Ultrastructure of somatic and meiotic nucleoids

.Ekrron Muox. Prmted ,n Great

0892.0354,'89 $000+0.50 t 1989 Pergamon Press plc.

RPI... Vol. 2. pp. 191.-229, 1989 Bntam All nghts reserved.

ULTRASTRUCTURE

OF SOMATIC

AND

MEIOTIC

NUCLEOIDS

M. V. GLAZKOV N. 1. Vurilor Institute of General Genetics. Acudrmy of Sciences of rhr USSR, G&kin .str. 3. B-333 Moscow

117809, U.S.S.R.

Abstract-h this review emphasis is placed on the contribution of transmission electron microscopy to the analysis of spread chromosomes and nucleoids. Support is advanced for the DNA loop and rosette organization of meiotic and metaphase chromosomes and nucleoids. Extensive discussion is given to the biochemical treatments used for producing nucleoids and the effect of divalent cations and chelating agents on chromatin compactization (supercoiling). Detailed studies on nucleoids from hepatocytes are presented, with emphasis on the significance of DNA attachment to the internal nuclear matrix and to the nuclear lamina. It is firmly predicted that from the increasing knowledge of the structural organization of eukaryotic chromatin and the genome. a greater understanding of the functional roles of the various intranuclear structures will ultimately follow.

CONTENTS I.Introduction ............................................................................................. II. Interphase chromosomes .................................................................................. A. Ultrastructural analysis of interphase chromosome organization. ............................................ B. Polytene chromosomes of insects as a model of structure-functional organization of interphase chromosomes. C. The nuclease analysis of interphase chromosome organization .............................................. III. Loop organization of meiotic chromosomes ................................................................. IV. Loop organization of metaphase chromosomes. .............................................................. V. Nucleoids as an experimental model of DNA loop organization in interphase nuclei ............................. A. The biochemicalanalysis ofnucleoids....................................................................20 B. The electron microscopic analysis of nucleoids............................................................ VI. Nucleoids of rat liver cells ................................................................................. A. Sedimentation and fluorescent analyses of nucleoids ....................................................... B. Nucleoid morphology at different isolation steps .......................................................... C. Intercalating agents and nucleoid morphology ............................................................ D. CL?’ ions and nucleoid morphology.....................................................................2l E. Disulfide bonds and nucleoid morphology ................................................................ F. The hepatocyte nucleoids core .......................................................................... G. DNA association with residual nuclear structures ......................................................... VII. Nucleoids ofmeiotic cells .................................................................................. VIII.Conclusion...............................................................................................22 Acknowledgements........................................................................................22 References...............................................................................................22

I. INTRODUCTION The genetic material of eukaryotes is organized into multicomponent complexes, chromosomes. Various DNA-DNA, DNA-protein, and possibly protein-protein and DNA-RNA interactions are involved in eukaryotic chromosome organization. In this review particular attention has been focused on the loop organization pattern of genes, since structural and functional units of eukaryotic chromosomes are suggested to coincide at this level of organization.

...

197 I98 198 199 202 202 203 204 4 206 207 207 209 210 0 210 215 218 221 2 6 6

The discovery of the loop organization of nuclear DNA significantly contributes to the elucidation of mechanisms of differential gene activity in eukaryotic chromosomes as well as those governing the structural rearrangement of chromosomes themselves. Indeed, linear separation of chromosomes into individual structural units (domains) makes their differential compactization (supercoiling) or decompactization possible. and that leads respectively to repression or derepression of the relevant genes. Rearrangement of chromosome structure may be represented as different

combinational variants of their constitutional structural units. The advancement of molecular genetic methods has permitted us to suggest that differential gene activity is also regulated via some other mechanisms. apart from compactization-~decompactization of separate chromosome regions. Abundant data on complex eukaryotic genome organization. discovery of regulatory DNA sequences, elucidation of the role of supercoiling in DNA function suggest evidence for the existence of other. finer mechanisms of genetic activity regulation. Individual nuclear DNA sites have been suggested to be directly involved in the architecture of eukaryotic chromosomes. It has become clear that investigation of the structur+functional organization of cukaryotic chromosomes requires a new model. to be tested by further experiments. In the second half of the 1970s intensive studies were initiated using nuclear matrix and nucleoids as such models. Nucleoids are the nuclei devoid of histones and the major part of nonhistone proteins. but retaining total nuclear DNA in their composition. DNA of nucleoids is supercoiled and organized into loops (Cook and Brazell. 1975. 1976). Nuclear matrix is. in a simplified form, nucleoids which are almost completely devoid of DNA (for a more strict definition of nuclear matrix see Berezney. 1979). The simplified composition of nucleoids and nuclear matrix compared to native nuclei could substantially facilitate investigation of the loop organization of nuclear DNA. However, the conditions employed for isolation and analysis of these residual nuclear structures arouse certain doubts with respect to the entire preservation of some supernucleosomic organization levels of chromatin within the corresponding residual structures. The problem is connected to the lack of information about all the factors involved in the organization of nuclear DNA. This. in its turn, may call forth doubts as to whether the DNA loop structures revealed in nucleoids and those actually existing in native nuclei are structurally equivalent. The intent of this review is to analyze data available in the literature and our own experimental results obtained from eukaryotic chromosomes. using different methods and chromosome models.

What is the real contribution of nuclcoid and nuclear matrix studies into the sum of our knowledge about the structure functional organization of eukaryotic chromosomes’? Where is the source of the contradictory results obtained with ditYercnt models utilized for nuclear DNA organization investigations? These are two specific questions which will be discussed in depth in this relicu.

II. INTERPHASE

CHROMOSOMES

At the present, several levels of chromatin organization in interphase nuclei have been revealed by electron microscopy; IO nm. 75~ 30 nm. and 100 200 nm fibrils (Igo-Kemenes (‘1 (11.. 19X2: Cartwright cl ul., 1982; Hancock and Boulikas. 1982; Butler, 1983; Zatsepina LJI trl.. 1983a; Chentsov rt 01.. 1984; Nag]. 1985; Gornung ct r/l.. 1986a,b; Tsanev and Tsaneva. 1986). The electron microscopic preparations of ;I 10 nm chromatin fibril appear as “beads-on-nstring’.. The “beads” correspond to the nucleosome core particle, whereas the “string” is the internucleosomic DNA region. At the nucleosomal level. the DNA is regularly wound around the core particle containing two molecules of each of the four histones: H2A, H2B. H3 and H4 (Mirzabekov, 1980; Igo-Kemenes 1’1 (11.. 1982: Cartwright t’t ~1.. 1982; Butler. 1983; Thomas. 1984; Nag]. 1985). There exist two main models of organization for the 25-30 nm chromatin fibril: nucleomeric OI superbead (Kiryanov of ~1.. 1976; Holier t’/ trl.. 1977) and solenoid (Finch and Klug. 1976). According to the nucleomeric super-bead model, a 25530 nm fibril is formed by globules brought together into close proximity to each other (nucleomers, superbeads). Each of these particles consists of 6GlO nucleosomes. The solenoid model contends that a 25S30 nm fibril is formed due to coiling of the filament. Each turn of the solenoid is suggested to contain 6.~7 nucleosomes. It should be noted that the electron microscopic results indicated that the major part of interphase

Ultrastructure

of Somatic

chromatin in the nucleus is represented by 25-30 nm fibrils (Finch and Klug, 1976; Chentsov et al., 1984; Tsanev and Tsaneva, 1986). 10 nm fibrils have been detected when nuclei or isolated chromatin are incubated in the presence of chelating agents or in medium with a low content of divalent cations (Du Praw, 1965; Thomas, 1984; Chentsov ef al., 1984; Tsanev and Tsaneva, 1986). Some authors have recently described globules of 100-200 nm diameter, detected in interphase chromosomes of animals and plants (Zatsepina et al., 1983a; Gornung et al., 1986a,b). Zatsepina et al. (1983a) consider these structures, called chromomers (elementary chromomers), to be level of another universal supernucleosomic chromatin organization. During gradual decrease of divalent cations in solution the chromomers acquire a rosette-like structure. Similar rosette structures have been found in unicellates (Martinkina et al., 1983). The rosettes consist of the electron-dense core and radial loops of DNA fibrils 25-30 nm in diameter (Fig. 1). Once divalent cations have been entirely removed from the medium, the 100-200 nm globules unwind into nucleosomic chains. According to the estimate of Zatsepina et al. (1983a), one chromomer contains about 50 kb of DNA. In interphase nuclei, chromatin has numerous sites of attachment to nuclear membrane (Davies, 1968; Franke et al., 1973; Brasch and Setterfield, 1974; Onishchenko and Chentsov, 1974; Hancock and Boulikas, 1982; Gerace, 1986; Katsumata and Lo, 1988). Chromatin association with nuclear membrane is mediated through a thin protein layer, the nuclear lamina, in continuity with the nuclear pore complexes (Gerace, 1984; McKeon, 1987). Attachment of chromatin to nuclear lamina is suggested to provide an ordered spatial organization of chromosomes in the nucleus, which is necessary for correct realization of their genetic functions (Heslop-Harrison and Bennett, 1984; Hochstrasser and Sedat, 1987; Allen et al., 1988). Electron microscopic analysis of interphase nuclei by Miller’s method (Miller and Beaty, 1969) revealed 25-30 nm chromatin fibril loops attached to the nuclei periphery (Tsanev and Tsaneva, 1986). So, depending on the conditions of nuclear isolation and chromatin analysis, two types of loop

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Nucleoids

199

structures formed by the 25-30 nm chromatin fibrils can be revealed in interphase nuclei. Small chromatin loops of the rosette structures have been detected in solutions of medium ionic strength in the presence of divalent cations. In the nuclei isolated in buffer with low ionic strength, in the absence of or at very low concentrations of divalent cations, large chromatin loops attached to the nuclei periphery have been found. Mention should be made about the apparent absence of DNA loops from nuclei in situ; loops are observed only after chromosome decondensation with various agents. B. Polytene Chromosomes of Insects as a Model of Structure-Functional Organization of Interphase Chromosomes Microscopic analysis of the structure-functional organization of interphase chromosomes has encountered severe obstacles in many cell types. Nevertheless, a unique model for investigating the functional organization of interphase chromosomes is now available, derived from polytene chromosomes in salivary gland cells of insects. Polytene chromosomes are the product of endoreplication at early larval embryogenesis (Fig. 2a). The polytene chromosomes have a specific band pattern of condensed parts (discs or bands) and decondensed ones (interbands). The discs are a cluster of individual chromomers (Beerman, 1972; Lefevre, 1974; Korge, 1987). Sorsa (1972, 1973) demonstrated that chromomers are formed by chromatin looping. It is noteworthy that in his experiments Sorsa used a strong deproteinizing agent, urea. Looping of chromatin fibrils at the cytological level corresponds to puffing and appears to be a structural manifestation of transcription and replication processes (Beerman, 1972; Lefevre, 1974; Lima-de-Faria, 1975; Korge, 1987). Using the method of differential chromatin decondensation (see Section II.A), Zatsepina et al. (1983b) showed that chromomers of Drosophila zGrilis polytene chromosomes have a rosette-like organization (Fig. 2B), the same as chromomers of interphase and metaphase chromosomes in Chinese hamster cells. The long DNA loops are

M. V. Glazko\

Fig. I. Rosette structures in animals and plants. Decondensing chromomera of Chinese hamster (from Zatsepma (‘I ui. 19X%1) (a) and Allium W/XI (from Gornung cl ul.. 1986a) (h). Decondensing chromomers consist of an electron-dense core and radial loopa of DNP fib&. Rosette structures of histone-dcplctcd chromosomes of rat (from Prusov <‘( trl.. 19x3) (c) and .4/lrw~ WI)U (d). Rosette loops of histone-depleted chromosomes arc represented by DNA threads. Scale mal-ku\ induxtc 0.2 burn

Ultrastructure

of Somatic

and Meiotic

Nucleoids

Fig. 2. Polytene chromosomes of Drosophila. A fragment of a polytene chromosome of Drosophila melanogusrer following dispersion and additional extension (from Ananiev and Barsky, 1985) (a). Scale marker indicates 2 pm. A decondensing chromomer of Drosophila Crilis (from Zatsepina et ul., 1983b) (b). Scale marker indicates 0.2 pm. Polytene chromosomes consist of alternating chromomers and interchromomeric regions. A partial chromomer decondensation reveals the rosette-like organization.

201

thought to form in chromosomes exposed to greater chromomer deproteinization, after removal of protein fasteners that maintain the rosette structures intact (Prusov pr ul.. 1983). Cytogenetic analysis of polytene chromosomes suggests that chromomers or a chromomer plus an interchromomeric region are not only structural units, but possibly correspond to functional chromosome units such as the replicon. transcription. recombination unit (Beerman, 1972; Lefevre. 1974: Zhimulev and Belyaeva. 1975; Korge, 1987). On average, a Drosophil~~ chromomer consists ol 30 kb, and an interchromomeric region contains about 2 kb of DNA (Beerman, 1972; Lima-deFaria. 1975; Kalisch fr rrl., 1986). Since the formation of the polytene structure of salivary gland cell chromosomes is related to embryogenesis in insects (Beerman, 1972). one can suppose that chromosomes in other types of interphase nuclei have a structure which can be described schematically as the alternation of chromomers and interchromomers.

(‘I t/l.. 1982; Cartwright ct ~1.. 1982; Hancock. 19X7: Butler. 1983; Reeves, 1984). Weintraub ( 1984) isolated nuclease-rc~istant chromatin particles from the cell nuclei of chicken. The DNA size in these particles was 20 40 kb. The particles were also shown to contain transcriptionally inactive genes (Weintraub, 1984). However. the nuclease method alone does not allow us to analyze organization of supernucIcOsomic structures where transcriptionally inacti\c chromosome domains are compactized. This rcquires the electron microscopic analysis ofchromatin fractions which are resistant to nuclca\c digestion and contain transcriptionally inactive genes. It is considered that a 100 nm chromomct of interphase chromosomes, having a roscttc-like organization pattern, will be a possible candidate for such investigations. This supposition IS not contradictory with the main principle of structure functional organization of polytene chromosomes in insects. although. according to Zatsepina 1’1(I/. (1983a). the rosette-like structures of interphahc chromosomes form heterochromatic region\.

III. LOOP Nuclease digestion of chromatin in nuclei has greatly contributed to the elucidation of genetic organization (Weintraub and Groudine, 1976). This method is based on the lower accessibility of DNA within the supernucleosomic level of chromatin organization to nuclease digestion, compared to that at the nucleosomic level. The method convincingly demonstrates that transcriptionally active (or potentially active) and nonactive sequences of interphase chromosomes differ in the degree of nuclear DNA compactization. Both the gene-containing chromosome sequences and flanking regions undergo structural changes. The size of the nuclease-sensitive domains of interphase chromosomes that contain transciptionally active genes averages JO-50 kb (Igo-Kemenes ct d.. 1982: Reeves. 1984). Transition into the active state is suggested to be accomplished through looping of an interphase chromosome domain, while inactivation is presumably achieved by means of loop compactiration into a supersolenoid (Igo-Kemenes

ORGANIZATION OF MEIOTIC CHROMOSOMES

In meiosis the principle of loop organiratlon of nuclear DNA persists (Risley c’t trl.. 19X6). At early meiotic stages. as in somatic interphase cells. nuclear lamina is the main element of the nucleus involved in the spatial organization of chromosomes (Gambino c’t trl.. 1981). In electron microscopic specimens of spermatocyte nuclei. prepared by the method of Miller. 25 ~30 nm chromatin fibrils attached to the nuclei periphery are visible. The size of these loops is close to that ofchromatin loops in interphase nuclei of somatic cells (Rattner c’f (11.. 1980). Light microscopy of meiotic prophase I demonstrates the chromomeric organization of leptotenc chromosomes. Similarity in sizes of these chromomers and those of polytene chromosomes in insect5 has been shown (Lima-de-Faria. 1975). During this stage. a synaptonemal complex (SC) begins to form. Along each homologue an axial strand i\ formed. to be later transformed into ;I lateral

Ultrastructure

of Somatic

element of SC. At pachytene, the SC is completely formed and chromatin loops appear to be associated with SC lateral elements (Moses, 1968; Comings and Okada, 1970, 1978; Westergaard and von Wettstein, 1972; Gillies, 1975; Keyl, 1975; Rattner et al., 1980, 1981; Moens and Pearlman, 1988). Chromatin loops that are anchored to SC contain in yeast about 20 kb, in mouse and rat about 120 kb, and in grasshopper up to 600 kb of DNA (Moens and Pearlman, 1988). Simultaneously in prophase I, dissociation of nuclear lamina occurs (Stick and Schwarz, 1982, 1983; Jerardi et al., 1983). So, at pachytene I, SC appears the main intranuclear structure which provides spatial organization of the chromosomes. Light microscopic analysis has also revealed globular structures within pachytene chromosomes. These globules have been termed pachytene chromomers. The number of pachytene chromomers per chromosome is not high; by size and DNA content they substantially exceed chromomers at the leptotene stage (Lima-de-Faria, 1975). At the end of prophase I (late pachytene-early diplotene) chromosomes of many organisms acquire a specific morphology owing to which they have been given the name “lampbrush chromosomes” (Callan, 1986). This type of chromosome has long been used as an experimental model for gene transcriptional studies in eukaryotes. Analysis of the ultrastructural organization of lampbrush chromosomes has shown that transcription occurs at their lateral loops (Sommerville et al.. 1978; Callan, 1986; Gall et ui., 1983). It was initially suggested that during transcription the lateral loops continuously extend and then are drawn back into a chromomer (Snow and Callan, 1969). However, later evidence has been obtained indicating that most probably no movement of loops occurs through a chromomer (Sommerville et uf., 1978; Gall et al., 1983; Macgregor, 1987). The ultrastructurally revealed organization of meiotic chromosomes. in particular of lampbrush chromosomes, has played a prominent role in the development of the principle of chromatin loop organization in other cell types. For example, the principle of radial arrangement of chromatin loops with respect to SC, the initial supposition about movement of chromatin loops through chromo-

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Nucleoids

203

mers in lampbrush chromosomes during transcription had a certain effect on interpretation of DNA organization in histone-depleted interphase and metaphase chromosomes (see below). Nevertheless, to date the homology of the loop-domains of somatic cell chromosomes and the loops of lampbrush chromosomes remains questionable, because the mechanism of chromosome restructuring during transition of cells from mitosis to meiosis is unknown. I think that some observations make the identity of loops of interphase chromatin (as well as metaphase chromosomes) and those of lampbrush chromosomes rather doubtful. Firstly, the size of chromatin loops anchored to SC as a rule exceed those found for loops of interphase or metaphase chromosomes. Lampbrush chromosome loops frequently contain several transcriptional units (Sommerville et al., 1978; Callan, 1986; Macgregor, 1987) whereas in interphase nuclei, loops of nuclear DNA are known to have only one transcriptional unit (Hancock, 1982). The chromomer sizes at pachytene are higher than those in interphase of somatic cells or at early meiotic stages (Lima-de-Faria, 1975). It is supposed that pachytene chromomers are equivalent in G-bands of metaphase chromosomes (Ambros and Sumner, 1987) while G-bands are a sum of elementary chromomers (Comings, 1978; Zatsepina et ul., 1983a). Thus, a chromomeric loop of lampbrush chromosomes is presumably formed by a chromatin fibril of several elementary chromomers. Secondly, in Triton lampbrush chromosomes, transcripts from a cluster of histone genes were shown to have sequences homologous to satellite DNA which is not observable in somatic cells. Satellite sequences in the Triton genome are flanking each of the histone gene clusters (Diaz and Gall, 1985). The above observations indicate that both the extension of the transcriptional unit and very likely the size of DNA loop in lampbrush chromosomes, are larger than in chromosomes of somatic cells.

IV. LOOP ORGANIZATION OF METAPHASE CHROMOSOMES The DNA organization interphase nuclei has been

in histone-depleted studied in parallel

with the structure of histone-depleted metaphase chromosomes. The electron microscopic preparations of histone-depleted metaphase chromosomes demonstrated by Laemmli et al. are clearly represented by the central element, a chromosome scaffold, which is surrounded by DNA loops (Paulson and Laemmli, 1977; Laemmli rt al.. 1978; Earnshaw, 1988). The scaffold remains stable under the treatment with 2 M NaCI. polyanions (dextran sulfate, heparin) but dissociates in 4 M urea; it is resistant to DNase and RNase. Histone-depleted chromosomes were shown to contain a low amount of nonhistone proteins (Adolph et ul., 1977a.b). and metal-protein interactions were found to play a significant role in scaffold stabilization (Lewis and Laemmli. 1982). The radial loops bound to the scaffold are probably completely devoid of proteins (Adolph et ~1.. 1977a); their length is some 30-90 kb (Paulson and Laemmli. 1977). Nonhistone proteins of the scaf-fold are supposedly associated with DNA nucleotide sequences at the base of each loop (Marsden and Laemqli, 1979). Subsequently (Earnshaw et ul., 1985; Earnshaw and Heck, 1985; Gasser et ul.. 1986). one of the main scaffold proteins was identified as DNA topoisomerase 11. The identification of DNA topoisomerase II associated with the base of the DNA loop has great significance. as it is indicative of the structural role of the enzyme responsible for maintenance of the topological state of DNA within the nucleus (Gasser and Laemmli, 1987). It should be noted. however, that there are certain doubts about the continuity of the protein scaffold in metaphase chromosomes (Okada and Comings. 1980; Hadlaczky rt ul., 1981a.b. 1982; Nasedkina and Slesinger, 1982). Indeed it has been suggested that the scaffold may result from aggregation of the proteins located at the bases of nuclear DNA loops. At present there are also other models of metaphase chromosome (Zatsepina et trl., 1983x Rattner and Lin. 1985: Nokkala and Nokkala, 1986). The model proposed by Zatsepina et al. (1983a) seems to be very perspective. In this model. a metaphase chromosome is a hierarchy of discrete structures: nucleosomes, nucleomers (6---lO nucleosomes). chromomers (3040 nucleomers). Tandem

chromomers form a chromonema (about 100 nm thick) which, in turn, constitutes the body of a mitotic chromosome. Evidence in favor of this model is the experimentally proved globular (chromomeric) organization of a 100 nm chromonema and the rosette-like organization of 100 nm chromomers in metaphase chromosomes of Chinese hamster (Zatsepina et ul.. 1983a). The rosette-like organization has also been detected in minute chromosomes of mouse cells (Hamkalo c’t r/l.. 1985). and in histone-depleted metaphase chromosomes (Okada and Comings, 1979). The discrete arrangement of chromonema can account for subunit structure of metaphase chromosomes observed in the studies mentioned above. However. the subunit structure of 100 nm chromatin fibrils in metaphase chromosomes was not reported in all studies (Nokkala and Nokkala. 1986; Rattner and Lin. 1987). Meanwhile. the model of Laemmli c’t (I/. has played an important role in originating the concept about loop organization of histone-depleted interphase chromosomes. The radial arrangement of chromatin in meiotic chromosomes in prophasc I constituted the ideological basis for this model (Earnshaw and Laemmli, 1984).

V. NUCLEOIDS AS AN EXPERIMENTAL OF DNA LOOP ORGANIZATION IN INTERPHASE NUCLEI

MODEL

To analyze the DNA loop organization in nucleoids, sedimentation and fluorescent methods arc currently used. Centrifugation of nucleoids in a neutral sucrose gradient containing intercalating agents or nucleoids pretreated with different nuclease doses, subjected to X-rays or UV light decreases the rate of their sedimentation. The process depends on the amount of the agents employed. and sedimentation occurs in a biphasic manner (Cook and Brazell. 1975, 1976: Ide c’t d., 1975; Benyajati and Worcel, 1976; Nakane (‘I trl.. 1978; Mullenders et al.. 1983). Analysis under the fluorescent microscope has demonstrated that interphase nuclei incubated in buffer containing

Ultrastructure

of Somatic

ethidium (or other intercalating agents) are fluorescent, of a rounded shape, with sharply, outlined edges. The nucleoid is also of a round configuration, but instead of having distinct edges it has a so-called luminous halo (Cook and Brazell, 1976, 1978; Warren and Cook, 1978; Vogelstein et al., 1980; Hancock and Hughes, 1982; BuongiornoNardelli et al., 1982). The results of sedimentation and fluorescent nucleoid analyses were interpreted in terms of the model according to which linear DNA of interphase nuclei is supercoiled and forms a series of topologically detached loops fastened at their bases with the help of proteins resistant to 2 M NaCl extraction. Within the frame of this model, a decrease in sedimentation rate or an increase of the nucleoid luminous halo diameter under the effect of the applied agents (see above) are attributed to relaxation of supercoiled DNA loops that form the halo (Fig. 3). This concept was named the loop-domain principle of DNA organization in interphase nuclei (Benyajati and Worcel, 1976; Igo-Kemenes and Zachau, 1978). The term “domain” designates a chromatin region between two sites of anchorage at residual nuclear structures (nuclear matrix), these sites being thus protected from nuclease digestion (Igo-Kemenes and Zachau, 1978). The size of loop-domains in eukaryotic nuclei can be estimated from nucleoid 1978; sedimentation characteristics (Hartwig, Mullenders et al., 1983; Schwarz et al., 1984) radius measurements of the nucleoid luminous halo (Vogelstein et al., 1980; Buongiorno-Nardelli et a/., 1982) or the amount and size of DNA fragments cleaved off with nucleases and anchored

Fig. 3. A scheme of nucleoid organization. DNA loops are suggested to form the nucleoid halo, and loop bases are likely to be anchored to nuclear matrix.

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Nucleoids

205

to nuclear matrix (Igo-Kemenes and Zachau, 1978; Razin et al., 1979; Berezney and Buchholtz, 1981; Mullenders et al., 1983; Tas et al., 1985). The size of the DNA loops, experimentally determined from the length of DNA fragments cleaved off with nucleases from nuclear matrix, averages 35-85 kb (Igo-Kemenes et al., 1982). Sedimentation experiments give as a rule higher estimates of this value. For instance, in HeLa cells the size of the supercoiled DNA domain, estimated from sedimentation characteristics of X-ray treated nucleoids, amounts to 4 x lo8 Da. Alternatively, the DNA loop size determined from the analysis of the length of the fragments released from nucleoids after radiation-induced single-strand breakage is 4.8 x 1O’Da (Mullenders et al., 1983). This discrepancy between the sizes of DNA loops and supercoiled domains of nuclear DNA is somewhat unclear. Data reported (Warren and Cook, 1978; Razin et al., 1979) suggests that the size of nuclear DNA loops remains unchanged throughout the cell cycle, whilst other evidence indicates certain differences in DNA loop sizes (supercoiled domains) at different stages of the cell cycle, during embryonic development, or following tumor cell transformation (Pinon and Salts, 1977; Pinon, 1978; Hartwig, 1982; Buongiorno-Nardelli rt al., 1982; Schwarz et al., 1984; Linskens et al., 1987). Supercoiled DNA loops are apparently attached to the nuclear periphery (Hancock, 1982; Hancock and Boulikas, 1982; Nelson et al., 1986). During transcription or replication nuclear DNA interacts with elements of the internal matrix. Transcribed or replicated genome regions are thought to pass through transcription or replication complexes, fixed on the nuclear matrix (Jackson et al., 1984; Razin, 1987). The genetic material is inactivated due to condensation of loop-domains and formation of supersolenoids (Hancock, 1982; IgoKemenes et al., 1982; Cartwright et al., 1982; Butler. 1983). It seems that the loop-domain model for the structureefunctional organization of genetic material as it exists at the moment still has a number of vague points. First, it is not clear what is the relationship between supercoiled domains (size estimates are based on nucleoid sedimentation and

fluorescence studies) and DNA loops (size estimates from experiments on DNA release from nuclease-treated nucleoids). Are these structures equivalent or not? Why does the size of the former usually exceed the size of the latter? Second. there is abundant evidence suggesting that nuclear DNA is anchored to the nuclear periphery (i.e. nuclear lamina). The bases of nuclear DNA loop-domains have been shown to be fastened with residual nuclear proteins but no data are available indicating that these are polypeptides of nuclear lamina. Moreover. at metaphase or meiotic prophase I, lamina is absent, although the loop organization of nuclear DNA is retained. This obscurity may be attributed to the fact that the ideology of biochemical investigations on nucleoids (sedimentation, fluorescence, viscosity tests) has been erroneously built on the experimental results and theoretical substantiation of physico-chemical analysis (Bauer and Vinograd. 1974) of circular supercoiled and relaxed DNA molecules of prokaryotes. To what extent can the data obtained with covalently closed DNA molecules be considered applicable to nuclear DNA of eukaryotes? We are still uncertain whether all factors involved in maintaining the loop structures of nuclear DNA in eukaryotes are known. The cytological method of nucleoid analysis can be used as the criterion of”nativity” of isolated structures. In this case the chromatin state in the nucleus should be under control in the process of nucleoid isolation and analysis. The effect of isolation conditions and different agents on the chromatin structure should be taken into account. Though even in such a case the “criterion of verity” is not absolute.

The HeLa nucleoid spread on a water surface consists of a core and a surrounding network of thick and thin fibrils (McCready ef ~1.. 1979). The majority of thick fibrils are assumed to be aggregated DNA fibers. Thin fibrils resemble supercoiled DNA molecules of prokaryotes. This finding, together with the reported physicochemical characteristics of nucleoids (Cook and Brazell. 1975. 1976) and electron microscopic

results of the morphology of DNA fibers tn the nucleoid halo. allowed the authors to conclude that nuclear DNA is organized as a series of large (about 220 kb) supercoiled loops (McCready ot trl.. 1979. 1982; Jackson pt u/.. 1984). Supercoiled DNA of the nucleoid halo has often the form of collapsed toroidal and interwound superhelices. Treatment of nucleoids with X-rays. a low concentration of ethidium bromide. and the untwisting enzyme leads to relaxation of supercoiled DNA fibers (McCready et trl.. 1979). A similar morphology has been shown for DNA fibers in nucleoids of yeast cells (McCready rt trl.. 1977). Hancock and Hughes (1982) showed electron microscopic preparations of histone-depleted interphase chromosomes of mouse. in which DNA loops with sizes of 10 to 180 kb (53 kb on average) were distinctly visible, In the distal regions of these loops RNA transcripts have also been detected (Hancock and Hughes, 1982). However. I consider that the structures presented are not native. hut rather dispersed nucleoids, as they lack the specific morphology (a core and a halo). All the above cited works have one very important observation in common. i.e. that histonedepleted nuclei of interphase cells have bcrn found to contain large DNA loops. But it is interesting to note that all these studies employed similar conditions for nuclear isolation and nucleoid analysts. namely: prior to histone extraction. the nuclei were kept in a buffer with low ionic strength and a low concentration of divalent cations. and the nucleoid analysis was performed in a buffer containing EDTA. One should bear in mind that under these conditions chromatin in the nucleus is represented by homogeneous 25 30 nm tibrils (see Section I1.A) and that this suggests destruction of higher level\ of native chromatin organization and the lack of the corresponding structures in histonedepleted interphase chromosomes. Besides large DNA loops. structures containing small DNA loops (below 5 kb) have also been revealed in histone-depleted nuclei. These arc the rosette structures. Rosette structures were initially found in preparations of partially deproteinized chromatin in bacteria and fish and later in other tissues (reviewed by Leon and Macaya. 1983). but were referred above to artefact structures created

Ultrastructure

of Somatic

during the process of preparing electron microscopic specimens. Indeed, purified DNA was shown to be capable of forming structures which resemble rosettes, provided that certain conditions were used for sample spreading (for references see Leon and Macaya, 1983). However, the centers of “true” rosette structures and rosette-like structures formed by purified DNA were found to differ morphologically (Okada and Comings, 1979). The fear of the artefactual origin of rosette structures was apparently the cause of delay, not only in the study of rosette structures themselves, but also in thorough consideration of the possibility of their actual existence in eukaryotic nuclei. Strong evidence for their existence in nuclear DNA is given by Okada and Comings (1979). Additional support is provided by the data indicating the appearance of breakages in DNA loops of rosettes during mild treatment of nuclei with restrictases or endonuclease (Prusov rt al., 1983; Leon and Macaya. 1973). Rosette structures were observed in nuclei extracted with NaCl (Sonnenbichler, 1969; Comings and Okada, 1976; Leon and Macaya, 1983) ammonium acetate (Okada and Comings, 1979) and polyanions (Prusov et al., 1983). The native state of chromatin in the nucleus is the factor which determines the detection of rosettes. The latter have been revealed in nuclei where chromatin contains 100 nm globules (see Section II.A), whereas in those with 10nm and/or 25530 nm chromatin fibrils, no rosette structures have been observed (Prusov et al., 1983). The presence of rosettes is also dependent on the degree of chromatin deproteinization (Okada and Comings, 1979; Prusov et al., 1983). Long DNA loops (corresponding by size to the loop-domains) are suggested to appear following extensive deproteinization of rosette structures and removal of protein “fasteners” maintaining the rosette center intact (Prusov et al., 1983). Rosette structures of histone-depleted chromosomes, on the cytological level, are likely to correspond to chromomers (Sonnenbichler, 1969; Prusov et al., 1983). VI. NUCLEOIDS During chromatin

OF RAT

LIVER

CELLS

the electron microscopic analysis in situ great significance is attached

of to

and Meiotic

207

Nucleoids

the conditions under which nuclei are kept. These conditions include: ionic strength of the buffer, concentration of divalent metals ions and their composition, the presence of agents affecting disulfide (SS) bonds in proteins. The above factors can change the chromatin state from “native” to diffuse and vice versa (for details see Glazkov, 1988a). In this connection, the uniformity of conditions chosen for nucleoid isolation and analysis is especially noteworthy: utilization of a buffer with low ionic strength for nuclei isolation, nuclei treatment with 2 M NaCl followed by centrifugation through a sucrose layer containing 2 M NaCl and EDTA. At the same time, low ionic strength of buffer and chelating agents are known to cause dramatic changes in the chromatin structure in nuclei, bringing about a completely diffuse decondensed state (Galcheva-Gargova et al., 1982). So the question arises as to whether nucleoids isolated according to the above protocol can serve as a reliable model for investigating the DNA loop organization in nuclei? Higher levels of chromatin organization are, almost certainly, liable to be destroyed during the change to the diffuse state. In order to check this supposition, sedimentation, fluorescent and electron microscopic analyses of nucleoids isolated under different conditions have been carried out. Moreover, nucleotide sequences associated with nuclear matrices isolated under conditions similar to those used for nucleoids have also been partly analyzed. A. Sedimentation

and Fluorescent qj Nucleoids

Analyses

In earlier sedimentation and fluorescent studies of nucleoids, Cu’+ ions were shown to stabilize the loop organization of nuclear DNA, whereas 2-mercaptoethanol, on the contrary, was found to destroy it (Lebkowsky and Laemmli, 1982a,b; Lewis et ul., 1984). Stabilization of intranuclear structures takes place once the nuclei are treated with nonionic detergent, i.e., during the liberation of nucleoids from external nuclear membrane. In view of this, the buffer used for nucleoid isolation from rat hepatocytes (Glazkov, 1986a) contained either CL?+ or Mg’+ ions at the stage of nuclear treatment with Triton X-100, and in some cases the

buffer contained 2-mercaptoethanol. Experiments on nucleoid sedimentation in a linear sucrose gradient containing EDTA (Fig. 4) demonstrated that the nucleoids isolated in the presence of Cu’ ’ ions has a high sedimentation rate (nucleoids of type I), while those isolated either in the presence of Mg’* ions. Mg’+ ions + 2-mercaptoethanol. or CL? + ions + 2-mercaptoethanol are characterized by a lower sedimentation rate (nucleoids of type II). These results are generally accepted to indicate (see Section V.A) a more compact state of nuclear DNA in nucleoids of type I compared to type II nucleoids. However, if the sucrose gradient employed for nucleoid sedimentation contains Mg’+ ions instead of EDTA. no transition of nucleoids of type II to nucleoids of type 1 is observed; in other words, in the absence of EDTA nuclear DNA is not decompactized (Fig. 5). These findings clearly indicate the important role of divalent cations in stabilizing the compact higher order state of nuclear DNA

c

0

.

iI\.

c8t

b

Fig. 5. Sedimentation of rat hepatocytc nucleo~ds 1115 JO”,, sucrose gradients (see legend to Fig 4) in the pwencc of 5 mu MgCl:. Nuclei were treated uith Trlton X-100 111 buKer m the presence of 5 mM MgCll (a). 5 rnM MgC‘l, + Ii mhl ?-mercaptoethanol (b). or 2 mu CuSO, (c) Nucleo~d\ ulth ;I low wdimcntation rate in the EI)TA-cvnt~llning ~ucrosc gradient (see Fig. 4) have I higher wdimentation rate in the Mg’ ’ -containing pradienl

b

Froctlon

Fig. 4. Sedimentation of rat hepatocyte nucleoids in a 5 vZO”/~sucrose gradient in the presence of I0mh1 EDTA (SW 50.1, 5000 rcwmin. 40mm. 4 C). Nuclei were treated wth Triton X-100 in the buffer containing 50 mM Tris HCI pH 7.4. 100 rn~ NaCI. I rn~ PMSP, in the presence of 5 mM MgCI, (a). 5 mM MgCI, + 2-mercaptoethanol (b). 2 mM CuSO, (c), or 2 mhf CuSO, + 15 mM ?-mercaptoethanol (d). The degree of supercoiling (compactization) of DNA loops is thought to be greater I” nucleoids wtth higher sedimentation rates compared to those with lower sedimentation5 rates. The direction of sedlmentatlon in the tigures is from right to left.

The fluorescent analysis performed to reveal the supercoiled DNA state in nucleoids confirmed the results of the sedimentation analysis (Fig. 6). Moreover, the fluorescent studies demonstrated the lower contribution of disulfide bonds to maintenance of the compact state of nuclear DNA compared to divalent cations. Thus DNA can be present in nucleoids In diffcrent degrees of compactization, depending upon the isolation conditions used. It is still to bc detcrmined to what extent the compact state of nuclear DNA revealed by sedimentation and fluorescent nucleoid analyses depends upon nuclear DNA supercoiling. In other words. are the theoretical propositions formulated for these methods on the basis of covalently closed prokaryotic DNA

Ultrastructure

u

of Somatic

and Meiotic

Nucleoids

209

e

Fig. 6. Fluorescent visualization of Mg’+ ions (a. b, c, d) or Cu’+ 2-mercaptoethanol f EDTA (d. h). of the nucleoid luminous

rat hepatocyte nucleoids. Nuclei were treated with Triton X-100 in a buffer containing ions (e, f, g, h) in the presence of EDTA (b. f), 2-mercaptoethanol (c. g) or Nucleoids were treated with ethidium bromide (2 pg/ml). An increase in the diameter halo is suggested to evidence relaxation (decompactization) of DNA loops.

molecules entirely true with respect to the complex organization of eukaryotic nuclei? To check this we undertook a series of electron microscopic analyses at each step of nucleoid isolation and studied the effect of various agents on nucleoid morphology (Glazkov, 1986b).

Fig. 7. Rat hepatocyte

nuclei treated

with Briton

B. Nucleoid Morphology

at D!fJrent

Isolation Steps

The first step in the nucleoid isolation procedure was treatment of nuclei with nonionic detergent. Figure 7 shows Triton X-100 treated nuclei in a buffer with average ionic strength in the presence

X-100 in buffer containing

5m~

MgC&.

Scale marker

indicates

I pm.

of Mg’+ ions. During this isolation step, the nuclei remain intact and retain their spherical shape. The result of the second step of nucleoid isolation (treatment with 2 M NaCI) is demonstrated in Fig. 8a. The main event at this stage is the release from the nucleus of the hbroglobular network to form the nucleoid halo. Nucleoid treatment with DNase leads to disappearance of the halo. and is indicative of its DNA composition. Globular elements of the nucleoid halo have a diameter of about 50&150 nm. DNA loops 0.550.7 pm long anchored to globules are vizualized thus suggesting their rosette-like organization. No other DNA structures have been detected in the halo. The third step of nucleoid isolation is centrifugation through a sucrose gradient containing EDTA. In the electron microscopic analysis of nucleoid morphology. this step was replaced by treatment of the nuclei with 2 M NaCl in the presence of EDTA (Fig. 8b). At this step, essential changes in the nucleoid halo take place; the globular elements disappear, and thick DNA fibrils are broken into thin elementary DNA fibers. No loop DNA structures have been detected in the nucleoid halo in these preparations.

C.

Interculuting

Agents

und

Nucleoid

Morpholog),

Proof for the existence of supercoiled DNA domains in nucleoids can be obtained from experiments on nucleoid titration with ethidium bromide. The theory behind these experiments is as follows: ethidium bromide (at certain concentrations) intercalates into supercoiled loopdomains. causes their relaxation and leads to a decrease in the rate of nucleoid sedimentation in the neutral sucrose gradients. Figure 9 shows nucleoids treated with ethidium bromide at different concentrations. It can be seen that the increase in ethidium bromide concentration renders the nucleoid halo more friable; the diameter of both the fibrils and the halo globules becomes greater. Large loop DNA structures are not revealed in these preparations. The friable halo of the nucleoids is likely to be the reason of the lowered rate of sedimentation. A correlation between the

degree of friability of the nucleoid halo and intercalator concentration has been noted.

the

Lebkowski and Laemmli (1982a.b) demonstrated ions stabilize intranuclcar that Cu” and Ca” structures and nuclear DNA compactization. This was concluded from the results of sedimentation and fluorescent nucleoid analyses using HeLa cell nuclei. Let us consider which structures are preserved when Cu” ions are utilized in the nuclcoid isolation procedure. Figure IOa shows nucleoids of rat hcpatocytes isolated from Triton X-100 treated nuclei in butfer containing 2 mM Cu’+. These nucleoids have ;I distinctly typical morphology: a cot-c and radial fibrils with halo-forming globules. The globule diameter is SO I50 nm. A gradual increase in (‘II’ ’ ion concentration (3. 3, S WI) in the butfcr at the time of nuclei treatment with Triton X-100 leads to aggregation of the core and the nucleoid halo elements, and at ;I Cu2 concentration of 5 mM the nucleoids are destroyed. Treatment of C‘LI‘ nucleoids with EDTA causes insignificant changes in their halo (Fig. lob). Hence. the LISC 01‘ C‘LI’ ions in the nucleoid isolation procedure preserve\ the globular elements which have ;I roscttc-like organization.

Disulfide bonds have been suggested (Kaufman rt ul.. I98 I: Lebkowski and Laemmti. I982a.b: Lewis et (I/.. 1984) to play an important rote in DNA loop organization in the nucleus. This conclusion has been based upon the experimental results indicating that nucleoid treattnent with 2-mercaptoethanol decreases the rate of their sedimentation and increases the diameter of the luminous halo. As mentioned above. these biochemical characteristics indicate relaxation of the loop structure of nuclear DNA. However. the data in Figs 4 6 strongly indicates that the above events take place only in the presence of EDTA. Electron microscopic analysis has shown that the nucleoids isolated in the presence of Mg” or ions and treated with 2-tnercaptoethanol CL? ’

Ultrastructure

of Somatic

and Meiotic

Nucleoids

Fig. 8. Rat hepatocyte nucleoids isolated in the presence of Mg *+ ions (a) with subsequent EDTA treatment (b). The nuclei extracted with 2 M NaCl leads to the release of fibroglobular material to form the nucleoid halo. Once the nucleoids are treated with EDTA, the globular elements disappear from the halo. The halo is formed by thin DNA only. Scale markers indicate 1 pm.

211

M. V. Glazko\

Fig. 9. Rat hepatocyte nucleoids isolated in the presence of Mg” wns treated wth ethidium bromldc at cvnccntrativn~ I ~~g.‘rnl (a). 2gg:ml (b), ?O,~(g:‘ml (c). The nucleoid not treated with ethidium bromide (d). Nucleoid treatment with ethidium bromide makes globular and Gbrillnr halo elcmerrts more friable. Scale markers mdxxte I /lr~?

Ultrastructure

Fig. 10. Rat hepatocyte

nucleoids

of Somatic

and Meiotic

213

Nucleoids

isolated in the presence of Cu’+ ions (a) with subsequent EDTA treatment serve to stabilize globular elements in the nucleoid halo.

(b). Cu’+ ions

M.

V.

Glazko\

,I

_H’. ..’ I

‘.

.

,ri . I

Ultrastructure

of Somatic and Meiotic Nucleoids

have a typical morphology; i.e. a core and radial fibrils with globules of 50-150 nm diameter (Fig. 11). Treatment of these nucleoids with EDTA brings about dramatic changes in their morphology (Fig. 12). No 50-150 nm globules have been actually revealed in the halo of nucleoids of both divalent cation types, and the halo itself becomes “fluffy”. Moreover, in the nucleoids isolated in the presence of Mg’+ ions and subsequently treated with 2-mercaptoethanol and EDTA, the cores have been noticed to be destroyed which strongly suggests the destruction of intranuclear structures. It follows from the electron microscopic analysis, that the agents breaking disulfide bonds in proteins cause decompactization of nuclear DNA only in the presence of chelating agents. The biochemical analysis and electron microscopy of nucleoids permit us to draw the following conclusions: (1) the rate of nucleoid sedimentation in the sucrose gradient and the diameter of the luminous nucleoid halo are determined by the chromosomal material released from the nucleus following the nuclei extraction with 2 M NaCI; (2) the structures containing DNA loops in the nucleoid halo are the rosette globules; (3) nuclear DNA decompactization revealed by sedimentation and fluorescent methods is morphologically manifested by the disappearance of the 50-150 nm globules from the nucleoid halo. F. The Heputocyte

Nucleoids

Core

In the above sections, the structure of the nucleoid halo has been emphasized. What regions of interphase chromosomes are involved in the formation of the nucleoid halo? Treatment of nuclei with 2 M NaCl causes dissociation of histone from DNA and of many nonhistone proteins which participate in chromatin organization on the nucleosomic and supernucleosomic levels. This induces the hitherto tightly compactized nuclear DNA to straighten out, like a mainspring of the clock, and eject itself from the nucleus. The first to spring out of the nucleus are apparently the interphase chromosome regions which are initially located immediately under the nuclear envelope. In the nuclei of animal cells, it is the transcriptionally

215

inactive chromosome regions that are, as a rule, located under the nuclear envelope surface (Brash and Setterfield, 1974). The nucleoid halo is for the most part likely to be formed by these transcriptionally inactive regions of interphase chromosomes. Transcriptionally active domains probably remain in the nucleoid core. A fragment of a rat hepatocyte nucleus subjected to 2 M NaCl treatment is shown in Fig. 13 (Glazkov, 1985). Intranuclear structures have been preliminarily stabilized with Cu’+ ions (see sections above). It is clearly seen that the preparation contains a residual fibroglobular network’ with large nuclear DNA loops associated with it. This network is probably located in the nucleus under the inner nuclear membrane/nuclear lamina and anchored to this, while DNA loops are directed inside the nucleus. Rosette structures have been detected in the residual network and large DNA loops. The latter contain at their ends fibroglobular material that is morphologically identified as RNA transcripts, thus DNA regions localized at the loop ends are identified as transcription sites (Fig. 14). Transcripts are located exclusively at the ends of the loops of nuclear DNA; the loop bases are free from RNA transcripts. The size of the DNA transcription sites approximates to 13 kb. This value is close to the mean size of pre-mRNA. The average transcription domain (the DNA region from one rosette to the next one or to the loop base which comprises the transcribed DNA region) is about 26 kb. The presence of RNA transcripts at the ends of large loops and their absence in rosettes suggest that rosette structures are a mechanism for packaging transcriptionally inactive genome domains. A significant feature of DNA organization in liver cell nuclei appears as the frequently observed pattern “loop in loop” (Fig. 13); the bases of the “inner” loops are, as a rule, located at the rosette structures of the “outer” loop. This phenomenon can be attributed to local polytenization of separate genome regions containing actively transcribed nucleotide sequences, which is a particular case of gene amplification. Rat hepatocytes are likely to have several amplified genes, rather than one. judging from the differences in the lengths of both the loops themselves, and transcribed regions in

Ultrastructure

of Somatic

and Meiotic

Nucleoids

217

Fig. 13. A fragment of the core of a rat hepatocyte nucleoid. Nuclei were treated with Triton X-100 in the presence of 2 rnM CuSO,. Large DNA loops contain RNA transcripts at their ends. Rosette structures are present both in large DNA loops and the fibroglobular network. Scale marker indicates 1 /cm.

different loops. Electron microscopy of rat liver cell nuclei has succeeded in revealing “loop in loop” amplified regions of nuclear DNA with a maximum size of about 300 kb. Such extended regions are apparently formed by means of fusion of individual replicons (amplicons) whose size averages some 20 kb (Glazkov, 1986~). It should be noted that the large loops of nuclear DNA found in histone-depleted nuclei of rat hepatocytes do not conform to the generally acknowledged notion about a loop-domain of interphase chromosomes. The difference is that in the revealed loop structures the loop bases are not drawn together as has been postulated in many reports (Igo-Kemenes and Zachau. 1978; Hancock, 1982; McCready et ul., 1982; Jackson

et af., 1984; Nelson et al., 1986). I consider this difference very important as it enables one to suggest another interpretation of interphase chromosome organization in the nucleus (Fig. 15). According to the proposed model, the structure of histone-depleted interphase chromosomes can be represented as a system of alternating globular and interglobular elements (by analogy with the structure of polytene chromosomes in insects (see Section I1.B). The globules have the rosettelike organization. Chromosomes of interglobular regions are apparently anchored to the nuclear lamina. Replication or transcription units appear as a DNA fragment comprising a rosette structure. The rosette structure looping potentiates replication or transcription of this genome region.

218

M. V. Glwko\

nuclear structures in the genetically. thoroughly studied region of the Dro.vophilu rllc,ltrrlo,~tr.vtc.r genome (320 kb long). Wide-range diffcrcncch have been demonstrated in loop-domain lengths (26 1I? kb). Moreover. from 5 to X unrelated genes have been found in large loop-ciomain~. Some of the latter contain more than one trarlscribed gene. The results of this study seem to hc clearly explicable in terms of the rosette organi/ation of nuclear DNA. The loop-domains containing several unrelated genes arc evidently the looped genome regions with several rosette \tructurcs. each containing one gene (functional unit). The genome region contained in the rosette btructurc can be transcribed independent of any ad.iaccnt regions.

I:lg. 14. Iragmenta of the core of a rat hepatocyte nuclcod \howng the end of a large DNA loop with RNA transcripts. Scale marker indicates I pm.

It has yet to be specified whether the sites of anchorage to nuclear lamina are present in each inter-globular region or whether their number is essentially less. Mirkovitch ct trl. (1986) mapped nucleotide sequences associated with residual

Fig. IS. A xhcme ol’ the structure Ilnctional chromosome organizntwn m an interphase nucleus The mterphasc chromw some structure I\ represented as alternation ofchromomcr~ (2) and inter~hromomeric rcgons. An interphase chromosome IS likely to bc anchorcd to nuclear lamina al interchromomeric regions (I ). Transcription (3) or replication (3) require uw \*indinp of rosette structures to l’orm large chromatin luops

One of the important aspects yet to bc elucidated from studies on the loop organization of chromosomes in interphase nuclei is the specificity of the DNA sequences associated with residual nuclear structures. An interphase nucleus is ~~~s~~med to have 60,000~125.000 sites of DNA attachment to the nuclear matrix (Razin c’t trl.. 1979: Hcre/nq and BuchholtL, 19X1). At present, the concept of two types of DNA association with nuclc’~~matrix is widely accepted (Cook and RraLcll. 19x0: .lackson (‘t ~1.. 19x4: Rarin. 19X7). The lirst type I\ supposed to maintain the intcgritb c)I‘ the lo<)p organization of nuclear DNA. This association ha been given the term “structural”. 01 perni;tncnl association. and the DNA sites ~ncolvcd arc’ termed “structural” attachment sitcs. Here an as\ociation of specific repeated nucleotide scy~~encc~ to nuclear lamina is implied. The \ccond type 01‘ DNA association with residual nuclear structure\ provides for genetic material functioning: traiiscription. replication. condensation. and deccjndensation of nuclear DNA. This aaociation 15 suggested to have ;I temporal, or “functional” character. The DNA sites in\,olved in this a\scG ation wsith nuclear matrix are called “functional”. or temporal attachment sites. In this USC. associa tion of random nuclcotide sequence with clement\ of the internal nuclear matrix i\ implied.

Ultrastructure

of Somatic

Before discussing the results of analysis of the nucleotide sequences associated with residual nuclear structures from rat hepatocytes, mention should be made of certain stages important for isolating residual nuclear structures, i.e., nuclear matrix. Two variants of nuclear matrix isolation procedures are most commonly used today: one, when nuclear DNA is digested with nucleases (DNase I, micrococcal nuclease, restriction enzymes), and the other when nuclear proteins are extracted with 2 M NaCl and other deproteinizing agents. The nuclease action is frequently terminated by adding EDTA into the reaction mixture, followed by centrifugation through a layer of sucrose (glycerol) containing 2 M NaCl and EDTA. It is noteworthy that the results obtained from analyses of nucleotide sequences associated with nuclear matrix are highly contradictory. This discrepancy is usually accounted for by differences in the conditions employed for nuclear matrix isolation (for more details see Glazkov, 1988b). The nuclear matrix has been isolated from rat hepatocytes under conditions similar to those used for nucleoid isolation, i.e., nuclei were treated with Triton X-100 in the buffer containing CL?+ or Mg’+ ions. Nuclear DNA was subjected to DNase I digestion prior to nuclear protein extraction with 2 M NaCl. and the reaction stopped with EDTA. The results indicated that nuclear matrices. from isolation conditions equivalent to those yielding nucleoids with globular elements, contained 2 types of DNA fragments that differed in length: “long” (about 12 kb) and “short” (about 1.8 kb). The nuclear matrices of nucleoids devoid of globular elements contained “long” DNA fragments only. So, the analysis of nucleoids and nuclear matrices of hepatocytes revealed the following correlations: nuclear matrices of the nucleoids characterized by a high sedimentation rate and a small diameter of the luminous halo were found to have 2 types of DNA fragments associated with residual nuclear structures, whereas in nuclear matrices of the nucleoids with a low sedimentation rate and a large diameter of the luminous halo, only one type of chromosomes fragment associated with residual nuclear structures has been detected. It is possible to isolate “short” DNA fragments from nuclear matrix in the absence of the CL?’

and Meiotic

Nucleoids

219

ions, used to stabilize the intranuclear structures. Figure 16 demonstrates electrophoretograms of DNA associated with hepatocyte nuclear matrices isolated in the presence of Mg’+ ions. In one case (Fig. 16a), DNase I digestion was terminated with EDTA and, following 2 M NaCl extraction, the nuclei were centrifuged through a layer of sucrose containing EDTA. In the other case (Fig. 16b), EDTA was not used at all. These data indicate that preservation of “short” fragments in the nuclear matrix directly depends on the presence of EDTA during nuclear matrix isolation (Glazkov, 1986a). A typical nuclear matrix consists of 3 components: the nuclear pore and lamina complex, a residual nucleolus, and a residual fibroglobular network called “internal matrix” (Berezney, 1979). a

b

Fig. 16. Electrophoretic distribution of DNA associated with nuclear matrices of rat hepatocytes that were isolated in the presence of Mg’+ ions. In one case (a) nuclear DNA digestion with DNase I was terminated with EDTA, whereas in the other one (b) EDTA was not used. The data indicates the “loss” of one type of DNA fragments associated with residual nuclear structures under the effect of DNase I inhibition with EDTA. Electron microscopic analysis of these nuclear matrices indicates that internal alements are “lost” when EDTA is used in the process of nuclear matrix isolation. DNA associated with the nuclear matrix devoid of internal elements is represented by “long” fragments only (I), the nuclear matrix that contain internal elements has both “long” and “short” (2) DNA fragments.

The question as to which structures of the nuclear matrix are stabilized with divalent cations is closely connected with another: which nuclear matrix structures are involved in association with the “long” and “short” DNA fragments, respectively? In Fig. 17 the nuclear matrices of hepatocytes that were isolated in the presence of Cu’ + and Mg’ ’ ions, using EDTA. are shown. The evidence suggests that ions of divalent metals stabilize the internal matrix elements. Thus, the “long” DNA fragments isolated from the nuclear matrix arc mostly represented by the nuclear pore complex and lamina, while the “short” ones come from the nuclear matrix internal elements (Fig. 18). The restriction analysis of the “long” and “short” fragments associated with nuclear matrix has demonstrated that they contain different repeated nucleotide sequences (Glazkov, 1986a). In addition, a difference in the content of BI- and B2-like repeats for two types of DNA, one associated with the nuclear matrix containing internal elements and the other associated with the nuclear matrix missing these elements. has been detected. (B-repeats are short DNA repeats dispersed

a

throughout the genome of rodents.) The former DNA fragment is much more enriched with B-like repeats than the latter (Glazkov. 1988~). What is the nature of the globular elements ot’ the internal nuclear matrix? It is interesting to note that rosette-like structures of nuclear DNA in nucleoids and globular elements of the internal matrix are found (or not found) under identical experimental conditions. The cores of rosette-like structures and globular elements of the internal matrix are of similar sizes (25 30 run). In view of this, I believe the globular elements of the internal matrix to be nucleaseand salt-resistant conponents of globule-rosettes. i.e.. the protein core\ of rosette DNA structures. Hence. the “long” and “short” DNA fragmcnta of nuclear matrix are likely to be structural regions of interphase chromosomes. The “long” fragments provide for interphase chromosome association with the nuclear periphery and are probably involved in the spatial chromosome distribution within an interphase nucleus, whereas the “short” fragments possibly contribute to the architecture of nuclear DNA rosette structures (Fig. IX).

b

d

E

(_I_ :i ,,

i:LL 1 ’

) _,;:,_II

t

c-1

-1

m-2

‘-’

e

Fig. 17. A protocol of isolation and electrophoretic analyslr for DNA associated wth rcs~dual nuclear structures 111rat hepatocytes. Divalcnt cations stahilizc rosette structures of nuclear DNA (a): in the ahsencc of divalent cations (e) the roscttc structure5 unwind. During nuclear DNA digestion with DNasc I (h. f) nucleotide sequences associnted with nuclear I;mlln;L (I ) and rosette structure cores (2) are retained. Hence, the nuclear matrix Isolated in the presence of dwalent catlons contain\ two types of DNA fragments (c). while the nuclear matrix isolated in the absence of divalent cations has only one type of DNA fragment (g). DNA fragments associated with nuclear lamina and rosette structure cores differ in length (d. II)

Ultrastructure

a

of Somatic

and Meiotic

Nucleoids

-

Fig. 18. Thin sections of rat liver nuclear matrix. Nuclei were treated with Triton X-100 in a buffer in the presence of 2 rnM CuSO, (a) or 5 mM MgCl> (b). The nuclei were digested with 50 pg/ml DNase I and 20 pgiml RNase. The reaction was stopped with 10 mM EDTA and the nuclei was extracted with 2 M NaCI. Cu’+ ions serve to stabilize “internal matrix”. Scale markers indicate 0.5 urn.

VII. NUCLEOIDS

OF MEIOTIC

CELLS

Globule-rosettes are specific elements of histonedepleted interphase chromosomes of somatic cells. Are these structures preserved in other chromosome types? To check this, we used testis cells of immature rat males as a model system (Glazkov, 1987). Considering the ploidy differences, testis cells can be subdivided into several fractions: spermatogonia, spermatocytes of prophase I, and spermatids. Figure 19 shows a nucleoid of a spermatogonium. Morphologically, nucleoids of spermatogonia are very similar to nucleoids of hepatocytes: a core and radial DNase-sensitive fibrils with globules. The globule diameter, the same as in hepatocytes, is 50-150 nm. DNA loops bound to globules are often vizualized, in other words, the 50-150 nm globules have a rosette-like organization pattern.

The morphological similarity revealed between nucleoids of spermatocytes and hepatocytes can be attributed to similar spatial arrangement of nuclear DNA in these cell types. In hepatocytes and chromosomes are anchored to spermatogonia, nuclear lamina (see Section II). Spermatocytes of prophase I are devoid of nuclear lamina. Chromosomes remain linked to nuclear membrane through their telomeric regions alone (Gillies, 1975: Moses et al., 1985) whilst chromatin loops are anchored to the synaptosomal complex (SC) (see Section III). At pachytene I, SC is an intranuclear structure that provides for spatial organization of nuclear DNA. This, probably, accounts for the specific morphology of nucleoids of spermatocytes of prophase I (Fig. 20). In these nucleoids, fibrils with globules in the halo are arranged spherically, not radially. When nucleoids of spermatocytes of prophase I are treated with EDTA, a partial decondensation of globules is

M. V. Glazko\

observed and their rosette-like organization becomes apparent (Fig. 20b). The loop size in these rosette-like structures does not exceed 1 I .S klm. Figures 21 and 22 demonstrate fragments of histone-depleted chromosomes of spermatocytes of prophase I. Their globular organization as well as the rosette-like organization is clearly visible. There are regions within chromosomal strands where proteins are entirely missing. This allows LIS to detect four DNA threads within one chromosomal strand, that corresponds to the double DNA amount (4C) in nuclei of meiotic prophase I at homologous chromosome conjugation. Apart from nucleoids, preparations of histoncdepleted meiotic nuclei were found to contain discretely located rosette structures of nuclear DNA. It is noteworthy that the DNA loop size is different in the nuclei of hepatocytes (about 0.62 11m). spermatogonia (about 0.49 pm). spermatocytes of prophase 1 (about 0.56 pm), and spermatids (about 0.34 /em). Hence. the roscttelike globules are obviously the structural elements of both interphase and meiotic chromosomes.

Today there are hardly any investigators ~110 have doubts about the unity of structure and function in biological organization. This notion i\ especially important with respect to eukaryotic chromosomes. Polytene chromosomes of insect\ and meiotic lampbrush chromosomes u’cre historically the first models used for structure functional studies of eukaryotic chromosome organization. the cytological and cytogenetic methods being the main tools in these investigations. The concept ol the chromomer as a structure functional unit of eukaryotic chromosomes and the discovery of it4 loop organization is of fundamental significance. histone-depleted interphase and Later, when metaphasc chromosomes were chosen as an expcrimental model for structureefunctional studies of genetic organization, molecular biological methods were widely applied, The cytological aspect in these investigations became secondary. Little account. II’ any at all. has been taken of cytological data which make obvious the dependence of the revealed

Ultrastructure

of Somatic

and Meiotic

Nucleoids

Fig. 20. Nucleoids of rat spermatocytes of prophase I. Nuclei were treated with Triton X-100 in the presence of Cu” ions. The nucleoid halo in the absence of EDTA treatment (a) contains dense globular elements. Once the nucleoids are subjected to EDTA treatment (b), rosette-like organization of globules becomes apparent. Scale markers indicate 1 I’m.

223

Fig. ?I. of Cu-

A nuclcold of a rat spermatocyte of prophase 1(a fragment) Nuclei were treated with +mm, The isolated nucleoids are treated with EDTA. The globular structure of meiotic orpamzation

of globules are clearly vislhle. Scale marker

chromatin structure upon the experimental conditions for its isolation and analysis (Aaronson and Woo. 198 1: Galcheva-Gargova et d.. 1982: Chentsov ct ui., 1984; Dixon and Buckholder, 1985; Prusov Pt ul.. 1983: Darzynkiewicz rt ui.. 1987). What features of structural organization of cukaryotic chromosomes have been revealed by different research methods and how are they correlated with the results of studies on the organization of histone-depleted chromosomes’? Analysis of the organization of chromatin and histone-depleted interphase chromosomes suggests the presence of numerous sites of anchorage to nuclear envelope (nuclear lamina). According to the loop-domain principle of DNA organization in the nucleus, supercoiled loop-domains are anchored to nuclear lamina. The data reported on DNA organization in histone-depleted chromosomes gave rise to the notion of the loop-domain as the structure functional unit of eukaryotic chromosomes (see Section V.A). Cytological and cytogenetic studies provide abundant evidence that elementary chrotnomers appear as structural units of eukaryotic

Trlton X-100 in the presence chromosome and roscttc-llkc indicates 0.5 ~(rn

chromosomes (see Sections II and Ill). Under the action of strong deproteinizing agents, elementary chromomers form a large chromomeric loop. Given the experimental conditions are less rigid. :I rosette-like organization pattern of elementary chromomers is also observed. Rosette structures found in the histonc-depleted and partially deproteinized interphase. metaphase. and meiotic chromosomes enable us to specify the ultrastructural chromosome organization in more detail. But, as a result, the attachment of chronic+ merit loops (loop-domains) to nuclear lamina. postulated by the loop-domain principle of nuclear DNA organization. becomes rather unclear. This obscurity can be avoided if one supposes that ;tn interphase chromosome is anchored to nuclearlamina at inter-rosette (interchromomeric) regions. The above supposition may have a number of consequences. Firstly, nuclear DNA is likely to be associated with two intranuclear structures, the rosette structure cores and the nuclear lamina. Secondly. the differences in sizes of supercoiled domains and DNA loop-domains appear more comprehensible (see Section V.A). The supercoiled domain revealed in sedimentation experiments is

Ultrastructure

Fig. 22. A fragment

of a rat spermatocyte

of Somatic

prophase

nucleoid

probably a chromosome region located between two neighboring anchorage sites at nuclear lamina and may contain several chromomers (rosette structures). DNA of rosette structures is supercoiled (see Section VI). Under ordinary conditions of isolation and sedimentation analysis of nucleoids (see Section V.A), the unwinding of rosette structures takes place. The supercoiled state of DNA previously maintained and limited by individual loops of rosette structures is now transmitted to inter-rosette regions within two adjacent sites of nuclear DNA anchorage to nuclear lamina. Thirdly, nuclear DNA rearrangement during the cell transition from interphase to metaphase or meiosis may be represented as different combinations of the same structural units, elementary

and Meiotic

Nucleoids

(see legend to Fig. 21). Scale marker

225

indicates

0.5 pm

chromomers, induced by stage-specific factors. When the cells enter mitosis, chromomers tandemly approach each other and form a chromonema, that is followed by the formation of a mitotic chromosome (see Section IV). The observed DNA loops extending outward from the scaffold of a metaphase chromosome are apparently loops of the unwound rosette structures. This assumption can also account for the subunit structure of metaphase chromosomes reported by some authors (see Section IV). During transition of cells into meiosis, nuclear DNA is possibly associated with SC at the same sites which are used by chromosomes at interphase for anchoring to nuclear lamina. The loops, observed in chromosomes at meiotic prophase I under the conditions

M. V. Glarko\

726

of rosette destruction (see Section III), are likely to be equivalent to interphase chromosome regions located between two anchorage sites at nuclear lamina. Within the framework of the above suppositions, one can explain both the larger size of DNA loops at meiotic prophase I compared to that of elementary chromomers, and the presence of several transcription units in one meiotic loop of nuclear DNA (see Section III). The persistent succession of rosette structures in different chromosome types (interphase. metaphase, and meiotic) can account for the similarity revealed in residual nuclear proteins (Wray rt N/.. 1980; Wunderli et c/l.. 1983; Jerardi et ul.. 19X3; Adolph. 1984) and associated DNA nucleotide sequences (Razin CI L/I.. 1979; Mirkovitch rl ~1.. 1988). It seems that the rosette model of chromosome organization is capable of explaining many experimental findings and is in good conformity with the cytological and cytogenetic data on eukaryotes. Also the organizational pattern of transcriptionally inactive regions of interphase chromosomes as rosette structures is not contradictory to the nuclease analysis results on interphase chromatin organization (see Section 1I.C). I believe that further progress in the structure functional investigations of eukaryotic genome organization will be closely connected with elucidation of the genetic content of the rosette structures and a more profound analysis of the function of these intranuclear structures.

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