J.Cell Sci. 24, 81-93 (1977) Printed in Great Britain
81
CHROMATIN BEHAVIOUR DURING THE MITOTIC CELL CYCLE OF SACCHAROMYCES CEREVISIAE C. N. GORDON Department of Molecular Biology and Biocliemistry, University of California, Irvine, California 92717, U.S.A.
SUMMARY Chromatin behaviour during the cell division cycle of the yeast Saccharomyces cerevisiae has been investigated in cells which have been depleted of 90 % of their RNA by digestion with ribonuclease. Removal of large amounts of RNA from the yeast nucleus before treatment of the cells with heavy metal fixatives and stains permits chromatin to be visualized with extreme clarity in thin sections of cells processed for electron microscopy by conventional procedures. Spindle pole bodies were also visualized by this treatment, although the associated microtubules were not. Chromatin is dispersed during interphase and occupies the non-nucleolar region of the nucleus which is known to be Feulgen-positive from light microscopy. Because spindle microtubules are not visualized, direct attachment of microtubules to chromatin fibrils could not be verified. However, chromatin was not attached directly to the spindle pole bodies and kinetochore differentiations were not observed in the nucleoplasm. During nuclear division chromatin remains dispersed and does not condense into discrete chromatids. As the nucleus expands into the bud, chromosomal distribution to the daughter cells is thought to result from the separation of the poles of the spindle apparatus with attached chromatin fibrils. However, that such distribution is occurring as the nucleus elongates is not obvious until an advanced stage of nuclear division is reached and partition of the nucleus is nearly complete. Thus, no aggregation of chromatin into metaphase or anaphase plates occurs and the appearance of chromatin during mitosis is essentially the same as in interphase. These observations indicate that the marked changes in the topological structure of chromatin which characterize mitosis in the higher eukaryotes do not occur in S. cerevisiae. INTRODUCTION Cell division in the yeast Saccharomyces cerevisiae has received considerable attention from a number of workers in recent years. Robinow & Marak (1966) discovered a spindle apparatus in the yeast nucleus and succeeding investigations have revealed broad areas of agreement on the structure of the spindle, its mode of formation and its behaviour during the mitotic cell cycle (Moens & Rapport, 1971 ; Byers & Goetsch, 1974, 1975 ; Peterson & Ris, 1976). On the other hand, the structure and behaviour of yeast chromatin during the cell cycle is still controversial. Wintersberger, Binder & Fischer (1975) recently described discrete, condensed bodies seen in smears of yeast sphaeroplasts as 'chromosomes'. The number of ' chromosomes' varied from cell to cell in smears of exponential cultures and it was assumed that this variation represented the topology of chromatin in different stages of the cell division cycle. Since most of the sphaeroplasts seen in their smears contain 6
CEL 24
82
C. N. Gordon
condensed bodies (e.g. fig. i{a) of Wintersberger et al. 1975) their results suggest that yeast chromatin is condensed during a substantial part of the cell cycle. By contrast, Peterson & Ris (1976), from studies using high voltage electron microscopy of thick (0*25-1 fim) sections and a surface spreading technique, concluded that yeast chromosomes 'do not condense and are not individually visible' during the mitotic cell cycle. On the basis of the number of microtubules counted in cross-sections of diploid and haploid cells they concluded that there was probably one non-continuous microtubule per genetic linkage group and that mitosis in yeast is essentially orthodox, except for the lack of chromosome condensation. Condensed chromosomes can be stained for light microscopy in yeasts which have been fixed at meiosis I by any one of a range of fixatives; however, none of these procedures shows condensed chromosomes in budding yeasts (C. F. Robinow, private communication). In the electron microscope, condensed chromosomes are not observed in thin sections of budding yeasts fixed with glutaraldehyde-osmium tetroxide (Robinow & Marak, 1966 ; Moens & Rapport, 1971 ; Byers & Goetsch, 1974, 1975); regions of electron-lucidity can be seen in the nuclei of permanganate-fixed cells and Yotsuyanagi (i960) and Williamson (1966) have argued that the lucid regions are 'chromosomes' or 'aggregated chromatin'. Lucid regions in permanganate-fixed cells are caused by the preferential leaching out of cellular components (Hayat, 1970). While it is possible that the lucid areas described by Yotsuyanagi (i960) and Williamson (1966) may have contained chromatin in the living cell the reaction with permanganate would preclude a clear judgement as to the state of chromatin aggregation before exposure of the cells to the fixative. The ratio of RNA to DNA in yeast nuclei is about 3, which is nearly 15 times higher than the values reported for animal cells (Molenaar, Sillevis-Smith, Rozijn & Tonino, 1970). The inability to see chromatin in thin sections of yeast fixed for electron microscopy by glutaraldehyde-osmium tetroxide might be due to this unusually high RNA to DNA ratio. RNA and DNA are similar in their uptake of electron-dense fixatives and stains and the large amount of nuclear RNA could compete with DNA for the binding of heavy metal compounds. This paper describes the results of a study of the yeast nucleus using a procedure in which about 90 % of the cellular RNA of yeast was removed by ribonuclease before exposure of the cells to heavy metal fixatives and stains. When such an RNA-depleted cell was processed for electron microscopy, electron-dense material could be seen with extreme clarity in the region of the nucleus known to be Feulgen-positive from light microscopy.
MATERIALS AND METHODS
Materials Saccharomyces cerevisiae strain SKQ zn was obtained from. Dr Brian Cox, Botany School, Oxford. This strain is a prototrophic diploid with the genotype a/a, ade 1/ +, + /ade 2, + /his 1. Bovine pancreas ribonuclease (RNase) was obtained from Worthington Biochemical Corp., Freehold, N. J. A stock solution of 10 mg/ml was heated at 80°C for 10 min to destroy deoxyribonuclease activity. Glutaraldehyde was obtained from Polysciences, Inc., Warrington, Pa., or Tousmis Research Corporation, Rockville, Md.
Chromatin behaviour in S. cerevisiae
83
Growth and harvesting of cells; fixation Cells were grown on YM-i medium (Hartwell, 1967) at 23 CC with shaking to a cell density of s x io 6 cells/ml. The cells were then harvested by low-speed centrifugation, washed once with water at room temperature and suspended for 3 h at room temperature in one of the following fixatives: Fixative A, consisting of 4 % glutaraldehyde, o-i M sodium cacodylate, pH 7, 1 mM CaCli; Fixative B, consisting of 4 % glutaraldehyde, 0-5 M sodium acetate, 1 mM CaCl,. The fixed cells were then chilled and either worked up immediately or stored for 1 -2 weeks at 4°C. Aliquots containing 1-5-6 x io 8 cells were processed for electron microscopy by one of the procedures described below.
Processing for electron microscopy Unless otherwise indicated all operations were carried out in a cold room maintained at 4°C. Procedure A. Cells fixed in fixative A were washed 4 times by alternate suspension and centrifugation in 0 1 M sodium acetate containing 1 mM CaClj,. The cells were then suspended for 30 min in a solution consisting of C2 M Tris, 002 M EDTA, pH 9 4 . After centrifugation, they were washed twice with o-i M sodium acetate, suspended in 4 % uranyl sulphate for 2 h, washed once in 0 1 M sodium acetate and dehydrated and embedded as described below. Procedure B. Cells fixed in fixative B were washed 5 times with 0 1 M sodium acetate and suspended in 5 ml 005 M Tris, pH 7 2 . RNase was added to a concentration of 100 /'g/ml and the cell suspension incubated at room temperature with shaking for 2 h. (During this period, the absorbance at 260 nm released in the supernatant was monitored; a maximum absorbance was reached in about 90 min). The cells were centrifuged and washed 4 times with OT M sodium, acetate, suspended in 4 % uranyl sulphate for 2 h, washed twice with 0 1 M sodium acetate and kept overnight at 4°C. The cells were then suspended in 1 % osmium tetroxide, incubated at room temperature for 15 man and centrifuged. After the cells were washed 4 times with water, they were suspended in 20 % ethanol and kept at room temperature for 30 min. The cells were then centrifuged and suspended for 2 5 h in a solution consisting of 4 parts ethanol plus 15 parts ethylene glycol. After centrifugation the pellets were processed as described below under Dehydration and embedding.
Dehydration and embedding Cells processed by either procedure were washed 3 times with ethylene glycol and once with propylene glycol by suspension of the cells with a glass rod, followed by centrifugation at 4500 rev/min in an angle-head centrifuge. The pellets were suspended in propylene glycol, stored overnight at room temperature, centrifuged, and suspended in 1 ml propylene glycol. To the suspension in propylene glycol was added 1 ml Spurr's epoxy resin (Spurr, 1969) of the following composition : ERL-4206, 10 g ; DER 736, 8 g ; nonenylsuccinic anhydride, 26 g ; dimethylaminoethanol, 0-2 g. The resulting 2-phase system was warmed briefly to 40-50 °C, stirred with a glass rod until it formed a single homogeneous phase, and then kept at 40°C for 1 h. Then 2 ml resin were added and after mixing with a glass rod the cell suspension was kept at 40 °C for an additional hour. The cells were then centrifuged, washed once with resin by suspension and centrifugation, suspended in fresh resin and kept at 40 CC for 1 h. After centrifugation, infiltration was completed by suspending the cells in fresh resin and incubating at 40 °C for 2 h. The cells were centrifuged and the pellets were scooped up on a spatula and placed in plastic embedding capsules filled with resin. The resin was polymerized at 70 °C for 16-24 h-
Electron microscopy Sections were cut with a diamond knife on a Sorval MT2-B microtome with the advance mechanism set at 8 c o nm (silver to silver-gold). The sections were mounted on mesh grids or on Formvar-covered 2 x 1 mm slots and allowed to dry. Staining with lead citrate was done in the following way. Lead citrate was prepared as described by Reynolds (1963) and diluted 100-fold with o-oi N NaOH. The grids bearing the sections were submerged in the diluted 6-2
84
C. N. Gordon
stain in a teflon container with a screw-cap top. The container top was screwed on tightly, excluding air, and the grids left in the stain solution for 2-3 h. Following this, the grids were removed from the stain, rinsed thoroughly with water and allowed to dry. Specimens were observed in a Philips EM300 electron microscope at 60 kV and with a 50fim objective aperture. Cells selected for observation and photography were sectioned along the long axis, thereby revealing the bud length and age in the cell cycle. Digestion of fixed cells with RNase; analysis for RNA and DNA Cells fixed with fixative B for 6 min were washed 5 times with o-i M sodium acetate and suspended in 005 M Tris, pH 7-2. RNase was added and the cell suspensions incubated with shaking at room temperature. The reaction was stopped by the addition of diethyl pyrocarbonate to a concentration of o-i % (Fedorcsak & Ehrenberg, 1966) and the reaction mixture centrifuged. The absorbance of the supernatant at 260 run provided an index of the extent of RNase digestion. The pellets were washed 3 times with 01 M sodium acetate and twice with C25 N perchloric acid at 4°C. The nucleic acids were extracted with 0-5 N perchloric acid at 70 °C. After clarification of the extracts by centrifugation, total nucleic acids were determined spectrophotometrically (Spirin, 1958) and DNA was determined by the diphenylamine reaction (Burton, 1956). RNA was then calculated by difference.
RESULTS
Effect of RNase on glutaraldehyde-fixed cells When yeast cells previously fixed with fixative B were washed free of excess glutaraldehyde and incubated with RNase, ultraviolet-absorbing material was released from the cells into the supernatant, reaching a maximum absorbance at 260 nm of Table 1. Release of ultraviolet-absorbing material by RNase from yeast cells fixed with glutar aldehyde Absorbance per cell (x io8) at 260 nm released into the supernatant after incubation with RNase for concentration, fig/ml
5 min
40 min
OI
o •21
i-o 2O-O
o '35 8• i c
o-55 4'49
ioo-o
8•37
9-10
— —
— —
o-o I-C + O - I % D P *
925
150 min
1050
min
243
955
8-89 — — 0-30 0-30
9-07 — — — —
• RNase was added to the cells containing o-i % diethyl pyrocarbonate (DP). about 9 x i o " 8 per diploid cell. Typical results are shown in Table 1. The release of absorbance depends both on enzyme concentration (for a given incubation time) and on time of incubation (for a given enzyme concentration). Relatively little release occurs in the absence of enzyme (row 5). Furthermore, no absorbance is released in the presence of diethyl pyrocarbonate (row 6). The latter is a known inhibitor of RNase (Fedorcsak & Ehrenberg, 1966).
Chromatin behaviour in S. cerevisiae
85
Table 2 shows the nucleic acid content of unfixed cells, of fixed cells not treated with RNase, and of fixed cells treated exhaustively with RNase (ioo/tg/ml, 2 h). (In the latter case, the release of absorbance at 260 nm was monitored to verify that the RNase limit digest had been reached.) Table 2 shows that RNase removes about 90% of the cellular RNA from glutaraldehyde-fixed cells but does not affect the DNA content of these cells. In addition, glutaraldehyde does not in itself affect the DNA or RNA content of cells. Table 2. Nucleic acid content of yeast cells Nucleic acid content (pg/cell) of Glutaraldehyde-fixed cells Nucleic acid
Unfixed cells
( - ) RNase
( + ) RNase
RNA 258, 261 253, 256 0-23, 023 DNA 0-035, 0036 0-037, 0038 0-037, 0-037 The results of duplicate determinations are shown.
Aldehyde fixation is known to affect the permeability properties of membranes, permitting access to the cell by molecules whose size or other properties would normally result in their exclusion (Hayat, 1973). The results of Tables 1 and 2 show that the RNase molecule (molecular weight, 13 800) can pass the cell wall and membrane, gaining entrance to the cytoplasm. Because most of the cellular RNA of yeast is cytoplasmic, these data do not prove that RNase entered the nucleus and digested nuclear RNA. However, evidence that nuclear RNA has been affected derives from prior studies by Molenaar et al. (1970). These workers purified yeast nuclei, fixed them with glutaraldehyde and digested the fixed nuclei with 30 /tg/ml RNase for 30 min; 84% of the nuclear RNA was released by this treatment. This value is similar to that obtained in this work (Table 2) and indicates that the nuclear envelope of glutaraldehyde-fixed cells does not act as a barrier to RNase. Ultrastructure of the untreated nucleus
Cells prepared for electron microscopy by procedure A have not been treated with RNase. Fig. 1 shows the nucleus of such a cell with a small bud in early interphase. Prominent ultrastructural features are the nucleolus, which occupies much of the nuclear volume and straddles one side of the nuclear envelope (Robinow & Marak, 1966; Sillevis Smitt, Vlak, Molenaar & Rozijn, 1973), and ribonucleoprotein particles, which are slightly but significantly smaller than the ribosomes in the surrounding cytoplasm (Mundkur, 1961 ; Gordon, 1977). Previous light-microscopic studies have indicated that the non-nucleolar region is Feulgen positive and hence is presumably the locus of most of the chromatin (Robinow & Marak, 1966). Later in the cell cycle the nucleus migrates into the bud and is partitioned between parent and progeny cells. Untreated cells at successive stages of the cell cycle were carefully observed. No internal changes in the chromatin-containing (non-nucleolar)
C. N. Gordon
Fig. i. Control cell (not digested with ribonuclease) in early interphase. The dashed line demarcates the nucleolus (no) from the chromatin-containing Feulgen-positive region of the nucleus, x 40000. Fig. 2. Control cell in the process of nuclear division. The nucleolus (no) remains intact and is partitioned between parent and daughter cells, x 40000.
Chromatin behaviour in S. cerevisiae
spb
Fig. 3. Ribonuclease-digested cell in interphase. Compare to Fig. 1. The dashed line demarcates the chromatin-containing region (ch) from a region which is largely devoid of electron density and which was the site of the nucleolus before ribonuclease digestion. X 40000. Fig. 4. Ribonuclease-digested cell with the nucleus just prior to its entrance into the bud. The section cuts through most of the spindle and shows one spindle pole body (spb). (The SPB on the opposite side of the nucleus is not contained in this section.) x 40000.
88
C. N. Gordon
region could be observed before, during or after nuclear migration. Fig. 2 is an example of a migrating nucleus with the persistent nucleolus partitioning itself between parent and daughter cells (Robinow & Marak, 1966; McCully & Robinow, 1973) and the ribonucleoprotein particles dispersed throughout the nucleus. The non-nucleolar region is diffuse, with no indication of chromatin condensation. Effect of RNase on nuclear ultrastructure Interphase. Robinow & Marak (1966) have shown by light microscopy that the Feulgen-positive region in the nucleus of interphase cells is closest to the bud, whereas the Feulgen-negative nucleolus is usually in an opposite or lateral position. Fig. 3 shows the nucleus of an interphase cell prepared by procedure B. The dashed line delineates electron-dense material (ch) in a region adjacent to the bud from a region of lower electron density. Based on a comparison of randomly selected sections of RNase-digested and of undigested interphase cells, it was concluded that the position of enhanced contrast in the nuclei of RNase-digested cells could be correlated with that of the Feulgen-positive region and that the electron-dense material in this region was chromatin (Gordon, 1977). Comparing Figs. 1 and 3 it is evident that the ribosomes, nuclear particles and nucleolus are lost as cytological entities. Chromatin acquires distinct stainability and has a diffuse character, showing neither condensation nor preferential accumulation in a particular region of the nucleus. (The latter was verified by observing a series of consecutive sections through most of the nucleus.) Nuclear division. Wintersberger et al. (1975) have reported that yeast chromatin is condensed into discrete chromosome-like structures. Peterson & Ris (1976), while maintaining that such condensation does not occur, describe the aggregation of chromatin during nuclear division into narrow regions between the spindle poles termed 'metaphase' and 'anaphase plates'. Cells processed for electron microscopy by procedure B were examined for these or other possible changes in chromatin morphology during nuclear division. Fig. 4 shows the nucleus of a cell which has formed a complete spindle (Moens & Rapport, 1971). The section cuts through one spindle pole body or SPB (term proposed at the First International Mycological Congress [Aist & Williams, 1972 ; Kubai, 1975]), whereas the SPB on the opposite side of the nucleus is not contained in this section. (The microtubules which emanate from the SPBs are not visualized clearly by this preparative procedure.) Note that the chromatin is uniformly dispersed throughout the nucleus, with much the same character as interphase chromatin (Fig. 3). Neither condensation into chromatids nor aggregation into a 'metaphase plate' midway between the spindle poles is evident. Note also the lack of direct attachment of chromatin to the SPB. Fig. 5 shows a stage later in nuclear division after the nucleus has entered the bud. A region of reduced electron density surrounds the SPB (dashed line), suggesting lack of direct attachment. The chromatin remains dispersed throughout the nucleus with no indication of aggregation into an 'anaphase plate'. Fig. 6 shows a cell in which partition of the nucleus is nearly complete. The
Chromatin behaviour in S. cerevisiae
1
Fig. 5. Ribonuclease-digested cell shortly after entrance of the nucleus into the bud. The dashed line indicates a chromatin-free region surrounding the SPB. x 40000. Fig. 6. Ribonuclease-digested cell at an advanced stage of nuclear division. The arrows indicate the neck of the dividing nucleus which is free of chromatin and has the same electron density as the cytoplasm, x 40000.
9°
C. N. Gordon
8 Fig. 7. Ribonuclease-digested cell in which partition of the nucleus is complete and cytokinesis has begun, x 30000. Fig. 8. Ribonuclease-digested cell in which cytokinesis is complete and cell wall separation has begun, x 30000.
Chromatin behaviour in S. cerevisiae
91
chromatin is still dispersed but is now localized at opposite poles of the dividing nucleus, while the narrow intervening neck is devoid of chromatin (arrows, Fig. 6). Figs. 7 and 8 show the terminal stages of cell division. The chromatin remains dispersed and has essentially the same cytological characteristics as in previous stages.
DISCUSSION
The formation and behaviour of the spindle apparatus in S. cerevisiae is well documented. The SPBs and associated microtubules are readily visualized in cells treated with glutaraldehyde-osmium and this has made possible a detailed analysis of spindle behaviour in serial thin sections (Moens & Rapport, 1971 ; Byers & Goetsch, 1975). Additional features of chromatin segregation in yeast have recently emerged from studies of Peterson & Ris (1976). Chromatin fibrils become attached directly to spindle microtubules without recognizable kinetochores. As nuclear division proceeds, uncondensed chromatin fibrils are drawn to opposite poles of the spindle apparatus. Peterson & Ris (1976) observed a region of enhanced electron density near the ends of the non-continuous microtubules and considered this to be an aggregation of chromatin into metaphase and anaphase plates. While the overall aspects of nuclear division which emerge from this work are compatible with their model, I fail to observe any preferential aggregation of chromatin into discrete regions. Their conclusion that such aggregation occurs is, in my opinion, not justified by the micrographs used to support this contention (figs. 12-14 °f Peterson & Ris, 1976). Only a small region of the nucleus between the spindle poles is shown and their identification of electron-dense material in this region as chromatin is unconvincing. Chromatin in yeast nuclei which have been lysed by osmotic shock has the appearance of knobby fibrils about 20 run in diameter (Peterson & Ris, 1976). Occasional dense granules of roughly this size could be seen in some of my micrographs of RNA-depleted cells and these may possibly represent individual chromatin fibrils cut in cross-section. In general, however, the cross-sections observed in RNAdepleted cells were considerably larger than 20 nm, suggesting that in situ, higher orders of folding occur. Two aspects of chromatin behaviour emerging from this work seem quite definite. First, condensation into the discrete chromatids with staining characteristics typical of higher eukaryotes does not occur in S. cerevisiae. Second, while some higher-order folding of the basic 20-nm fibril may occur in situ, the general topological structure of chromatin as visualized in the electron microscope, using these procedures, is cytologically indistinguishable throughout most of cell division. Only when cell division has reached a relatively advanced stage is a separation into 2 distinct groups evident. The results described in this work are in sharp conflict with the findings of Wintersberger et al. (1975) that condensed chromosomes are seen at some stages of the mitotic cycle of S. cerevisiae. The preparative procedure used by these authors was severe : cells were divested of their cell wall with snail-gut enzyme and placed on a glass slide. After heating with a bunsen flame, they were allowed to dry before fixation with ethanol-acetic acid. It is possible that artifactual aggregation into Giemsa-positive,
92
C. N. Gordon
electron-dense structures occurred on the slide as a consequence of heating and drying, since during this process the cells were unfixed and lacked the protection of a cell wall. Among lower eukaryotic organisms it is now clear that there are a number of deviations from the classically orthodox picture of mitosis in which chromosomes condense and align themselves between the poles of the spindle (reviewed by Kubai, 1975). Among the fungal species there is apparently a broad range of mitotic behaviour, including several species in which typically orthodox mitosis seems evident (Kubai, 1975). While a number of important details of nuclear division in S. cerevisiae remain to be worked out, at this point the available evidence favours the following scheme. At some stage in the cell cycle, chromatin fibrils become attached to non-continuous spindle microtubules (Peterson & Ris, 1976). The elongation of the spindle concomitant with movement of the nucleus into the bud provides the mechanism by which parental and progeny chromatin separate. Before and during the separation process the chromatin fibrils have the same topological structure as in interphase and do not undergo additional condensation or supercoiling. Nor does preferential aggregation into discrete regions of the nucleus occur. Whether mitotic behaviour of this type can be aptly characterized as 'orthodox' (Peterson & Ris, 1976) is semantical. I thank Dr Hans Ris for supplying me with a copy of the manuscript by himself and Dr Peterson prior to publication. This study was supported by grant BMS 73-06847Aoi from the National Science Foundation.
REFERENCES AIST, J. R. & WILLIAMS, P. H. (1972). Ultrastructure and time course of mitosis in the fungus Fusarium oxysporum. J. Cell Biol. 55, 368-389. BURTON, R. K. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the estimation of deoxyribonucleic acid. Biochem.J. 62, 315-323. BYERS, B. & GOETSCH, L. (1974). Duplication of spindle plaques and integration of the yeast cell cycle. Cold Spring Harb. Symp. quant. Biol. 38, 123-131. BYERS, B. & GOETSCH, L. (1975). Behaviour of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. J. Bact. 124, 511-523. FEDOHCSAK, 1. & EHRENBERG, L. (1966). Effects of diethyl pyrocarbonate and methyl methansulfonate on nucleic acids and nucleases. Acta chem. scand. 20, 107-112. GORDON, C. N. (1977). Ribonucleoprotein particles in the yeast nucleus. (Submitted,?. Cell Biol.) HARTWELL, L. H. (1967). Macromolecule synthesis in temperature-sensitive mutants of yeast. J. Bact. 93, 1662-1670. HAYAT, M. A. (1970). Fixation. In Principles and Techniques of Electron Microscopy, vol. 1,
pp. 61-62. New York : Van Nostrand Reinhold. M. A. (1973). Specimen preparation. In Electron Microscopy of Enzymes, vol. 1 (ed. M. A. Hayat), pp. 1-43. New York : Van Nostrand Reinhold. KUBAI, D. F. (1975). The evolution of the mitotic spindle. Int. Rev. Cytol. 43, 167-227. MCCULLY, E. K. & ROBLNOW, C. F. (1973). Mitosis in Mucor hiemalis. A comparative light and electron microscopical study. Arch. Mikrobiol. 94, 133-148. MOENS, P. B. & RAPPORT, E. (1971). Spindles, spindle plaques, and meiosis in the yeast
HAYAT,
Saccharomyces cerevisiae (Hansen). jf. Cell Biol. 50, 344-361.
I., SILLEVIS-SMITH, W. W., ROZIJN, TH., H. & TONINO, G. J. M. (1970). Biochemical and electron microscopic study of isolated yeast nuclei. Expl Cell Res. 60, 148-156.
MOLENAAR,
Chromatin behaviour in S. cerevisiae
93
B. (1961). Electron microscopical studies of frozen-dried yeast cells. II. The nature of the basophile particles and vesicular nuclei in Saccharomyces. Expl Cell Res. 25, 1-23. PETERSON, J. B. & Ris, H. (1976). Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae. J. Cell Sci. 23, 219-242. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. ROBINOW, C. F. & MARAK, J. (1966). A fiber apparatus in the nucleus of the yeast cell. J. Cell Biol. 29, 129-151. SILLEVIS-SMITT, W. W., VLAK, J. M., MOLENAAR, I. & ROZIJN, T H . H. (1973). Nucleolar function of the dense crescent in the yeast nucleus. Expl Cell Res. 80, 313-321. SPIRIN, A. S. (1958). Spectrophotometric determination of total nucleic acid content. Biokhimiya 23, 617-622. SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43. WILLIAMSON, D. H. (1966). Nuclear events in synchronously dividing yeast cultures. In Cell Synchrony (ed. I. L. Cameron & G. M. Padilla), pp. 81-101. New York : Academic Press. WINTERSBERGER, U., BINDER, M. & FISCHER, P. (1975). Cytogenic demonstration of mitotic chromosomes in the yeast Saccharomyces cerevisiae. Molec. gen. Genet. 142, 13-17. YOTSUYANAGI, Y. (i960). Mise en Evidence au microscope electronique les chromosomes de la levure par une coloration sp^cifique. C. r. Hebd. Se"anc. Acad. Sci., Paris 250, 1522-1524. MUNDKUR,
(Received 19 July 1976)
