Ultrastructure in chromatin

4 ULTRASTRUCTURE IN CHROMATIN BEAL B. HVDV

Division of Biology, California Institute of Technology, Pasadena, California

CONTENTS I. I1.

III.

IV. V.

INTRODUCTION

131

HYPOTHESES OF CHROMOSOME STRUCTURE

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1. 2. 3. 4.

132 133 133 137

The ultimate unit The multistranded hypothesis Evidence for a single-stranded chromatid The metaphase chromosome

OBSERVATIONS OF METABOLIC NUCLEI

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1. General considerations 2. Isolated nuclei

139 139

MISCELLANEOUS NUCLEAR INCLUSIONS

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SUMMARY

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ACKNOWLEDGEMENTS

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REFERENCES

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4 ULTRASTRUCTURE IN CHROMATIN BEAL B. HYDE Division of Biology, California Institute of Technology, Pasadena, California

I.

INTRODUCTION

The interpretation of electron micrographs of chromosomes or chromatin is inextricable from the large bodies of information concerning their mechanics, functions and chemistry. Reliance on models derived from techniques other than direct observation is more pronounced in the case of chromatin than for any other subcellular component because order and symmetry are rarely obvious in the fine structure of the nucleus. There are several reasons for this. Substantial evidence from a variety of sources suggests the unit structure of chromosomes is a strand 100A in diameter, which consists of two fibrils 35-40A_ in diameter (Miihlethaler, 1958; Steffensen, 1959; Ris, 1961; Taylor, 1962a, 1962b). These strands probably consist primarily of nucleohistone (Ris, 1962). Relatively enormous lengths of such strands must be packed into the volume of a nucleus or metaphase chromosome of ordinary size (Read, 1961 ; Steffensen, 1961). Such packing requires intricate folding and/or coiling at size levels varying from tens of angstrom units to a few microns. It is not surprising, then, that except for a few specialized cases, little progress has been made in detecting order in the size range between the 100A unit strand and the lower limit of resolution of the light microscope which is about 3000 A. In the so-called resting nucleus where chromatin is not condensed, other complications interfere with the recognition of order. Structure detected at the 100 A level or below must encompass the tenets of genetics which include, beside informational linearity, the mechanism for transferring information into messenger RNA. In addition the precise, semi-conservative self-duplication of DNA occurs at this stage and one must expect eventually to see the orderly evidence of this process. Technical limitations intrude at the 35-40A level. While these dimensions are within the resolving power of present-day electron microscopes they are approaching the limit to which complicated biological material may be reliably fixed and prepared for electron microscopy. This is one reason why the relationship between histone and DNA is not yet clear despite continued studies by both electron microscopy and X-ray diffraction (Huxley and Zubay, 1961 ; Luzzati and Nicolaieff, 1963; Zubay, 1963). Any satisfactory interpretation of the ultrastructure of chromatin must also encompass several other groups of findings. For example, the radiation sensitivity of chromosomes, their susceptibility to chemical mutagens and the repair mechanisms must be explained (Wolff, 1961). No serious explanation of the 131

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process of recombination or segregation has yet been possible at the ultrastructural level. Electron microscopy has not yet provided any insight into heterochromatin, first described in detail and given a functional basis by Heitz (1929), re-evaluated by Cooper (1959) and recently associated with biochemically repressed chromatin (Frenster, Allfrey and Mirsky, 1963). Following also from Heitz but with some recent ultrastructural evidence is the role of chromosomes in the assembly of the n ucleolus (Lafontaine, 1963; Jacob and Sirlin, 1963). This survey, therefore, will have to be selective and will concern itself with problems of chromosome structure which have been dealt with primarily by direct observation. First it will re-examine the solidity of the evidence that the tmit structure of chromosomes from organisms higher than bacteria or bluegreen algae is a strand about 100A across. Second, it will discuss the question whether the chromosome is polytenic or composed of one or perhaps two of these unit strands. Recent work on lampbrush chromosomes will be brought to bear on this question. Brief mention will be made of observations made on condensed chromosomes as well as chromatin from the interphase or metabolic nucleus. It further seems worth while to collect together a list of recent miscellaneous observations of nuclear structures (other than the nucleolus) which may be related to the function of chromatin. Attention will be directed primarily at cells with relatively unspecialized chromosomes or chromatin. Observations of the giant polytene Dipteran chromosomes or the nuclei of sperm, for example, will not be dealt with directly.

II. HYPOTHESES OF CHROMOSOME STRUCTURE I. The Ultimate Unit

The earliest cytological studies of fine structure consisted largely of measurements of the diameter of strands seen in a variety of nuclear types from a variety of biological materials. By 1957 several workers (de Robertis, 1965; Marquardt et al., 1956; Kaufmann and De, 1956; Nebel, 1957) had described filaments in the 30-40 A range, which, as was repeatedly pointed out, is not much larger than the diameter of purified calf thymus DNA (Hall and Litt, 1958). In addition a larger group of observations indicated a strand of a diameter ranging rather widely around 100,~ to be common to almost all nuclei or chromosomes. Ris (see reviews in 1959, 1961 and 1962) has shown that 100~, strands seen in preparations of isolated calf thymus chromatin are composed of two smaller strands if they are appropriately extracted. He (1959) has demonstrated that saline-versene which he considered to remove nonhistone protein will reveal two subunits of 40 A within the 100 A strands. These subunits were interpreted to correspond to the 30 A strands of purified calf thymus nucleohistone characterized by Zubay and Doty (1959). Such strands have also been reported to compose most of the chromatin fraction isolated from pea nuclei (Birnstiel and Hyde, 1963). Ris (1962) was also able to demonstrate two subunit

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strands by extraction with 0.1 i HC1 which is known to extract histone from chromatin. A variety of fixatives other than osmium tetroxide have been tested for preservation of chromosomal ultrastructure (Rossner, 1961 ; Bloom and Leider, 1962; Resch and Peveling, 1962). Goodman and Spiro (1962) show particularly fine detail in the fibrils of Drosophila salivary gland chromosomes following a combination of Carnoy's and osmium tetroxide fixation. Robbins (1961), Claude (1962) and Davies and Spencer (1962) have experimented with the conditions of osmium fixation but no one set of conditions has yet been agreed upon as standard. Glutaraldehyde, which has been shown to preserve plant cytoplasm so well (Ledbetter and Porter, 1963), has not yet been extensively tested on the nucleus. However, nearly all the fixatives mentioned above do, at least sometimes, provide an image quite similar to that produced by osmium tetroxide alone. It does not seem likely then that the present concept of the ultimate unit of chromosome structure will be altered by a new fixative or fixation procedure. 2. The Multistranded Hypothesis

Developing from the above conclusions another generality was advanced. It has been noticed that chromosomes consisted sometimes of fibrils in the 200-250~, range as well as in the 500/~ range (Ris, 1957). Moreover, photographs of chromosomes isolated and shadowed, as well as thin sectioned, were interpreted to indicate that a single chromosome consisted of many parallel strands. This multiplicity of threads combined with the series of sizes ranging from 40 • to 500 ~, suggested to Kaufmann and De (1956) that the early prophase chromosomes of Tradescantia are composed of "as many as 64 identifiable subsidiary strands, assumedly arranged as intertwined pairs to form a hierarchy of pairs of pairs". This idea was supported by the observations of Yasuzumi (1955), Grasse et al. (1956), Nebel (1957), Ris (1957) and Chardard (1958). It was generalized by Kaufmann et al. (1960) and ingeniously systematized by Steffensen (1959, 1961) and Read (1961). As a result of Steffensen's model to explain the duplication of a multistranded chromosome this hypothesis has been tagged as the "rope hypothesis". 3. Evidence for a Single-stranded Chromatid

From the beginning, however, certain considerations weighed against the multistranded hypothesis. Geneticists and chromosome cytologists alike were reluctant to face the genetic and mechanical complexities inherent in a multistranded chromosome. Observations supporting a single-stranded chromatid are still fragmentary but while they do not yet lead to a complete understanding of all chromosomes in all stages of meiosis and mitosis they are tending to converge. Although highly interrelated they must be taken up successively in what is hopefully a logical sequence. Several reviews and critical discussions together with models for chromosomes consisting of single-stranded chromatids have been published (Freese, 1958; Gall, 1958; Swift, 1962; Taylor, 1962a and b).

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To return to strand diameters it can be stated that a fibril of desoxyribonucleohistone size has been observed by a sufficient number of investigators in a sufficient variety of cell types to consider it established as a regular component of chromatin. Interpretations of the 100A strand vary, however, and it is quite possible that more than one kind of 100 A strand exists, depending on the stage of duplication of the chromosome as well as its functional state. Amano et al. (1956), for example, interpreted the 100A strand observed in the metabolic chromatin in plasma cells, monocytes and leucocytes of the mouse as a helix composed of a single 20-30 A strand. On this basis he suggested a model of a chromatid which assumed that strands of all sizes to the light microscopic level were composed of a single 20-30 A strand coiled in a series of helices each with a successively larger gyre diameter. Bopp-Hassenkamp (1959) came to a similar interpretation of the meiotic chromatin of plants, but as will be seen below her observations were incomplete. Genetic considerations based on the overwhelming evidence for linear genetic information residing in a linear DNA molecule always lead to the hope that the chromosomes of higher plants and animals might, like the chromosomes of bacteria and viruses, be composed of a single nucleohistone strand or at least linear segments of nucleohistone joined by segments of protein into a single strand. Thus the chromatid of classical cytogenetics, which arises by selfduplication of the chromosome, would also be single stranded and all complexity seen in electron micrographs above the 40A or possibly 100A level would be the result of coiling or packing of the single strand. Recent work by Gall (1963) strongly supports the above hope. The system, recently reviewed by Callan (1963), is the vertebrate lampbrush chromosome. The logical basis for the recent experiments was laid in a balanced and thoughtful review (Gall, 1958). The results are deemed crucial enough to merit discussion in some detail. The lampbrush chromosomes of Triturus viridescens are in the diplotene stage of meiosis and thus are paired although joined by chiasmata at only a few points. Each chromosome possesses an axis along which chromomeres are located. From the chromomeres project paired loops. Stretching experiments indicate the chromosome to be composed of two continuous strands (Fig. 1). The dimensions of these chromosomes are relatively enormous. The length of the intact structure may equal l mm and that of a fully extended chromatid probably is of the order of 5 cm (Gall, 1958). Gall reports the interchromomeric axis to have a diameter of 200-400A in electron micrographs suggesting that the chromatid is 150-200 A. A particular pair of these chromosomes was placed in a solution of DNAase and the rate at which the loops, as well as the interchromomeric axis, broke was recorded. It was assumed that for a break to occur, all the D N A chains in a strand would have to be hydrolyzed at very nearly the same level. The kinetics of the breakage strongly indicate that loops consist of a single D N A double helix, while the interchromomeric axis consists of two D N A double helices. Thus the data suggest the basic linear component of the chromatid to be either a continuous strand of DNA or segments of D N A joined end to end by protein or some other type of linker.

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IN CHROMATIN

It is highly unlikely that the lampbrush chromosomes of vertebrates are unique, or that however specialized functionally (Izawa, Allfrey and Mirsky, 1963b), their structure is fundamentally different from that of smaller, more condensed chromosomes of the same and other organisms. It is also important to point out that Ris (1961) examined Triturus lampbrush chromosomes fixed in several ways and interpreted the ultrastructure of the loops to be "coiled 200 A

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@ FIG. 1. Above--drawing of a segment of lampbrush chromosome from an oocyte of the newt, Triturus. Pairs of lateral loops project from an axis of Feulgen-positive chromomeres. Below--postulated structure of a pair of lampbrush chromosome loops. The loop consists of ribonucleoprotein matrix surrounding a very delicate DNA fibril. (From Gall, 1963c•) fibrils associated in two bundles which themselves are coiled". These conclusions juxtaposed with those of Gall indicate that dimensions alone are not good indications of the basic genetic structure of these chromosomes• This is probably particularly true when they are genetically active as is the case with lampbrush chromosomes and the chromatin of the metabolic nucleus. A chromosome structure related to that of the lampbrush chromosome is the synaptinemal complex (Fig. 2) first described in the meiotic prophase nuclei of salamander by Moses (see a later review 1958b). Such complexes have been recently described in all other animals and plants (Chardard, 1962; Ris, 1962)

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where meiosis has been carefully observed.* Their interest lies in their direct association with synapsis (Moses, 1958a), genetic crossing over and their resemblance and probable structural similarity (Grun, 1958) to lampbrush chromosomes. So far, like lampbrush chromosomes they have been convincingly demonstrated only in meiotic prophase. Moses (1958b), Sotelo and TrujilloCenoz (1960), and Chardard (1962) all believe that before the pairing process begins, individual chromosomes have taken their pairing form, i.e. with a main axis having lateral projections or loops, and that the synaptinemal complex is a product of the union of two such chromosomes. Moreover, Roth (1958) showed that the core, characteristic of the synaptinemal complex, persists until metaphase in the snail. Two recent studies of the synaptinemal complex (Nebel and Coulon, 1962a and 1962b; Coleman and Moses, 1963) describe the effect of enzymatic hydrolysis on their structure. The former authors fixed the testes of the pigeon in formol-phosphate and extracted them with RNAse, DNAse, and trypsin. Their observations suggested to them that the lateral loops were largely hydrolyzed by DNAse, became contracted with RNAse while the axis remained relatively resistant to all enzymes separately or together. Coleman and Moses emphasize, on the other hand, that DNAse extraction does not materially affect the structure of the synaptinemal complex. Their extraction of DNA was carefully checked by spectrophotometric and histochemical methods and they conclude that DNA alone is not by itself, in fixed material, responsible for the structure of the chromosome. Both sets of authors agree on the probable concentration of protein along the axis. Gay (1963) also using proteolytic enzymes as well as DNAse on fixed and unfixed chromosomes concluded that chromosome structure is not dependent on D N A alone. However, the fact that the unfixed isolated lampbrush chromosomes are so susceptible to fragmentation by DNAse (Gall, 1963) could be explained by the hypothesis of Huang and Bonnet (1962). Their experiments (see also Barr and Butler, 1963; Huang, Bonner and Murry, 1963) show that certain histone fractions when carefully complexed with D N A suppress the capacity of DNA to support RNA synthesis in in vitro systems. Native nucleohistone does support R N A synthesis to some extent. Extrapolation of these findings to chromosomes suggests that a chromosome region which is supporting RNA synthesis may not be occupied by histone. Such might be the case in an intensely active RNA synthetic system such as the lampbrush chromosome (Gall and Callan, 1962). Further support for such an explanation comes from the work of lzawa, Allfrey and Mirsky (1963a). They showed that the loops of isolated lampbrush chromosomes can be made to retract in the presence of substances (actinomycin D and certain histone fractions) which are known to complex with D N A and to suppress the synthesis of RNA. * Bopp-Hassenkamp (1959) observed meiotic prophase nuclei in several higher plants but failed to see the synaptinemal complex in any of them including the lily in which it has been demonstrated by Ris. Her hypothesis as well as that of Marquardt (1956) concerning the ultrastructural organization of meiotic chromosomes can apply at best, therefore, only to the lateral projections of the synaptinemal complex.

FIG. 2. Synaptinemal complex from primary spermatocytes of the pigeon. Black and white arrow points to typical tripartite structure associated with paired meiotic chromosomes. Black arrow points to lateral projections interpreted by some authors as loops. (From Nebel and Coulon, 1962a.) \C36,500.

FIG. 3. Stereoscopic pair of photographs of area of chromatin in cell of pea root tip meristem. Chromatin and "interchromatin" areas are present. Helical strands of 60~, size may be seen. Arrows indicate strands which are double when observed with 2X stereoscopic viewer. (Viewer may be obtained from Abrams Instrument Corp., Lansing, Michigan, U.S.A.) Fixed in 1 per cent buffered OsO4, embedded in Epon and post-stained with uranyl acetate and lead hydroxide. ~ 79,000. FiG. 4. Stereoscopic pair of photographs of area from nucleus isolated from 3-day embryonic axis of pea. Arrow indicates strands which are clearly double when observed with stereoscopic viewer. Pellet fixed in 1 per cent unbuffered OsO4 embedded in Epon and post-stained with lead hydroxide. /79,000.

FIG. 5. Pattern of strands thought to be composed of DNA from preparation of isolated nuclei suspended in 4M ammonium acetate and prepared for electron microscopy by the method of Kleinschmidt. 35,000. FIG. 6. Figures formed by strands of D N A obtained from preparation of week-old suspension of isolated pea nuclei in 3 M ammonium acetate and prepared by the method of Kleinschmidt. The strands making up the smallest flower-like figure (upper left) is approximately 8 Ft in length. Difference in strand diameter in the two photographs in Figs. 5 and 6 is thought to be due to differences in the shadowing process rather than representing any effect of aging or salt concentration. :,: 37,000.

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structure in thin section of nucleus from pea root FIG 7. Honeycomb-like meiFistem cell. Nucleolus lies at top half of photograph, chromatin at the bottom. Fix ed in buffered 0~04, embedded in Epon and post-stained with uranyl acetate and lead hydroxide. x 89,000. FIG;. 8. Nuclear body from thin section of pea root meristem cell prepared as in Fig . 7. Note resemblance to nucleolar interior (lower left). Differences between nut :leolar body and nucleolus are closer packing of dense granules and lack of peripheral particulate area. ;. 15,000.

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Reports of chromosomes with axial or lampbrush structure in other than meiotic cells are scattered. Nebel and Coulon (1962) claim that chromosomes in all stages of mitosis in the onion and barley cell show lampbrush structure but have not as yet documented these observations. Yasuzumi and Sugihara (1962) present several electron micrographs of mitotic prophase chromatin in Ehrlich ascites tumor cells which appear to have a paired axial structure. As an alternative to thin sectioning Gall (1963) spread nuclei of Triturus erythrocytes, human metaphase chromosomes and grasshopper meiotic prophase chromosomes on a water surface to bring the entire chromosome as nearly as possible into one plane. He examined preparations made in this way both shadowed and stained. He observed only uniform strands about 400-600 A in diameter with a 150 A core of slightly greater density. Bader et al. (1963) observing thin sections of cultured human metaphase chromosomes also report two 400A axial components. Schl6te and Schin (1962) have interpreted ultrastructure in thin sections of grasshopper spermatids, however, to indicate a combination of both polytene and lampbrush structure. These findings must await further confirmation but it now seems possible that the chromosome may possess a fine structure quite appropriate to the chromosome postulated by the requirements of genetics, chromosome mechanics and the self-duplication of the chromosomes. 4. The Metaphase Chromosome

The metaphase chromosome is of particular interest in the developmental anatomy of chromatin for two reasons. First, it is generally considered to be genetically inert (Mazia, 1961) and, like the nucleus of the sperm head, represents a convenient package for transport of genetic material. Therefore its ultrastructure could be predicted to be relatively free of any accessory machinery associated with the synthetic activity of chromatin. The silhouettes of electron scattering material of metaphase chromosomes which are the data of electron microscopists, should represent the informational and fundamental structural elements of chromatin alone. Secondly, the chromosome occupies its smallest volume at metaphase and each chromosome is separable from the others. These conditions also apply to anaphase and possibly also to early telophase, although at the latter stage there is good evidence that functional activity is again resumed (Lafontaine and Chouinard, 1962). The packing of the ultimate genetic fibrils in the metaphase chromosome then will be partly a function of the structure of the chromosome as a whole, i.e. whether it is a bundle of strands or whether it is perhaps of the lampbrush type. The packing, which is largely a matter of coiling will also be a function of the relationship at the molecular level between the DNA and the histone and perhaps other protein or even lipids (LaCour and Chayen, 1958a and b; Busch and Davis, 1958). Zubay (1963) has speculated that histone may not only be wrapped around DNA but also may form cross links between the gyres of DNA coiled at the next level above the Watson-Crick helix. Most reports of mitotic and meiotic metaphase chromosomes have yielded no evidence for an axial or lampbrush structure. Porter (1958), for example, shows metaphase chromosomes which are composed of a mesh of strands without order.

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Chardard (1960) presented several electron micrographs of thin sections of meiotic metaphase chromosomes of orchids. These sections reveal only many coiled 100A strands also without higher organization. Similar pictures of mitotic metaphase chromosomes of onion (Albersheim and Killias, 1963) and cultured mouse sarcoma cells (Dales, 1960) have been published. Dales presented in the same report evidence that metaphase chromosomes will disintegrate when digested with DNAse. Barnicot and Huxley (1961) show electron micrographs of whole metaphase chromosomes from cultured human tissue fixed in aceticalcohol but do not find any recognizable order just below the resolution of the light microscope. Somers (1963) working at the light microscope level has shown that metaphase chromosomes of cultured Chinese hamster cells may be uncoiled by removing Mg and Ca from the suspending medium. Such modified chromosomes will be interesting to observe at the level of resolution of the electron microscope. Chorazy et al. (1963) working also at the light microscope level with isolated unfixed mammalian metaphase chromosomes confirm the importance of Ca in maintaining their structure. In addition they report that urea and deoxycholate will disperse the condensed chromosomes. The enzymes pepsin and RNAse separately or together fail to disrupt the metaphase chromosomes, while chymotrypsin, trypsin or DNAse cause rapid (10-20 min) and complete disintegration. This kind of experiment indicates strongly that condensed chromosomes owe their structure almost exclusively to a complex of DNA and a basic protein. It is clear nonetheless that certain cations play a role in the structure of chromosomes (Steffensen, 1961) perhaps by affecting the tertiary structure of nucleohistones (Anderson, 1956; Peacocke, 1960). Observations of the nuclei of several protozoan species have provided tantalizing images of chromosome structure. Comment upon them is included in this section because the chromosomes remain at least partially coiled even in the interphase nucleus. Cleveland (1949) described one example of this kind of coiling cycle at the light microscope level in certain parasitic protozoans. At the electron microscope level, helical, Feulgen-positive structures have been described in the interphase nucleus of amoebae (Pappas and Brandt, 1958, and Mercer, 1959). The helices are arranged, bristle-like, around a central axis. The strand forming the helix is 120 A in diameter and appears to be double. The nuclei of several dinoflagellates have also been studied. These chromosomes (Giesbrecht, 1962, and Ris, 1962) resemble those of bacteria in appearance and also in their lack of an associated basic protein (Ris, 1962). Dodge (1963) interprets his electron micrographs of Prorocentrum chromosomes as showing coiled bundles of strands. The unit is a 150A strand composed of a pair of 40-60A fibrils. Afzelius (1963) shows somewhat similar chromosomes in the nucleus of Noctiluca but does not interpret their structure. However, several of his chromosome images as well as those of Dodge and Pappas and Brandt are at least as indicative of a lampbrush type of structure as they are of a multistranded structure.

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III. OBSERVATIONS OF METABOLIC NUCLEI 1. General Considerations

Electron microscopy of the metabolic or interphase nucleus has provided relatively little information about chromosome structure. Aside from the fact that at this stage chromatin appears to be dispersed into strands of a size not far above the resolution of the electron microscope for biological material, this is the stage at which DNA is reduplicated and also the stage at which the genetic function of the chromosomes is carried out. It is not unreasonable to suspect that the extended, uncoiled structure of interphase chromatin reflects, as it does in the case of puffs in giant dipteran salivary chromosomes and the loops of lampbrush chromosomes, its activity. The machinery for DNA synthesis, messenger RNA synthesis and structures associated with interchange between chromatin and nucleolus (Rho and Bonnet, 1961) as well as nucleus and cytoplasm will always have to be borne in mind when interpreting the ultrastructure of interphase chromatin. All these activities must be carried on without altering the linear integrity of the ultimate genetic informational unit. The closest approach to a correlation of one activity of the interphase nucleus--the synthesis of DNA--and its ultrastructure is a recent study by Hay and Revel (1963). By autoradiographic techniques these workers located the regions of the interphase nucleus in which H a thymidine was incorporated. The ultrastructure of chromatin in these regions was carefully studied electron microscopically. The conclusion is reached that the actively synthetic DNP of the resting nucleus as well as the condensed chromosome is a three-dimensional mesh made of strands 50-70,~ in diameter. In the interphase nucleus the DNP gel is more dispersed and associated with a granular component; that in the anaphase chromosome is more compact, denser and without a granular com~ ponent. No attempt is made to relate the strands of the gel with any of the higher orders of organization of the chromosomes. Yasuzumi (1960) shows a similar mesh in the nucleus of amphibian erthyrocytes. 2. Isolated Nuclei

Another approach to the study of the interphase chromatin has been to observe the ultrastructure of isolated nuclei (Crawley and Harris, 1963; Maggio et al., 1963; Hyde, 1963). Certain possible advantages might be expected from studying isolated nuclei. Already a good deal of information has accumulated about the synthetic capacities of nuclei isolated in various ways from several organisms (Mirsky and Osawa, 1961 ; Birnstiel et al., 1962; Rho and Chipchase, 1962; Chipchase and Birnstiel, 1963; Allfrey, 1963), indicating that the isolation process does not noccssarily destroy their organization. Secondly, so far as chromatin is concerned, the isolation process may be expected to remove soluble substances in the nuclear sap which in in situ nuclei might obscure details

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of ultrastructure. On the other hand it is far from clear in what ways the various isolation procedures alter the nucleohistone complexes of the interphase nucleus. Some beginnings on this problem have been made by Crawley and Harris (1963). Their procedure for isolation of Hela cell nuclei causes swelling of the nuclei although they conclude that the swelling is not osmotic. Thirty per cent of the dry mass of the nucleus is lost during isolation as judged by interferometry. By using models they conclude that the fine structure of their nuclei is entirely filamentous--no granules are present. Their filaments are 30-50 A in diameter in methacrylate embedded material while they measure 20-30/~ in Araldite embedded material. Maggio et al. (1963a, b) have prepared beautifully clean pellets of nuclei isolated from guinea-pig liver. The electron microscope image is quite similar to that of Crawley but the filamentous material is of larger diameter--50-100/~. However, a subfraction of the nucleus, which they interpret as being chromatin appears to be a mass of filaments 25-30 A in diameter. In this laboratory the ultrastructure of chromatin of isolated interphase nuclei of peas has been studied by stereoscopic technique (Hyde, 1963). We hoped that isolation might result in a loss of extra chromosomal material which might be expected to confuse the image in ordinary tissue sections. Figures 3 and 4 are respective stereoscopic photographs of thin sections of in situ and isolated chromatin of the pea embryo. Without recourse to a stereoscopic viewer one can see in both a mesh of strands quite like that described by Hay and Revel and others. In Fig. 3 it may be seen that the strands commonly appear double not only in the chromatin area, but also in the "interchromatin" regions. Where doubleness is clear the strand measures approximately 60/~ in diameter. Most strands, however, are coiled to some extent and are, therefore, thicker and are not clearly double. In Fig. 4, an area of chromatin from an isolated nucleus, the chromatin mesh is much looser. Indeed, almost the entire chromatin mass of the nucleus is a homogeneous mesh of this kind. With the exceptions to be described below and in a later section, no distinction can be made between chromatin and interchromatin areas (Swift, 1959) and no differentiation of the chromatin into chromocenters (Peveling, 1961; Rossner, 1961) or heterochromatin (BoppHassenkamp, 1959; Yasuzumi, 1960) is evident. Where doubleness is apparent, the strands are about 75/~ in diameter but the paired, slightly helical substrands measure, as they do in in situ nuclei, 20-30/~ in diameter. These observations confirm Ris's (1959) findings concerning the structure of nucleohistone extracted from thymus nuclei. In addition to the strands which compose most of the nucleus Ris (1962) has described dense fibrils 250 A in diameter which are both DNAse and RNAse resistant. Such strands are also evident in isolated pea chromatin particularly around the nuclear bodies (Sankaranarayanan and Hyde, unpublished) to be described in Section IV. Ris suggests these fibrils are "residual" chromatin and points out their continuity with the more common 100 A strands.

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A further attempt to study the chromatiu of isolated pea nuclei utilizes the method developed by Kleinschmidt (1962) for studying the DNA of viruses and bacteria. If the DNA of interphase nuclei could be spread essentially on one plane, information on two questions might be obtained. First, how long are the DNA molecules in chromatin and second, what relation do the molecules have to one another ? Extensive twisting or tangling might give support to the polytene hypothesis of chromosome structure. This was accomplished by injecting suspensions of nuclei isolated by the method of Rho and Chipchase (1962) into 1, 2, 3, and 4 M solutions of ammonium acetate which can be expected to remove most of the histone from the DNA molecules. Cytochrome C is added and the suspension is spread on the surface of distilled water. The appearance of the preparation varies with the molarity of the ammonium acetate and the age of the suspension (Ris and Chandler, 1963). Figure 5 shows a typical pattern of strands obtained from a flesh suspension of nuclei in 4 M ammonium acetate. The DNA strands are at least tens of microns long and run more or less linearly in different directions. The strands show no orderly relation to one another. Figure 6 shows a typical pattern obtained from nuclei suspended in 3 M ammonium acetate. Such figures can be obtained from any sample of DNA prepared by the method and are not characteristic of pea chromatin. They may, however, give some idea of the length of the DNA molecules although such figures are frequently catenated to the extent that measurement is difficult. In addition the number of free ends is difficult or impossible to ascertain. Measurement of such figures (assuming each with two or fewer free ends in a single strand) suggests that the DNA can break into finite lengths. Until more is understood about the changes caused by the Kleinschmidt procedure, however, no reliable deductions about the arrangement of DNA in the chromosomes can be made from patterns observed in these preparations. Nonetheless, the DNA in Fig. 5 is remarkably free of twisting or tangling. This suggests long lengths of DNA in the chromsome are easily separable from other strands. Other observations have been made on the nucleoprotein gel of nuclei. The purpose has been to determine, if possible, how the histone or non-historic protein contributed to lateral or end-to-endlinking of DNAmolecules. Anderson and Fisher (1960) have studied viscosity changes in rat-thymus nuclei suspended in 1 MNaC1 induced by DNAse, changes in pH and X-rays. Their results are in accord with the idea that DNA molecules are strongly linked end-to-end by protein and more weakly crosslinked laterally by protein. Dounce and Sarkar (1960) also conclude that DNA molecules are held together end-to-end by covalently linked non-histone protein. Zubay (1963) on the basis of X-ray diffraction patterns and electron micrographs, has proposed that oriented nucleohistone gels are composed of longitudinal DNA molecules with lateral histone bridges lying at 60 ° to the DNA molecules and lying in the large groove. He extends this model to chromatin by suggesting that super coils of DNA are stabilized laterally by similarly oriented histone bridges.

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IV. MISCELLANEOUS NUCLEAR INCLUSIONS From time to time in recent literature there have appeared articles and comments upon structures in the nucleus, other than the nucleolus. These structures, quite diverse in appearance, do not resemble ordinary chromatin, yet do not appear to be unusual or pathogenic in nature. These structures fall into two general, probably not mutually exclusive, groups. The first group (Buvat, 1963) is small and appears to represent specialized forms of chromatin. Rudsinska and Porter 0955) reported a honeycomb-like structure for certain chromatin bodies of the macronucleus of the protozoon, Tokophrya infusorium. Actually the structure is that of a honeycomb with elongated cells, the greatest diameter of which is about 280 A. Such a structure has also been described in the large egg nucleus of the gametophyte of Pinus laricio by Camefort (1959). However, the dimensions are reported to be slightly smaller. We have observed such a structure (Fig. 7) a few times in cells of the apical meristem of the pea Pisum sativum. The repeating period across a longitudinal section of the honeycomb is about 250 A which is within the same range as that described in Pinus. The fine structure of the honeycomb appears to be fibrillar and is not unlike the neighboring c ~ o m a t i n in appearance except that it is more densely packed and ordered. Nothing is known of the chemical nature of these structures, however, and it is too early to speculate on their meaning. However, a honeycomb of similar dimensions has been described by Yasuzumi (1957) in the spermatid nuclei of the grasshopper. Here the entire nucleus is of this structure and it seems quite certain that it represents one form of chromatin. The second group includes particles or clusters of particles in some cases definitely proven to be RNA-containing, which may be products or centers of nuclear synthetic activity. Four types of particles have been discussed in some detail recently by Bernhard and Granboulan (1963) and Swift (1963) The first are RNA-containing particles approximately 150 A in diameter, widely described in both plant and animal nucleoli. They probably owe their origin to the activity of the chromosomes (Lafontaine, 1958; Lafontaine and Chouinard, 1963, and Jacob and Sirlin, 1963). In the nucleoli of peas they are slightly smaller than cytoplasmic ribosomes but appear to be similar in being composed of fine fibrils (Hyde and Sankaranarayanan, 1964). Another group of particles have been named "perichromatin granules", by Watson (1962). These granules are about 300 A in diameter and are separated from the chromatin in which they are usually partly or wholly embedded by a clear zone about 250 A thick. They have been observed in several species of animals and in plants. Watson suggests they may contain both D N A and RNA. A third particle, the "interchromatin granule", has also been described from both plants and animals. These lie in the spaces between masses ofchromatin, often in large dusters. They vary from 200-500/~ in size and are susceptible to RNAse. The fourth type of granule occurs in clusters and is RNAse resistant. They measure only about 100A in diameter.

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Neither the function nor the origin of the last three types of particles is known. Weber and Frommes (1963) have called attention to what may be another class of nuclear bodies. These bodies are somewhat smaller than nucleoli and several may be present in a single nucleus but they do not occur in the nuclei of all cell types. In the ox, they are spherical and have a diameter of about 1/~ and contain in a central core dense particles 100-300/~ in diameter. Similar structures have been seen in other animals and Weber and Frommes suggest they are not unlike the prenucleolar bodies described in plants by Lafontaine (1958) and Lafontaine and Chouinard (1963). The latter structures appear next to telophase chromosomes and fuse to form the nucleolus. Falk (1962) has described similar bodies in the interphase nuclei of Allium cepa. Although in Falk's photographs the bodies strongly resemble nucleoli, he suggests they might be akin to "puffs" in Dipteran salivary chromosomes. The same structure (Fig. 8) occurs in the root meristem cells of peas (Birnstiel and Hyde, 1963 ; Bouck, 1963; Hyde, 1963 ; and Sankaranarayanan and Hyde, unpublished). These structures are sometimes associated with the nucleolus and sometimes lie amongst the chromatin of the interphase nucleus. They are spherical and contain dense granules of 350-500 A in diameter and while their fine structure bears a resemblance to the interior of the nucleolus, they are always distinguishable from the nucleolar substance. Enzymatic extraction of preparations of isolated nucleoli which also contain these bodies suggests that they owe their structure to both RNA and DNA (Sankaranarayanan and Hyde, unpublished). These fragmentary observations of intranuclear structures suggest that ultrastructural studies may make an appreciable contribution to knowledge of nuclear function. They also support the hope that structures associated with genetic activity of chromatin may be distinguished from structures associated with stable genetic information.

V. SUMMARY A survey of recent studies of the ultrastructure of chromatin from higher plants and animals suggests that a unit strand of 50-120 A in diameter is common to all. Variation in the diameter measurements may be attributed to variations in the extent of coiling of this strand. The unit strand is composed of two substrands which are also helically disposed and which are of nucleohistone diameter (20-30/~). The substrands are easily separable from one another over long distances ( > 10/0. The arrangement of long lengths of the unit strand in the chromosome remains unknown. Nevertheless experimental studies of fine structure in lampbrush chromosomes and thin sections of meiotic chromosomes of both plants and animals increasingly suggest that the chromatid consists of one unit strand. Reports of various granules and specialized regions of the chromatin which are thought to be sites of functional activity are also discussed.

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ACKNOWLEDGEMENTS T h e w o r k f r o m this l a b o r a t o r y r e p o r t e d in this article was s u p p o r t e d by National Institutes of Health, Public Health Service Grant No. GM-03977. T h e a u t h o r is i n d e b t e d to P r o f e s s o r s J a m e s B o n n e r a n d A l a n J. H o d g e for advice and encouragement.

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