Tuatara: Volume 12, Issue 2, July 1964
The Cell Nucleus
The Cell Nucleus
The chromosomes of the cell nucleus, and mitosis, the process of cell division, are apt to dominate one's thinking when confronted with the term ‘cytology’. This is by no means the fault of the reader or hearer alone for before the advent of the electron microscope cytologists were largely engaged in a study of chromosomes; the cytoplasm was often regarded as having little if anything new to offer its investigators. The reason of course was that the resolving power of the ordinary light microscope was limiting. It was not until the discovery of the electron microscope less than 20 years ago that a break-through came and careful attention was turned again to other-than-nuclear structures. To the cytologist the electron microscope increased resolution beyond belief bringing startling new fields for research into discernment of the human eye. Today the balance is tipped, and the cell cytoplasm with its now revealed array of organelles and membrane structures is the centre of attention of many cytologists. In fact the chromosomes of the nucleus are often looked on as too large for the electron microscope, and it is only very recently that electron microscopists have begun looking anew at these unique structures.
In respect of new discoveries mention must be made too of the great contributions that phase-contrast and birefringence microscopes, and the use of radiography have recently given cytology. To be able not only to see various components in a living cell but also to ‘see’ certain molecules in this cell and to follow others in their cellular passage and metabolism has been of tremendous importance.
‘Cytology’ from the Greek Kutos meaning ‘a vessel’ is the study of cells. Karyology is a specialised branch of cytology dealing with the cell nucleus. This branch of science is over a century old. Since the nucleus was discovered by Robert Brown in the early 1830's, and soon after recognised as a normal and characteristic cell component, much literature dealing with this subject has of course been published. I am attempting here to give an overall picture of the nucleus as it is seen today, and to give a brief outline of what is known of the functioning of the nucleus and its importance to the cell as a unit. Emphasis page 82 is given to important findings of recent years on issues that were previously in doubt or unknown. The article will be divided into two parts. Part 1 will deal with the general features of the nucleus and the structure of its components. Part 2 will cover the cell in division — mitosis — and will appear in a following issue of Tuatara.
Nearly all living cells possess a nucleus, for this body is the controlling centre, the ‘brain’, of the whole cell. A few living cells such as human red blood corpuscles have no nucleus. These, however, are very specialised cells; their nuclear loss during differentiation of a specialised structure and function is parallelled by a loss of nearly all the major functions of protoplasm. More correctly, perhaps, it should be said that most cells possess nuclear material, for a number of organisms are devoid of the distinct structure we generally associate with the term nucleus. Bacteria, for instance, have a less dense core (under the electron microscope) of material surrounded by a jacket of cytoplasm; the core is the nuclear material and may be equated with chromatin, the major functional and structural component of nuclei in general. Viruses (not strictly cells) consist simply of nuclear material surrounded by a protein coat.
With a microscope, a slide and a coverslip it is easy to see, if only to see, a cell nucleus. With your fingernail scrape a portion page 83 of the cells lining your palate and smear them onto a slide. Cover the cells with a drop of saliva and a coverslip, and examine under the microscope. The large epithelial cells have a small, but quite distinct central nucleus.
The living nucleus cannot always be seen as easily as those of palatal epithelial cells, for the nucleus commonly exhibits optical properties identical with those of the cytoplasm. Under the phase-contrast microscope, however, particularly at a time when the cell is preparing to divide, the living nucleus is readily visible. This fact has enabled important comparisons to be made with stained material, and these comparisons have shown that careful fixation and staining give a reasonably clear and correct representation of the internal structure of the nucleus.
The form of the nucleus is generally ovoid (Fig. 1) though various diverse shapes arise in cells with specialised metabolic functions. Larval insects, for example, have much branched nuclei in their cocoon spinning gland cells.
There may also be more nuclei in a cell than the usual one. Multinucleate organisms as some fungi and algae and most voluntary muscle cells have many nuclei distributed throughout their cytoplasm. The giant amoeba Chaos has many nuclei in its single cell, while such unicellular ciliates as Paramecium commonly have a large macronucleus and a number of small micronuclei. Human red blood cells we have already noted are enucleate. The number of nuclei possessed by a cell is probably closely related to the mass of surrounding protoplasm, since within certain limits a definite nuclear surface area: cytoplasmic volume ratio must be maintained for continued functioning of the cell as a whole.
The position of the nucleus is quite variable and is largely determined by the physical features of its surrounding cytoplasm. In a young cell it ordinarily occupies the centre of the cell (Fig. 1), but as the cell becomes vacuolated during differentiation it is commonly displaced, with the cytoplasm, to the side of the cell. Position is possibly related to function for it often lies in regions of high metabolic activity.
A nucleus not visibly undergoing division is referred to as a resting, interphase or metabolic nucleus. The term ‘resting’ implies inactivity, at least as far as cell division is concerned: in dealing with mitosis in the following article it will be clearly shown that this is not true; the ‘resting’ nucleus is indeed very actively associated with division. The term ‘interphase’ is descriptive though suggestive of a phase in which only certain features of cell division occur. This is true in some respects for we know today that ‘interphase’ is the principal stage of reproduction rather than division. The term ‘metabolic’ suggests that this phase is one of major metabolic activity in the nucleus. page 84 This is so, firstly in relation to its controlling action in the cell and, secondly, in its activity in building up material essential to the dividing phases. Indeed there are indications that while the metabolic rate of the non-dividing nucleus is very marked, this activity ceases to a large extent once the cell is visibly dividing; and some aspects of the metabolic activity of the cytoplasm are comparatively low as well.
The combined term ‘interphase-metabolic’ used here refers to the nucleus of a young (meristematic) cell as distinct from that of a differentiated cell. The latter, though metabolic, (as above) appears incapable of normally entering division and, therefore, is not strictly interphasic.
Cells which have been appropriately killed (fixed) and stained show the nucleus to be composed principally of two phases, a nucleoprotein or chromatin phase dispersed throughout an essentially protein mass, the nucleoplasmic phase. The chromatin is generally responsible for the staining properties of the nucleus and imparts to it affinities for a wide variety of dyes. One of these dyes, a very important one, is basic fuchsin (Feulgen's stain); it is specific for deoxyribonucleic acid (DNA), the main acid portion of chromatin and that portion now known to be the hereditary material of an individual. The specificity of the dye has enabled research workers to make accurate estimates of the quantity of DNA in a particular cell; the results obtained were an important early pointer to the identification of this acid portion of chromatin as the carrier of genetic information.
During the early stages of nuclear division the chromatin of the metabolic nucleus becomes transformed into a fixed number of individualistic bodies. These are the chromosomes (Figs. 4-7). Also within the nucleus, one or two (sometimes more) rather large, deeply staining bodies known as nucleoli can usually be seen (Fig. 1). These bodies are formed at particular regions of the chromatin and it is probably best to regard these organelles as specialised portions (with specialised functions) of the chromatin phase.
The nucleus is bounded from the cytoplasm by a nuclear membrane.
The Nuclear Membrane
The electron microscope has been very useful in revealing the finer morphology of the nuclear membrane (nuclear envelope) (Fig. 2) and has given at least an indication of how it is formed after the nucleus has divided. Indications are that the membrane is a specialised cytoplasmic structure, and it was therefore described in detail in an earlier article in this series (Sampson, Tuatara 11/3). Apart from morphology, however, the puzzling question of its page 85 function is still largely unanswered. Micro-puncturing of the nuclear membrane is fatal (in contrast to similar treatment to the plasmalemma). Its presence as an intact structure is essential. Quite remarkably, though, it disappears during the early stages of cell division to be reformed round the daughter nuclei at the close of division. One can merely speculate carefully and note that the nuclear membrane is probably responsible for creating a specialised intranuclear environment, different from that of the cytoplasm, and on which the controlling actions of the nuclear components depend. The membrane pores would allow a controlled nuclear/cytoplasmic interaction which, as will be noted later, is so essential for nuclear functioning. There is probably some connection between the dissolution of the nuclear membrane during cell division and the very marked retardation of metabolic activity, which we have already noted is clearly evident at the onset of actual division.
The nucleolus (Figs. 1, 2 and 3) is often the only conspicuous organelle in the living meristematic cell. This fact led to the early discovery and description of the nucleolus as a major component of the nucleus.
When stained nearly all nuclei show the presence of one or a few nucleoli. The actual number depends not only on the species, but on the metabolic activity of the cell as well, for while the number is generally constant in meristematic cells, nucleolar fusion or budding paralleling differentiation and metabolic activity may considerably alter this number. In young cells where modification in number has not occurred this basic number of nucleoli is an indication of the number of sets (see later) of chromosomes present in the cell, and also a reflection on the mode of formation of the nucleolus. The nucleolus disappears (perhaps more correctly, disperses) at the earliest phases of cell division and is reformed (reorganises) in the closing phases. Reformation takes place at specialised regions on certain chromosomes of the complement (Fig. 3). The number of these so called organising chromosomes is constant under normal conditions and hence so is the number of nucleoli formed.
Under the electron microscope the nucleolus is seen as an aggregation of electron dense granules (Fig. 3). The granules are considered to be composed of ribonucleoprotein (i.e. protein plus ribonucleic acid). There is no membrane forming a boundary between the nucleolus and the remainder of the nucleus.
Electron micrograph studies on nucleolar formation have revealed that nucleolar material (prenucleolar bodies) first appear as ribonucleic (RNA) or RNA-protein granules scattered amongst page 86 the chromosomes. These bodies are organised (under unknown forces) into a distinct organelle by the activity of the nucleolus organising chromosomes (Fig. 3). The RNA and protein content of nucleoli is proportional to the number of organising centres present, and also this content is controlled by a number of genes in the chromosome complement. It seems probable also, that once formed the nucleolus organising centre retains a control on the metabolic activities of the nucleolus.
The precise origin of the prenucleolar bodies is still rather obscure, though it seems probable that the RNA of these bodies is produced from special loci on the chromosomes, and that their protein portion is derived from pre-existing proteins of the cell, formed before division. During organisation, RNA replication and nucleolar RNA synthesis of protein probably account for nucleolar growth.
Chemically, nucleoli have a very high content of protein with up to 6% RNA. This RNA content is 90% of the total RNA of the cell as a whole. Autoradiographic studies have shown that there is a constant turnover in nucleolus RNA (i.e. RNA is constantly being formed and then used). There is also an incorporation of amino acids into proteins as a result of nucleolar activity.
Fig. 2: Electron micrograph of a nucleolus (right of centre) from a spermatocyte of Pachyrhamma fasscifer × 24,000. Part of the nuclear membrance can be seen at lower left. (Kindly supplied by W. S. Betaud, D.S.I.R., Lower Hutt).
Fig. 3: Two nucleoli from a pollen mother-cell of Allium triquetrum. Notice the nucleolus organising chromosomes (two, closely synapsed) between the two nucleoli. The two nucleoli had been drawn together through the pairing of the homologous organising chromosomes to which the nucleoli were attached.
Quite dramatic progress in recent years has been made towards understanding the function of the nucleolus and its relationship to the cell as a whole. Many functions have hitherto been attributed to the nucleolus. To mention a few, the organelle was once considered to be of no functional use to the cell at all and was bound for eventual loss. Almost as an antithesis it was considered at one time to be the progenitor of the nucleus and hence of the cell as a whole. Other suggested functions included one as a food store for the nucleus, another as responsible agent of numerous activities of the nucleus during its division. A number of earlier workers, however, realised that a direct relationship existed between cellular metabolism and nucleolar activity. Thus protein synthesising embryonic cells, meristematic cells and specialised secretory cells characteristically have very large nucleoli (Fig. 1); differentiated cells reverting to a meristematic condition to repair damaged tissue show conspicuous enlargements of nucleoli; starved cells have smaller nucleoli than normal, and refeeding these cells causes the nucleoli to revert to normal size. Even before it was clearly realised that the nucleolus is associated with protein synthesis, these observations led to the suggestion that cancerous growth might be caused through a disruption of nucleolar controlled metabolism leading to an excessive production of cellular material, and hence to malignancy. Many cancerous cells have very large nucleoli. This hypothesis has by no means been proved but it is still a currently held view of some carcinologists. What might cause an over activation of nucleolar functioning presents the problem. Viral infection, chemical substances (smoking?), various radiations (atom bombs?) and other mutagenic agents are all known to affect in diverse ways the morphology of nucleoli and the synthesis of cytoplasmic proteins. The alternative, and the most commonly held explanation for the nucleolus/cancer relationship, is that disturbed metabolic activity in the cytoplasm affects nucleolar activity. In this view the root cause of cancer is to be found elsewhere in the cell rather than in the nucleolus.
The identification of a constant turnover of RNA in the nucleolus has given strong experimental support for the present day concept that the nucleolus is the site of synthesis of a major portion at least of cytoplasmic RNA, and is closely associated with cytoplasmic protein synthesis.
Of what importance to a cell is cytoplasmic RNA?
There are three major types of RNA present in a cell, distinguishable by their function, their site of synthesis, whether or not they are end products in themselves, and sometimes in their chemical make-up. Firstly, ‘structural’ or ‘particulate’ RNA is built into the framework of cytoplasmic organelles (e.g. ribosomes). ‘Carrier’ or ‘transfer’ RNA is not an end product page 88 in itself but functions in the cytoplasm in ribosomal synthesis of protein. In this currently held mechanism of protein synthesis a specific amino acid (proteins are long chains of amino acids) becomes attached to a specific carrier RNA molecule, and is then transported to the ribosome, coded into a particular position on this ribosome, and finally released into the cytoplasm along with other coded amino acids as a specific protein. The third RNA fraction is the coder for this mechanism of protein synthesis. This ‘messenger’ RNA is a direct product of DNA of the chromosomes, the heredity material of a cell. It is a replica of a particular segment of the chromosomal DNA (a gene) and contains the necessary information (genetic information) for coding a given sequence of amino acids to form a given protein. Once in the cytoplasm it becomes associated with the ribosomes and codes, as mentioned above, a particular amino acid sequence so to form a particular protein. The amino acids are those carried to the ribosomes by carrier RNA molecules.
Later a little more will be said concerning this protein synthesising mechanism. Its importance will be seen when it is understood that enzymes are proteins, that enzymes control metabolic activities of a cell, and that these activities ultimately control the very form a cell is to assume.
Autoradiography, particularly in sea-urchin eggs and certain dipterous salivary gland cells, has shown that a major portion of cytoplasmic tranfer RNA comes from the nucleolus and is synthesised within these organelles. The nucleolus may in other cases function as a transit or store or augmenter of chromosomal transfer RNA before its passage to the cytoplasm.
As a site of transfer RNA synthesis the nucleolus may be regarded as taking on an auxilliary function in protein synthesis by regulating the amount of carrier molecules supplied to the cytoplasm. In view of this numerous authors have suggested that the nucleolus is concerned principally with cell differentiation and growth, while the chromosomes are responsible for actual form by dictating through messenger RNA.
The relationship, whether direct or indirect, between nucleolus activity and cancer can now be more fully appreciated.
Much less is known regarding the synthetic site of particulate RNA. Sirlin, one of the chief present day workers on nucleoli, has pointed out that though positive evidence is small there is the possibility that the RNA incorporated with protein into ribosomal structure is manufactured in the nucleolus. Proof of this would undoubtedly strengthen the claim for an auxilliary role of the nucleolus in protein synthesis and thus in the role of growth and differentiation. Also, it has already been mentioned that amino-acids are taken up by metabolising nucleoli; perhaps both the RNA and protein fractions of ribosomes are of nucleolar origin.page 89
Very recent experiments suggest that the nucleolus may also augment and perhaps modify messenger RNA before it passes to the cytoplasm for its coding work.
In view of what has been said above it is rather difficult to attach significance to the disappearance of the nucleolus during cell division. Some authors have suggested that nucleolar material needs a constance reshuffling or constant reseeding for metabolic activity. Others have suggested that it disappears simply because its presence interferes with chromosome movements during mitosis. Chemical and irradiation data point to a possible relationship between the nucleolus and the spindle fibres (see later). From recent observations on the formation of spindle fibre material it does not seem likely that a simple exchange of material between the nucleolus and spindle occurs. The precise relationship has yet to be found out.
This discussion could well have been headed — The changing concept of the nucleolus. Indeed, the functional concept of the nucleolus has radically changed in recent years.
Interphase — Metabolic Chromatin
The chromatin phase of the nucleus is the cell's hereditary material. Genes determine, together with the environment, both th macroscopic and microscopic features by which individuals are distinguished one from another, and also the invisible molecular structure of their various components. Chromatin is a molecular complex of nucleoprotein and it may be questioned whether or not the nature of the genetic material can be narrowed down even further. Indeed it can, for, in contrast to 20 years ago, it is now known for certain that the nucleic acid rather than the protein portion of chromatin is the genetic material.
Four macromolecules constitute the principal building blocks of chromatin (1) a simple, low molecular weight basic protein, (2) a more complex high molecular weight acidic protein (often referred to as residual protein), (3) ribose nucleic acid (RNA) and (4) deoxyribose nucleic acid (DNA). Structurally, however, two of these are of greatest importance. Experiments with differential digestion of chromatin from interphase — metabolic nuclei have shown that the morphological configuration of the chromatin, as seen under the microscope, is due to nucleoprotein complexes formed by combination of DNA and residual protein molecules. If either the DNA portion is digested (by an enzyme deoxyribonuclease) or the residual protein (by an enzyme trypsin), chromatin structure is lost completely; removal of the RNA and the basic protein molecules have no such effect.
The DNA renders the nucleus ‘Feulgen positive’. Using the Feulgen staining technique and a variety of others it has been page 90 shown in recent years that the amount of DNA in nuclei from different tissues of an individual, and from tissues of different individuals of the same species, is remarkably constant. In fact, the amount of DNA present in a nucleus is a measure of the number of chromosomes present. Interesting, then, is the finding that reproductive gametes with half the somatic number of chromosomes have half the quantity of DNA as their corresponding somatic cells. Even more remarkable is the fact that not quantity alone but quality (there are many different types of DNA) in the cells of a given species is constant and remains constant under drastically varying metabolic and environmental conditions.
These findings were some of the important early pointers to the identification of DNA as the genetic material of an individual; if the characters of a species are to remain constant then so must also its determining genetic material.
In contrast, the protein portion of chromatin, both basic and residual though principally the latter, varies markedly in amount and quality from tissue to tissue and under changing metabolic and environmental conditions. Both types of protein molecules are linked to DNA as nucleoprotein complexes, and as we have seen the residual proteins impart structure to the chromatin. Functionally, however, little is known about the relationship of the protein to the genetic material though considerable evidence suggests it is concerned with the metabolism of the nucleus and cytoplasm, and perhaps also with the working of the genetic material.
Magnesium and calcium ions in small quantities are characteristic of the chromatin make up. The magnesium irons are linked to the DNA molecules at certain positions where they take the place of the protein molecules; these magnesium sites are concerned with nuclear production of energy compounds on which the functioning of the nucleus depends. There is good evidence to suggest that calcium is important for chromatin integrity.
Little is known of the RNA portion of chromatin though it is a definite structural component. The amount present is small compared with DNA and residual protein. Localised sites of RNA may be related to localised production of nucleolus material or other ‘special’ functions of the chromatin (The genetic material of some viruses [e.g. tobacco mosaic virus] is RNA, not DNA).
The chromatin of the interphase-metabolic nucleus is generally considered to be in a greatly extended and hydrated state, forming interlacing series or a network of fine fibres (Fig. 1). The electron microscope has thrown very little light on this aspect of nuclear structure. In some tissues the chromatin is readily visible after staining while in others it stains very faintly except for small scattered regions, the chromocentres. These chromocentres are generally considered to represent specialised regions of the chromatin designated as heterochromatin.page 91
The distribution of the chromatin during the metabolic phase does not seem to be at random. Indications are that certain parts at least are located in definite sites. Observations on the distribution of sex chromatin and chromocentres have suggested that position is related to interaction between the cytoplasm and chromatin.
Heterochromatin and Euchromatin
What seems to be a general feature of chromosomes is the presence within them of the two types of chromatin, heterochromatin and euchromatin. During a metabolic phase the heterochromatin is generally observable as darkly stained regions. The reverse is often the case in dividing cells for the heterochromatin regions of the chromosomes can only be observed with special treatments. The term ‘heteropycnosis’ is used in connection with this property of heterochromatin, i.e. it appears ‘out of phase’ with euchromatin both during mitotic divisions and metabolic phases.
Sex chromosomes (those that determine the sex of an individual), as the Y chromosomes of Drosophila, are composed entirely of heterochromatin. Otherwise heterochromatic regions on the autosome chromosomes are located adjacent to the centromeres (the chromosome's organ of movement), at the chromosome ends and at regions specialised for the formation of nucleoli.
The finding that sex chromosomes are generally heterochromatic gave rise to the early concept that heterochromatin is the basis of sex determination. The reverse seems more likely to be true, however, i.e. a change of chromatin to a heterochromatic state has accompanied the origin of sex chromosomes.
Very few genes have been located at heterochromatic regions and this and other facts have given the impression that heterochromatin is genetically inert and may be lost without severe detriment to the organism. The latter is probably true, though it now seems certain that heterochromatin is involved in the process of cell differentiation. The possible role of the nucleolus in differentiation has already been mentioned so it is interesting to note again that heterochromatic regions are often associated with regions of nucleolus formation. In maize too, certain chromosome regions (designated Ac and Ds) have been discovered which are undoubtedly concerned with genetic expression and hence affect differentiation; these regions are thought to be heterochromatic.
To account for its functional activity and staining phenomena, heterochromatin must differ from euchromatin in some general chemical structure. Nucleic acid starving experiments indicate a possible difference in DNA content but the exact nature of this or other possible differences is not understood.
Euchromatin is that part of the chromatin that is the true genetic material, concerned qualitatively with cell processes. It will be page 92 described in fuller detail in the section concerned with the nature of the gene.
Apart from chemical composition little is known of the ultra structure of the nucleoplasm in which the chromatin phase is distributed. Chemically it is largely protein. Structurally it is devoid of the numerous organelles and membranes present in the cytoplasm, and under the electron microscope appears as a finely granulated ground substance similar to that in which the mitochondria, microsomes, etc., of the cytoplasm are situated. A number of enzymes are present in the nucleoplasm and these are concerned with intranuclear synthesis of proteins, DNA, energy compounds, etc.
Metaphase Chromatin — the Chromosomes
During preparations for the process of cell division the chromatin of the metabolic nucleus becomes transformed and condensed into a number of discrete units known as chromosomes (cf. Figs. 1 and 5). At the crisis of cell division, metaphase, when the actual feat of division is about to begin, these transformations are generally complete, and as the chromosomes have become arranged in an orderly manner along the cell equator (Figs. 8 and 9), this phase of division serves as a useful point at which to describe the morphology of the chromosomes.
The number and morphology of the haploid chromosome set of a species is a character, indeed sometimes a useful taxonomic character, of that species. It is known as the species karyotype. An illustration of this aspect of chromosome number is found in the New Zealand species of Hebe (Koromiko, etc.), all of which were originally referred to the northern hemisphere genus Veronica. Frankel and Hair (1937), however, looked at the chromosomes of New Zealand veronicas and found that whereas the northern hemisphere veronicas were built up of haploid chromosome sets of 7, 8 or 9, those in New Zealand were of 20 or 21. This was an important finding and was largely responsible for the New Zealand veronicas being placed in a separate genus Hebe.
An organism's haploid set or complement of chromosomes is seen in its sexually reproductive bodies (sperm, ova; pollen, embryo sac; gametes in general). The zygote formed by fertilisation of a male and female gamete will then possess two identical chromosome sets which are described as being homologous with each other. They constitute the diploid complement. When mature, the organism produces reproductive gametes by a special page 93 type of division, meiosis, which halves the number of chromosomes so that each gamete has one set.
Some organisms are produced from the union of gametes that possess more than one chromosome set (identical or not). They, therefore, have multiple chromosome sets in their body cells. Such organisms are called polyploids.
The haploid number of chromosomes varies greatly from species to species though closely related species often show clear relationships between their different haploid sets. The New Zealand podocarps (Matai, Miro, Totara for example) are illustrative of this point. The seven New Zealand species of the genus Podocarpus show clear morphological differences one from another. Hair and Beuzenberg (1958) have shown that clear but related differences also exist in the number and morphology of the chromosomes of the species, and that morphological and cytological differences seem to parallel each other. In his book ‘Chromosome Botany’, Darlington gives a very interesting account of the importance of chromosome studies in relation to taxonomy and evolution.
The parasitic horse roundworm Ascaris, and the grassy herb Haplopappus, each have n = 2, 2n = 4* (Fig. 4). Many primitive organisms (e.g. our native Tmesipteris) have many hundreds of chromosomes and are undoubtedly polyploids. Man has 2n = 46 (Fig. 5).
Human chromosomes are about 4-6 microns in length**. This is about an average size. Many monocotyledon plant species such as the lily have quite large chromosomes, some being as large as 30 microns. Many fungi have very minute chromosomes, and even the nucleus containing them is very difficult to observe under the light microscope.
Chromosome size is at least partly under genetic control (i.e. is controlled by genes within the chromosomes themselves) and is a function of a series of coils that give a ‘body’ to the chromosome similar to a spring. Under normal treatment these coils cannot be clearly seen at metaphase but on treatment with nitric acid and other chemical agents they can be made to loosen out and become quite conspicuous (Fig. 6). Each turn in the coil is a gyre, and it is the number, compactness and diameter of the gyres that chiefly determine chromosome size.
* n denotes the haploid number, 2n the diploid number.
** 1 micron is 1/1000 of a mm.
We can ask then, how are the DNA units arranged in the chromosome so that the genes are aligned in the ‘observable’ linear fashion? A number of ‘chromosome models’ have been proposed in recent years in an effort to answer this question but as yet the problem is still unsettled. One possibility is that the DNA is present in a single, continuous strand. This would readily account for a linear arrangement of genes. However, measurements of the weight of DNA isolated from diverse nuclei suggest that the macromolecules are of a homogenous size, much smaller than that required to account for all the genetic material. The most recent models, then, are based on the assumption that the DNA is divided into many molecules connected in a linear series by protein material (matrix?) and arranged as a zig zag. No model yet proposed fits all the known cytological and genetical data, and the question is still open. Also, recent refined experiments indicate that perhaps the DNA molecules are larger than previously thought; that the DNA exists in a continuous strand has still not been entirely refuted.
Approached from the above angle, the electron microscope picture of the chromosome presents a new problem. Could the separate strands so seen be alignments of closely packed molecules rather than of DNA strands lying parallel to each other?
The coiled chromosome unit (chromonema as it is often referred to) is sometimes described as lying embedded in a mass of non-genetic material, the matrix. Some prominent cytologists refute the presence of this component. If a reality, its presence might account for the generally smooth outline the chromosomes present during cell division. It might function as a protective coat for the genetic material during cell division, or perhaps is related in some way to the formation or maintainance of the chromosome coils.page 95
Fig. 4: The four mitotic chromosomes of the horse roundworm Ascaris.
Fig. 5: The 46 chromosomes of man (From Ford et al in Nature, 181: 1565, 1958).
Fig. 6: Chromosome spirals in Tradescantia virginiana pollen mother-cell, pretreated with nitric acid. (Courtesy Dr. C. Darlington).
Fig. 7: Chromosomes from a root tip of Allium triquetrum pretreated with colchicine to accentuate the centromeres. The right arrow marks the centromeres of two sister chromatids, each bridged by the chromonema. The left arrow marks two satellites of sister chromatids.
Contrary to what has long been believed, the centromere is now recognised to be double at metaphase along with the rest of the chromosome. (Fig. 7, arrow and Fig. 14). Each ‘half’ is the structural and functional centromere unit of each chromatid.
A number of animal species and the wood rushes of the genus Luzula have so called ‘diffuse’ centromeres rather than ‘localised’ ones as described above. Little is known of the structure of such diffuse centromeres but their nature of being evenly extended along the whole chromosome is attested by the fact that if the chromosome is broken into pieces, each fragment behaves normally in cell division.
Fig. 8: The centromere positions (arrow) of chromosomes of Allium triquetrum pollen mother-cells. Notice how the chromatin is being drawn out as the centromeres move apart. Fig. 9: Satellites and satellite chromosomes (arrows) from a root tip cell of Allium triquetrum. Fig. 10: Centric granules of the centromere of Allium cepa. This chromonema bridging the granules is not completely in focus. (Courtesy Dr. A. Lima-de-Faria).
So called secondary constrictions add further morphological features to certain chromosomes and seem to be generally associated with nucleolus formation. The region of nucleolus organisation is commonly marked by a very distinct secondary constriction whose presence has given rise to the rather apt terms ‘satellite’ and ‘satellite chromosome’ (Figs. 7 and 9). The satellite is double, each half again corresponding to one chromatid (Fig. 7).
The presence of a constriction at the region of nucleous organisation is probably only a feature of metaphase (coiled) chromosomes, for at earlier stages of cell division where the chromosomes are much more extended the region is not at all constricted (Fig. 3), and in maize, is marked by a very conspicuous heterochromatic swelling. McClintock has shown that in maize this swelling is the actual region of nucleolus organisation. It seems likely that the metaphase constriction is a direct result page 97 of the presence of the nucleolus during the time the chromosome is being transformed into its coiled metaphase state, and that the region of nucleolus organisation is the region where the satellite joins the chromosome arm.
Having discussed the double structure of centromeres, satellites and chromosomes (chromatids) it is logical to suggest that there is at metaphase, not one, but two structurally and functionally complete chromosomes. This is probably so. The two have been formed from chromosome duplication during preparations for cell division, and the two will separate and become the chromosome units of the two daughter cells after division. What continues to bind the two chromosomes together at metaphase is a problem that is not fully understood, but we shall see in the following article that the presence of two centromeres bound together, as they are at metaphase, is an important feature governing the orientation of the chromosomes along the cell equator.
Salivary Gland Chromosomes
This short discussion on the structure of chromosomes would be incomplete without a mention of the salivary gland chromosomes of dipterous insects, not because they add much to our knowledge of chromosome structure, but because these enormous gene houses form the principal bridge between cytology, the study of cells, and genetics, the study of inheritance. As early as 1881 Balbiani discovered the giant chromosomes in insect salivary gland cells, but it was not until some 50 years later that their importance was realised. In the following 30 years Drosophila genetics, as it is often called, has received a tremendous amount of attention, attention that has been rewarded with knowledge which surely would have otherwise remained hidden or at least obscure for a long time.
These enormous chromosomes (Fig. 11) are found in the cells of salivary glands dissected from the third instar larvae of such dipterous insects as the common fruit fly (a species of Drosophila). The chromosomes are more than 100 times as long as the equivalent metaphase chromosomes taken from, say, ganglion somatic tissue, and the largest approach ½mm in length.
Careful attention will show that each chromosome is in fact a bivalent, for longitudinal pairing, homologue with homologue, has taken place during maturation of the salivary glands, so that cells show only the haploid number of chromosomes. (Do not confuse homologues with chromatids). But more important, the chromosomes show differentiation into a series of alternating chromatic ‘bands’ and achromatic ‘interbands’. Four features of these bands will be mentioned. (1) Individual bands differ greatly among themselves and can be readily recognised and page 98 identified. (2) The pairing of the homologous chromosomes is so exact that band pairs with band throughout; this feature will become significant when we study chromosome pairing during meiosis of germ cells in the following article. (3) The pattern of banding is a constant feature of a given chromosome segment so that particular regions can be identified from generation to generation. (4) Most significantly, certain bands have been associated with particular characteristics of the fly (eye colour, wing shape, bristle number, etc.), and have been shown to represent the actual sites of the genes controlling these characteristics. So vast has the study been that complex gene ‘maps’ have been constructed showing the position on these chromosomes of many of the genes that control the flies' characteristics. An example — the gene for ’white eyes’ is carried close to the tip of the X chromosome (Fig. 11).
Certain regions of the chromosomes are puffed up at various times as so called Balbiani rings. These appear to be genetic regions engaged in very active metabolism, the individual threads of the chromosomes being pushed outwards as loops. These puffs, then, are a very direct expression of gene activity.
The Mitotic Spindle
In material which has been fixed and stained, a series of fibrous elements can be seen at metaphase extending from the so called poles of the cell and spraying outwards over the equator on which the chromosomes are aligned. Careful observations will show fibres of two sorts (Figs. 12 and 13): those extending from pole to chromosomes (chromosome fibres), and (Fig. 15), those page 99 extending from pole to pole (continuous fibres). In many animal cells the poles are marked by a small, often minute granule termed the centriole (Fig. 13), and from this a series of astral rays are generally directed away from the equator into the cytoplasm (Fig. 12).
These four components, the continuous fibres, chromosome fibres, centrioles and astral rays (centrioles have not been identified in plant cells), constitute the mitotic apparatus. In the second part of this article we shall see how intimately these components are associated with both the division of the chromosomes into two nuclei, and the division of the cytoplasm to form two complete cells. The fibrous elements are organised as such just prior to metaphase of cell division from material preformed in the interphase-metabolic cell.
The fibre-like elements of the mitotic apparatus cannot be seen in the living cell and this fact had for many years thrown more than just a shadow of doubt on the reality of these structures; many cytologists considered until recently that the fibres were coagulation artifacts caused through fixation. It was not until 1952 when Mazia and Dan devised a unique method for isolating the intact mitotic apparatus from living sea urchin eggs, and the now famous studies a few years later by Inoue and Bajer* and others using the polarising microscope** to ‘see’ spindle fibres in living cells, that the controversial question of spindle reality was finally settled. There can be no doubt now that spindle fibres do exist in the living cell (Fig. 14) and that these structures are very similar in morphology and arrangement to those fibres seen in fixed cells.
In three dimensional view the spindle is in the form of two cones, base to base; hence the appropriate term spindle.
The electron microscope has recently added a great deal to our knowledge of the fine structure of the spindle apparatus. In particular, the centrioles are now known to constantly consist of a series of the nine tubular fibres arranged longitudinally in the form of a hollow cylinder. The centriole is visible just outside the nuclear membrane when the cell is not undergoing division. This single structure seen under the light microscope is in fact double under the electron microscope, with two ‘sister’ cylinder-like centrioles arranged closely at right angles to each other; the sister centrioles separate as the cell enters division and take up positions to mark the poles. During cell division they reproduce themselves as double structures.
* See Mazia, in The Cell, Vol. III, 1961.
** The polarising microscope makes certain oriented molecules (as spindle molecules) stand out against non-oriented molecules — the phenomenon known as birefringence — and so become visible to the eye.
Biochemical analyses and autoradiography studies have even further broadened our knowledge of these important fibrous structures. Chemically, spindle fibres have a high protein content of simple and characteristic type. RNA is also present and is thought to be built into macromolecules of nucleoproteins which together make up the spindle fibres. The spindle nowadays is considered to be a physical gel of RNA-protein molecules; the spindle fibres are regions of this gel in which the molecules are in a highly oriented state.
Fig. 12: Chromosome and astral fibres of the whitefish. Fig. 13: Centrioles marking the spindle poles in Ascaris megalocephola. Fig. 14: Birefringence of the spindle (arrow) in living endosperm cells of Caemanthus katherinae. (Courtesy Dr. S. Inoue). Fig. 15: Continuous fibres between separating groups of chromosomes in Allium cepa mitosis. Fig. 16: Possible mode of orientation of protein spindle molecules to form fibres.
During their experimenting with isolation techniques, Mazia and Dan found it necessary to ‘protect’ the spindle apparatus from dissolution by the use of oxidising agents that preserve disulphide (-S-S-) bonds. Once isolated the spindle could be put into solution by reducing agents known to break disulphide bonds. This and other findings have led to the suggestion that the spindle fibres are composed principally of small protein molecules linked end to end by disulphide bonds to form an elongate, oriented protein fibre (Fig. 16).
Electron micrographs have clearly shown that the spindle chromosome fibres connect to the centromeres of the chromosomes. This is important as there is an intimate relation between the spindle fibres, the centromere, and chromosome movement. Their connection at the other end to the centriole is still not fully resolved.
Our knowledge of the structure of the spindle is bound to increase greatly in future years. Indeed it must if we are ultimately to grasp the exact mechanics of cell division. How important this is to us can be realised when it is considered that excessive uncontrolled cell division is a visible effect of cancerous and other tumerous diseases.
Genetic Units — the Genes
It will be appreciated from a mere glance at the complexity of structural organisation, from unicellular organisms such as Paramecium to man, that a great many genetic units must be carried by an organism to bring about such diversity and detail. But what exactly is a gene? Can their existence be proved or are they entirely hypothetical? Where are they? What is their chemical composition? What imparts to each its individuality? How do genes work? Today we can to some extent answer all these questions and indeed the solving of them has been one of the most dramatic achievements of present day science.
We begin with Mendel, the founder of our present day concept of inheritance. Mendel recognised and showed experimentally that the genes are carried in an organism as discrete units, are passed on from generation to generation at the same time retaining their individuality, and together express themselves in the phenotype (form) of their bearer. Mendel was unable to say what or where precisely the units of inheritance were, and it was not until a number of years later that the chromosome theory of inheritance was put forward and proved correct beyond doubt. Many experiments on the large Drosophila chromosomes and those of maize plants, among others, have shown that the genes are carried on the chromosomes, and that with the chromosomes they pass from cell to cell, and (via gametic cells), from generation to generation through the process of mitosis.page 102
There are far too few chromosomes in an organism for these structures themselves to be the genes; the chromosome theory of inheritance recognises that the chromosomes each bear a large number of genes. The many experiments of crossing different strains of an organism (e.g., a fly with red eyes X a fly with white eyes) have made it possible to construct ‘maps’ showing the relative positions of particular genes carried by particular chromosomes of an organism (in Drosophila, maize, the bread mould Neurospora, bacteria, viruses and even in the sex chromosome of man). The chromosomes undoubtedly carry the genes.
We have noted that the chromosomes are complex chemical structures built principally of two macromolecules, protein and DNA. The question is, which of the two carry the genes, or do both of them?
To carry genetic information the molecules of a macromolecular chemical compound must have the potential of being organised into a large number of distinct combinations, each combination to correspond to a gene. Proteins are long chains of some 20 naturally occurring amino-acids, and these amino-acids can be linked together in many different combinations (to form different proteins). Proteins were for a long time considered to be the primary genetic material; different amino-acid combinations were considered to represent different genes. But starting with the experiments of Griffith using pneumonia-causing bacteria, and the ingenious experiments of a number of workers in 1952 using bacterial viruses*, the DNA portion of chromatin has been conclusively identified as the genetic material. Genes are DNA.
* These experiments are simply and clearly described by R. P. Levine in Genetics of the Modern Biology Series, 1962.
Genetic information is contained in the order of bases within the DNA molecule. Different sequences of bases will obviously give rise to chemically distinguishable forms of DNA. Studies have shown that whereas the X-ray diffraction patterns of DNA isolated from diverse organisms all fit Watson and Crick's model, their different nucleotide ratios vary greatly from species to species. These differences correspond to differences in the gene make-up of these different organisms. It will be appreciated that many different sequences of bases are possible; it is these that form the basis of gene diversity.
We come now to a brief consideration of gene expression and the genetic code.
Genes express themselves through enzyme formation. Enzymes control cellular chemical reactions, whether or not and at what rate they proceed. Enzymes are proteins, long chains of amino acids linked together in particular array. They are very specific in their activity, for a particular enzyme will control only one or one type of chemical reaction; enzyme type, activity and specificity is almost certainly directly related to the kinds and arrangement of amino acids in its protein make-up. Cells possess a very large number of these very important compounds.
Chemical reactions determine the structure of a cell and of its components, and the cell's activities; so genes indirectly (through enzymes) control cell form and function.
Genes express themselves by dictating to the mechanism of protein synthesis the type and position of each amino-acid within its molecular structure. An indication of the currently held view of how this is brought about has been given already in dealing with RNA, and Fig. 1 in Sampson's article in a previous issue of Tuatara illustrates the essentials of the mechanism. Many details remain to be solved, but these essentials have received very much experimental support.
The genetic code concerns the problem of what sequence of nucleotides within the DNA molecule determines a particular type and position of amino-acid within a protein. Some extremely ingenious experiments have been conducted in the last two or three years in an effort to solve the problem, and while it is far from fully solved some important aspects are understood. It seems likely that three successive nucleotides dictate one amino-acid, that a particular order of bases in these triplets of nucleotides dictates a particular amino-acid, and that successive non-overlapping triplets govern the order of amino-acids in at least sections of page 104 The protein chain. It has been shown, for instance, that the base sequence GUC codes for the amino-acid arginine. AUA for lysine, and similar triplets (codons) have been worked out for nearly all the twenty or so amino-acids that occur naturally in living organisms.
Fib. 17: Base — sugar — phosphate molecules of DNA linked longitudinally as chains. The chains are linked transversely by hydrogen bonds (•) across complementary bases (b and bc). S = sugar, P = phosphate, molecules. Fig. 18: Watson & Crick's model of the DNA molecule. The helix is formed by a sugar-phosphate backbone. Paired complementary bases are indicated by transverse bars. Fig. 19: Illustration of specificity imposed upon the DNA molecule by complementary base pairing. A = adenine, T = thymine. G = guanine, C — cytosine.
|Genetic DNA||Messenger RNA||Amino-acid|
|A) ——||U) ——||Arginine|
In a following issue of Tuatara the mechanism of cell division will be covered in some detail. The present article has been designed as a follow-up to Sampson's article on the cytoplasm, and at this point a brief word on the relation between the two will be made: while the cell can readily be ‘dissected’ for descriptive purposes, in life no clear cut distinction between cell components exists. The organelles of the cytoplasm are developmentally, structurally and functionally related to each other. I have indicated here some interraction between the components of the nucleus, and in dealing with protein synthesis an important nuclear/-cytoplasmic interaction has been noted. As well, some exacting experiments in nuclear transplantation, particularly in Drosophila, have indicated a remarkable reciprocal interaction between the cytoplasm and nucleus. For example, when contained within the cytoplasm of a young meristematic cell, certain genes are actively expressing themselves upon the cell form while other genes are relatively inert. But transplantation of such a nucleus into the cytoplasm of a differentiated cell brings about a marked change. Those genes expressing themselves previously while in the cytoplasm of a meristematic cell cease to do so, while other previously inert genes become very active. In some way the nuclear environment, the cytoplasm, partly determines which genes are expressed, and so have some influence in the development of the cell.
The cell is a structural and functional unit.
References and Suggested Articles for Further Details
Darlington, C. D. Chromosome Botany. Allen and Unwin, London (1956).
Frankel, O. H., and Hair, J. B. N.Z. J. Sci. Tech. 18, 669 (1937).
Hair, J. B., and Beuzenberg, E. J. Chromosome Evolution in the Podocarpaceae. Nature, 18 (1958).
Levine, R. P. Genetics. Modern Biology series. Holt, Rinehart, Winston (1962).
Sirlin, J. L. The Nucleolus. Prog. Bioph. and Biochem. Chem. 11 (1962).
Sutton, H. E. Genes, Enzymes and Inherited Diseases. Holt, Rinehart, Winston (1961).
Swanson, C. P. Cytology and Cytogenetics. 2nd. edit. Macmillan, London (1960).
Taylor, J. H. Chromosome Reproduction. Int. Rev. Cytol., 13 (1962).
Thomas, H. J. The Genetic Code. Amer. Sci., 51 (2) (1963).
White, M. J. D. The Chromosomes. 5th. edit. Wiley and Sons, N.Y. (1961).