J, Biophy. and Bioch. Cyt. 4: 5-11. 99) - 1960. The metabolic characteristics of nucleolar, chromosomal cytoplasmic ribo nucleic acid of Drosophila salivary glands. J. Biophy. and Bioch. Cyt. 8: 365-78. 100) Miller, O. L. 1962. Studies on the ultrastructure and metabolism of nucleoli in am phibian oocytes. 5th Int. Cong. Elect. Micros. 2: NN-8, -9. 101) Mirsky, A. E. and Ris, H. 1951. The composition and structure of isolated chromosomes. J. Gen. Physiol. 34: 475-92. 102) - and Osawa, S. 1961. The interphase nucleus. In Brachet, J. and Mirsky, A. E. The Cell, Academic Press, Inc., New York: 677-770. 103) Montgomery, P. O'B. 1963. Experimental approaches to nucleolar function. Exptl. Cell Res. Suppl. 9: 170-75. 150 E. H. J. O'Donnell Cytologia 36

104) Monty, K. J., Litt, M., Kay, E. R. M. and Dounce, A. L. 1956. Isolation and properties of liver cell nucleoli. J. Biophy. and Bioch. Cyt. 2: 127-45. 105) Mulnard, J. 1956. Presence d'inclusions Feulgen-positive dans les nucleoles larvaires de quelques Dipteres. Arch. Biol. 67: 485-98. 106) Muramatsu, M., Smetana, K. and Busch, H. 1963. Quantitative aspects of isolation of nucleoli of the Walker carcinosarcoma and liver of the rat. Cancer Res. 23: 510-18. 107) Northrop, J. H. 1926. The resistance of living organisms to digestion by pepsin or trypsin. J. Gen. Physiol. 9: 497-502. 108) O'Donnell, E. H. J. 1961. Deoxyribonucleic acid structures in the nucleolus. Nature 191: 1325-6. 109) - 1961. Feulgen-positive structures in the nucleolus. 1st Ann. Meet. Am. Soc. Cell Biol., Chicago: 156. 110) - 1962. Nucleolar Ultrastructure. I. Euglena gracilis. 2nd Ann. Meet. Am Soc. Cell Biol., San Francisco: 137. 111) Ohno, S., Trujillo, J. M., Kaplan, W. D. anp Kinosita, R. 1961. Nucleolus organisers in the causation of chromosomal anomalies in man. Lancet: 123-6. 112) -, Weiler, C. and Stenius, C. 1961. A dormant nucleolus organizer in the guinea pig, Cavia cobaya. Exptl. Cell Res. 25: 498-503. 113) Pelling, G. 1959. Chromosomal synthesis of RNA as shown by incorporation of uridine labelled with Tritium. Nature 184: 655-6. 114) Perry, R. P. 1960. On the nucleolar and nuclear dependence of cytoplasmic RNA synthesis in HeLa cells. Exptl. Cell Res. 20: 216-20. 115) -, Hell, A. and Errera, M. 1961. The role of the nucleolus in ribonucleic acid and protein synthesis. I. Incorporation of cytidine into normal and nucleolar inactivated HeLa cells. Biochem. Biophys. Acta 49: 47-57. 116) - 1962. The cellular sites of synthesis of ribosomal and 4s RNA. Proc. Nat. Acad. Sci. (U. S.) 48: 2179-86. 117) Porter, K. R. 1958. Problems in the study of nuclear fine structure. 4th Int. Cong. Elect. Microsc., Springer 2: 186-99. 118) Prescott, D. M. and Bender, M. A. 1963. Autoradiographic study of chromatid dis tribution of labelled DNA in two types of mammalian cells in vitro. Exptl. Cell Res. 29: 430-42. 119) Rattenbury, J. A. and Serra, J. A. 1952. Types of nucleolus reconstitution in telo phase and the question of the "nucleolar organizer". Port. Acta Biol. 3: 239-60. 120) Reitalu, R. 1957. The appearance of nucleoli and heterochromatin in mesothelial cells and cancer cells of ascites tumours of the mouse. Acta. Path. Microbiol. Scandinav. 41: 257-66. 121) Revel, J. P. and Hay, E. D. 1961. Autoradiographic localization of DNA synthesis in a specific ultrastructural component of the interphase nucleus. Exptl. Cell Res. Res. 25: 474-80. 122) Reynols, R. C., Montgomery, P. O. B. and Karney, D. H. 1963. Nucleolar "caps" -a morphologic entity produced by the carcinogen 4-nitroquinoline N-oxide. Cancer Res. 23: 535-8. 123) Rho, J. H. and Bonner, J. 1961. The site of ribonucleic acid synthesis in the isolated nucleus. Proc. Natl. Acad. Sci. (U. S.) 47: 1611-19. 124) Ris, H. 1956. A study of chromosomes with the electron microscope. J. Bioph. and Bioch. Cyt. 2 (Suppl.): 385-92. 125) - 1957. Chromosome structure. In McElroy, W. D. and Glass, B. A Symposium on the Chemical Basis of Heredity. Johns Hopkins Press: 23-62. 126) - 1961. Ultrastructure and molecular organization of genetic systems. Canad. J. Genet. and Cyt. 312: 95-120. 127) Schultz, J., Caspersson, T. and Aquilonius, L. 1940. The genetic control of nucleolar composition. Proc. Nat. Ac. Sci. (U. S.) 26: 515-23. 1965 Nucleolus and Chromosomes in Euglena gracilis 151

128) Sharma, A. K. and Sharma, A. 1958. Recent advances in the study of chromosome structure. But. Rev. 34: 511-49. 129) Sibatani, A., Kloet, S. R. de, Allfrey, V. G. Mirsky, A. E. 1962. Isolation of a nuclear RNA fraction resembling DNA in its base composition. Proc. Natl. Acad. Sci. (U. S.) 48: 471-7. 130) - 1963. Ribonucleic acids of cancer cells. Exptl. Cell Res. Suppl. 9: 289-329. 131) - Svilin, J. L. 1962. The Nucleolus. Prog. in Biophysics and Biophysical Chemistry 12: 25-66, 319-26. 132) - and Jacob, J. 1962. Function, development and evolution of the nucleolus. Nature 195: 114-7. 133) Sotelo, J. B. 1959. El comportamiento del nucleolonema en la mitosis. Estudio con el microscopio electronico. An. Fac. Med. Montevideo 44: 447-51. 134) - 1959. An electron microscope study on the cytoplasmic and nuclear components of rat primary oocytes. Zeit. f. Zellfors. 50: 749-65. 135) Speigelman, S. 1961. The relation of informational RNA to DNA. Cold Spring Harb. Symp. Quant. Biol. 26: 75-90. 136) Stern, H., Johnston, F. B and Setterfield, G. 1959. Some chemical properties of isolated pea nucleoli. J. Biophy. and Bioch. Cyt. 6: 57-60. 137) Sun, C. N. 1961. The occurrence of dense granules of unknown function in the nu cleoli of certain plant cells. Exptl. Cell Res. 25: 213-15. 138) Swift, H. 1959. Studies on nucleolar function. In Zirkle, C., A Symposium on Molecu lar Biology, Univ. Chicago Press: 266-93. 139) - and Adams, B. J. 1962. Simple methods for nucleic acid and acid mucopolysaccharide localization in electron micrographs. Proc. 5th Int. Cong. Elect. Microsc. 2: 0-4, -5. 140) - 1962. Nucleic acids and cell morphology in Dipteran salivary glands. In Allen, J. M. The molecular control of cellular activity, McGraw-Hill, New York: 73-125. 141) - 1963. Cytochemical studies on nuclear fine structure. Exptl. Cell Res. Suppl. 9: 54-67. 142) Taylor, J. H., Woods, P. S. and Hughes, W. L. 957. The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium labelled thymidine. Proc. Nat. Acad. Sci. (U. S.) 43: 122-8. 143) - and Woods, P. S. 1959. In situ studies of polynucleotide synthesis in nucleolus chromosomes. In Hayashi, T., Subcellular Particules, The Ronald Press Co., New York: 172-185. 144) Taylor, J. H. 1963 Control mechanisms for chromosome reproduction in the cell cycle. In Harris, R. J. C., Cell Growth and Cell Division, Academic Press, Inc., New York: 161-177. 145) Thorell, B. 1955. Nucleic acids in chromosomes and mitotic division. In Chargaff, E. and Davidson, J. N., The Nucleic Acids, Academic Press, Inc., Inc., New York 2: 181-98. 146) Ueda, K. 1958. Structure of plant cells with special reference to lower plants. III. A cytological study of Euglena gracilis. Cytologia 23: 56-67. 147) Venkataraman, P. R. and Coe, E. L. 1961. Incorporation of AMP into embryonic chick liver nucleic acids. Federation Proc. 20: 359. 148) Vincent, W. S. 1952. The isolation and chemical properties of the nucleoli of starfish oocytes. Proc. Natl. Acad. Sci. (U. S.) 38: 139-45. 149) - 1955. Structure and chemistry of nucleoli. Int. Rev. of Cyt. 4: 269-98. 150) - 1960. A biochemical approach to cell morphology. In Walker, P. M. B., New Ap - proaches in Cell Biology, Academic Press, Inc., New York: 89-94. 151) Weinreb, S. 1955. Ultraviolet polymerization of monomeric methacrylates for electron microscopy. Science 121: 774-5. 152) Weiss, J. B. 1962. Biosynthesis of ribopolynucleotides. Federation Proc. 21: 120-126. 153) Woodard, J., Rasch, E. and Swift, H. 1961. Nucleic acid and protein metabolism

Cytologia 30, 1965 11 152 E. H. J. O'Donnell Cytologia 30

during the mitotic cycle in Vicia faba. J. Biophy. and Bioch. Cyt. 9: 445-62. 154) Woods, P. S. 1960. Autoradiographic studies of ribonucleic acid metabolism with tritium-labelled cytidine. In Butterworth & Co., Ltd., London, The Cell Nucleus. 127-37. 155) Yankofsky, S. A. and Spiegelman, S. 1962. The identification of ribosomal RNA cistron by sequence complementarity. II. Saturation of and competitive interaction at the RNA cistron. Proc. Natl. Acad. Sci. (U.S.) 48: 1466-72. 156) Yasuzumi, G., Sawada, T., Sugihara, R., Kiriyama, M. and Sugioka, M. 1958. Elec tron microscope researches on the ultrastructure of nucleoli in animal tissues. Zeit. f. Zellfors. 48: 10-23. 157) - 1962. The fine structure of chromosomes of ascites tumor cells as revealed by electron microscopy. 5th Int. Cong. Electr. Microsc. 2: xx-2, -3 158) Zenkteler, M. 1959. Spostrzezesenia nad budowa jaderek. Act. Soc. Bot. Pol. 28: 477-85. 159) Zirkle, C. 1928. Nucleolus in root tip mitosis in Zea mays. Bot. Gaz. 86: 402-18.

Explanation of Plates I-XIV

Plate I

1, untreated control cell of Spirogyra. Late interphase. Aceto-carmin. 2,400•~. 2-4.

In vivo DNAse-treated cells of Spirogyra (2hr.). The arrows point to depolymerized nucleolar material in the shape of a contorted "tubule": lighter depolymerized areas are

in upper focal planes and darker depolymerized areas in lower focal planes. RNAse

resistant ground nucleolar material is Feulgen-negative and pyroninophilic. All cells at late interphase. 2,180•~.

Plate II

5-8. In vivo RNAse-treated cells of Spirogyra (2hr). The arrows point to Feulgen

positive filamentous structures inside the nucleoli which became noticeable after RNAse degradation of the ground nucleolar material. Intranucleolar structures show the gross

morphology of chromonemata. All cells at late interphase. 2,800•~.

Plate III

9, untreated control cell of Euglena. Late interphase. Nucleous (N); nucleolar indenta

tions (I) crossed by 100th fibrils. Long, electron-dense filaments (DF) of the first nucleolar component continuous with condensing chromonemata (Co). Short nucleolar fibrils (SF) of

the second nucleolar component closely associate with DF. 48,500•~.

Plate IV

10, untreated, incubated control cell of Euglena. Prophase. Nuclear membrane (M).

Ring-shaped nucleolus with enlarged indentation (I). Long arm of the nucleolar chromo

some (NC) and satellite region (Sa) of the nucleolar chromosome. The arrows point to

two long, electron-dense filaments-first nucleolar component-coiling in the nucleolus. 71,000•~.

Plate V

11, untreated, incubated control cell of Euglena. Late prophase. Bizarre nucleolus

showing long, electron-dense filaments (DF) continuous with the chromonemata of the

nucleolar chromosome (NC). Inside the chromosome body, the chromonemata (Chr) coil

and fold in diagonal with matrical proteins laid down around them. 87,000•~.

Plate VI 12, RNAse-treated cell of Euglena (4hr). Midprophase. RNAse-depolymerized chioroplast

11* 1965 Nucleolus and Chromosomes in Euglena gracilis 153

(CL). Nuclear membrane (M). Nucleolar RNAse-resistant electron-dense filaments (DF), short fibrils around them depolymerized by the enzyme. Electron-dense (ED) region of

the nucleolus continuous with the long arm of the nucleolar chromosome (NC). (DF) continuous with chromonemata of the satellite region (Sa) of the nucleolar chromosome.

Chromosomal matrix (Mx) around chromonemata depolymerized by the enzyme. 49,600•~.

Plate VII

13, RNAse-treated cell of Euglena (4hr). Prophase. Chromonemata (Chr) and long nucleolar, electron-dense filaments (DF) are RNAse-resistant. Matrical fibrils around (Chr)

and nucleolar short fibrils (SF) around (DF) were degraded by the enzyme. Nucleolar

material continuous with both chromatids of the nucleolar chromosome (NC). 62,200•~.

Plate VIII

14, RNAse-treated cell of Euglena (2hr). Early prophase. The enzyme depolymerized

certain regions of the chromonemata (Chr) and the short nucleolar fibrils. Electron-dense

region of the nucleolar material (ED) continuous with the chromonemata of the nucleolar chromosome (NC). Structures crossing the nucleolar lumen (S) show depolymerization

pattern similar to the depolymerization pattern of chromonemata. Cross section of a turn in the chromonemata (Cs). 69,500•~. 15, RNAse-treated cell of Euglena (2hr.). Midpro

phase. Nucleolar material compactly aggregated and continuous with the nucleolar chro mosome (NC). Long, nucleolar, electron-dense filaments RNAse-resistant. Short, nucleolar fibrils depolymerized by the enzyme. Structures (S) crossing the enlarged nucleolar

indentation are RNAse-resistant. 59,000•~.

Plate IX

16, DNAse-treated cell of Euglena (2hr). Early prophase. Nuclear membrane (M).

Chromonemata, certain regions of the long nucleolar electron-dense filaments and the structures crossing the nucleolar indentation (I) were degraded by the enzyme. Condensing chromonemata (Chr) continuous with nucleolar material. Small electron-dense (ED) region

in the nucleolar material. 62,500•~.

Plate X

17, DNAse-treated cell of Euglena (3hr). Midprophase. Chloroplast (CL). Chro monemata in condensing regions of chromosomes (Co), certain nucleolar components and

the structures crossing the nucleolar indentation were degraded by the enzyme. Electron dense regions of the chromonemata (DC) were less affected by the enzyme. Nucleolar

electron-dense region (ED). Nucleolar material continuous with nucleolar chromosome

(NC). 39,700•~.

Plate XI

18, DNAse-treated cell of Euglena (2hr). Prophasic nucleolus. The enzyme hydrolyzed

a nucleolar component, the chromonemata (Chr) and the structures (S) crossing the nu cleolar indentation. 81,000•~. 19, interphase nucleolus of Euglena cell treated by RNAse

(2hr) followed by ƒ¿-chymotrypsin (2hr). Long, nucleolar electron-dense filaments (DF) resistant to both enzymes. Short, nucleolar fibrils (SF) highly degraded. 100,400•~.

Plate XII

20, interphase nucleolus of a trypsin-treated (2hr) cell of Euglena. Long, nucleolar elec

tron-dense filaments (DF) resistant to the enzyme. Short, nucleolar fibrils depolymerized by the proteinase. 38,100•~. 21, condensing, prophasic chromosomes of a trypsin-treated

cell of Euglena. Chromonemata in the condensed regions (Co) and electron-dense regions

(DC) of the chromonemata resisted the enzyme. Matrical fibrils were depolymerized by 154 F. H. J. O'Donnell Cytologia 30

trypsin. 38,100•~. 22, Midprophase chromonemata of a Euglena cell treated by RNAse

(2hr) followed by ƒ¿-chymotrypsin (2hr). Electron-dense chromonemata (Chr) surrounded by depolymerized matrical material (Mx). 41,300•~ 23, early prophase cell of Euglena treated by DNAse (2hr) followed by ƒ¿-chymotrypsin (2hr). Nucleolus (N). Chromo

nemata (Chr) and long, nucleolar electron-dense filaments degraded by DNAse. Short,

nucleolar fibrils and matrical fibrils depolymerized by the proteinase. Pair of chro

monemata relationally coiled (PC). 62,000•~.

Plate XIII

24, incubated control cell of Euglena. Late metaphase. Nuclear membrane (M). After

dispersion of the nucleolar material, the intranucleolar electron-dense filaments (DF) are clearly visible still attached to the separating nucleolar chromosomes (NC). 38,100•~. 25,

incubated, control cell of Euglena. Anaphase. Nucleolus (N) is promptly organized by nucleolar chromosome (NC). Long nucleolar electron-dense filaments (DF) are continuous

with the chromonemata of (NC). 47,400•~. 26, incubated, control cell of Euglena.

Midtelophase. Nucleolus (N) is organized by the nucleolar chromosome (NC). Long nu cleolar electron-dense filaments are continuous with chromonemata of (NC). 51,000•~

27, ƒ¿-chymotrypsin-treated cell of Euglena. Late telophase. The enzyme degraded the

short fibrils in both nucleolar bodies (N), the matrical fibrils in the long arm of the nu

cleolar chromosome (NC) and in the satellite region (Sa) of the nucleolar chromosome. Long nucleolar electron-dense filaments (DF) are continuous with an electron-dense region

of the nucleolar chromosome (ED). 48,000•~.

Plate XIV

28, chick embryo-9 somite stage. Ventral-lateral yart of neural tube. Nucleolar electron dense filaments (DF). Osmic fixation. Epon. 38,100•~ (Dr. K. M. Lyser's electronmicro

graph.) 29, chick embryo-8 days old. Mantle layer of basal plante, spinal cord. Nucleolar electron-dense filaments (DF). Osmic fixation. Epon. 38,100•~. (Dr. K. M. Lyser's elec tronmicrograph.)