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 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 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. 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 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 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 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.

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 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.

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 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.

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 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

n denotes the haploid number, 2n the diploid number.

Human chromosomes are about 4-6 microns in length

1 micron is 1/1000 of a mm.

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.

At metaphase and earlier stages of mitosis each chromosome is split lengthwise into two chromatids (Figs. 5 and 14). The chromatids are the future chromosomes of the daughter cells produced by division. Further longitudinal division of these chromatid units is still a matter of much controversy. Chromatids that have separated at anaphase are sometimes seen to be longitudinally double, the two units representing the chromatids of the following division. In the mosquito Culex pipiens, as many as 16 sub-units have been observed, and under the electron microscope a number of workers have resolved bundles of fibril-like structures bound together in loose spirals. Each of these latter fibrils has been thought of as a nucleoprotein complex and it is tempting to consider the chromosome as a large bundle of DNA-protein complexes all arranged longitudinally and, at metaphase, the whole wound into a coil. Genetic studies, however, have shown clearly that genes are arranged as a continuous linear seri