9.2—
Nuclear Structure and Composition
In general, nuclei are spherical or disc-shaped, although occasionally they may appear lobed. They vary in size in different species but are usually within the range 1–10 µm in diameter (Fig. 9.1). Nuclei may be isolated by gently breaking cells in a suitable osmoticum, filtering the homogenate, for example through muslin, followed by low-speed centrifugation. Attempts are usually made to free the nuclei of contaminating cytoplasm, cell wall fragments and other organelles by centrifugation through sucrose. However, methods for obtaining high yields of intact, purified nuclei from higher plant cells are at present not very satisfactory. For certain purposes it is often easier to work with chromatin preparations which consist essentially of chromosomal and nuclear material obtained from broken nuclei.
The major part of the cellular DNA is present in the nucleus, arranged in a highly organized fashion within the chromosomes which at different times may show characteristic bands or loops (see Fig. 9.1). Chromosomes are composed of approximately equal amounts of DNA and basic proteins known as histones (see Table 9.7). In addition, much smaller quantities of RNA and acidic proteins are present which are probably of great importance in controlling gene expression. There is wide variation in the number of chromosomes observed within the nuclei of different species although in diploid cells of a single species the number is normally constant. During interphase the chromosomes are less highly condensed than at mitosis and are more difficult to detect. At this stage at least part of the DNA is being transcribed into RNA. It is possible to identify chromosomes on the basis of morphological features such as size, position of the centromere and the presence or absence of constrictions. In addition, certain
Figure 9.1
Phase contrast light micrograph of nuclei and isolated nuclear
contents from Acetabularia. These giant nuclei are unusually large;
the scale line represents 10 µm. Chromosomes (CH) and nucleoli (NUC)
can be seen within the nuclei (N) fixed in situ (a and c) and the appearance
of isolated chromosomes and nucleoli are shown in b and d. Note the large
aggregates of nucleolar material with an internal cavern system (c and d) and the
'lampbrush-chromosome-like' morphology of the chromosomes (b). (Fig. 9.1a, c and d
from Spring et al., 1974; 9.1b, previously unpublished micrograph from the same authors.)
areas of heterochromatin, which appear more highly condensed and therefore take up more stain, may be distinguished from the remainder of the chromosome, termed euchromatin. In certain cell types, such as embryo suspensor cells, giant polytene chromosomes occur. In such situations it is possible to study the structural organisation of chromosomes in much greater detail.
Recent developments have made it possible to identify individually each of the 46 chromosomes in man on the basis of characteristic banding patterns produced by certain staining procedures. These techniques and their application to the study of plant chromosomes have been reviewed by Vosa (1975).
Organelles known as nucleoli become visible within the nucleus during interphase. These are very prominent spherical bodies with different staining properties to the remaining part of the nucleus. They arise in association with specific regions, the nucleolar organizers, of certain chromosomes. Nucleoli are not surrounded by a membrane but they do have a highly ordered structure (Fig. 9.2). The central core consists of a region of fibrils 8–10 nm across surrounded by a region of dense granules 15–20 nm in diameter. Mutants which lack nucleoli are unable to synthesise ribosomes and it is now known that the genes coding for ribosomal RNA normally occur at the nucleolar organizer region of chromosomes. Here the DNA is transcribed into ribosomal-RNA (rRNA) which is assembled to form ribosomes in the outer region of the nucleolus by combination with ribosomal proteins. The nucleolus is most prominent, therefore, when synthesising large numbers of ribosomes. This explains the old observation that there is a general correlation between nucleolar volume and the rate of protein synthesis of cells.
The fact that nuclei are surrounded by a membrane was deduced from the observation that they are selectively permeable to small molecules. The nature of the double membrane may be readily observed with the aid of the electron microscope (Roberts & Northcote, 1971). It is often interconnected with the cytoplasmic membrane system, the endoplasmic reticulum, and is perforated by a large number of pores. This is demonstrated quite clearly in Fig. 9.3 which shows a surface view of a portion of the nuclear membrane of Acetabularia. The apparent diameter of a nuclear pore is of the order of 100 nm. However, the observation that colloidal particles of much smaller size do not readily pass through these pores suggests that their effective size is substantially less, probably due to the presence of a protein-containing matrix within the pore.
Figure 9.2
Ultrastructural details of the composition of the nucleolar subunits of Acetabularia
revealed by electron microscopy. Note the finely fibrillar texture in the internal zone
A and the dense packing of small granulofibrillar structures in zone B, extensions
from which deeply penetrate zone A (see also the insert ) and the very dense
packing of granules in the cortical zone C. The scale indicates 1 m m,
the scale in the insert denotes 2.5 µm. (From Spring et al., 1974.)