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".