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Module IV Nucleus
Structure and functions of interphase nucleus, Nuclear membrane, pore complex,
structure and functions of nucleolus
Chromosomes – Structure; Heterochromatin, Euchromatin, Nucleosomes,
Nucleus is the most important part of the cell situated in the cytoplasm. All the
cellular activities are controlled by it. Nucleus is a directing and organizing unit without
which the cell could not exist. It was discovered by Robert Brown (1831) in flowering plants
and is now recognized as the structure that contains the hereditary material of the cell. The
study of nucleus or karyosome constitutes karyology.
The location of nucleus varies in the cell depending upon the species. Usually it is
situated in the centre of the cell surrounded on all sides by cytoplasm. In green algae,
Acetabularia, it shows various positions, though mainly present in the basal part of cell.
Generally the nuclei are scattered in the cytoplasm.
Morphology:
1. Shape:
The shape of nucleus is variable according to cell type. It is generally spheroid but
ellipsoid or flattened nuclei may also occur in certain cells. The nuclear margins are generally
smooth but they may be lobulated bearing small infoldings of nuclear membrane as in
leucocytes. In certain white blood corpuscles the nucleus is dumbbell-shaped and exhibits
variation during life history stages. In human neutrophil, it is trilobed.
2. Number:
Mostly cell contains a single nucleus, known as mononucleate cell. Cells containing
two nuclei are known as binucleate cells (e.g., Paramecium), and cells of cartilage and liver.
Sometimes more than two nuclei (3 to 100 nuclei) are present in a single cell. Such cells are
called polynucleate cells. Such cells in animals are called syncytial cells (e.g., osteoblast) and
such plant cells are termed coenocytes (e.g., siphonal algae).
Cells having distinct nucleus are called eukaryotic types, whereas cells without
definite nucleus are called prokaryotic cells, (e.g., bacteria, etc.). The latter possess scattered
chromatin material (DNA) in the cytoplasm.
The mammalian erythrocytes (eukaryotic cells) possess no nuclei.
3. Size:
Size of nucleus is not constant and is generally correlated with DNA contents. The
nuclear size is a function of chromosome number, i.e., the size is variable depending upon the
number of chromosomes. Usually, there exists a ratio between the nuclear volume (Nv) and
the volume of the cytoplasm (Cv) for each cell type. It can be expressed by an equation called
nucleoplasmic index (NP) given by R. Hertwig which is as follows:
NP = Nv / Cv – Nv
This index is fixed for each cell and any disturbance of equilibrium of NP acts as
stimulus for cell division. Cells containing more than one set of chromosome are called
diploid or polyploid cells, having larger number of chromosomes.
Thus, cells with more than the diploid number of chromosomes have usually larger
nuclei. The nucleus of resting period, when it does not undergo any division, is called the
metabolic nucleus, while dividing nucleus, is called kinetonucleus or arbeitonucleus in
German language.
The nucleus consists of the following main parts:
(1) Nucleolemma or nuclear membrane (karyotheca) (2) Nuclear sap or karyolymph
or nucleoplasm (3) Chromatin network or fibres (4) Nucleolus (5) Endosomes.
1. Nuclear membrane (karyotheca):
The nucleus is separated from the cytoplasm by a limiting membrane called as
karyotheca or nuclear membrane. This membrane plays an important role for the transport of
the material between the nucleus and the cytoplasm. Nuclear envelope regulates
nucleocytoplasmic exchanges and coordinates gene action with cytoplasmic activity. It has
direct connections with the endoplasmic reticulum and during cell division, this nuclear
envelope develops as an extension of the endoplasmic reticulum applied to the nucleus and
subsequently modified. In the end of mitosis, ie, in telophase, the cisternae of the
endoplasmic reticulum gather around the chromosomes and by fusing together they form the
nuclear membrane.
Structure:
The nuclear membrane appears to be a double membrane having interruptions or
pores at intervals. The outer one is called ectokaryotheca and inner one is termed
endokaryotheca. Each of the membranes is about 75 to 90 A thick and both the membranes
enclose an intervening space is called as perinuclear space or cisterna.
Outer nuclear membrane is comparatively thicker than the inner membrane and has a
rough outline due to the presence of attached RNA particles which may form spirals, parallel
lines or crescents. The inner membrane is smooth having no ribosomes. The nuclear
membrane is followed by a supporting membrane, the fibrous lamina, of uniform thickness
(300 A thick).
Nuclear pores:
The nuclear membrane possesses a number of nuclear pores or annuli, which vary
from 40 to 145 per square micro-meter in nuclei of various plants and animals. Watson
(1959) stated the number of pores in mammalian cells as 10 percent of the total surface of the
nucleus. In amphibian oocytes, certain plant cells and protozoa the surface occupied by the
pores may be as high as 20 to 36 percent.
The nuclear pores are octagonal in shape, their diameter varies from 400-1000 A, and
they are separated from each other by a space of 1500 A. The nuclear pores are enclosed by
circular annuli. At the annulus the inner and outer membranes of the nuclear envelope fuse.
The pores and annuli collectively form the pore complex. Each annulus consists of
eight granules of about 15 nm, which are present on both the nuclear and cytoplasmic
surfaces. Inside the pore is a central granule. Fine fibrils (about 30 A in diameter) extend
from central granule to the peripheral granules, forming a cartwheel structure.
A less defined amorphous annular material is present in the opening itself. This
material is digested by trypsin and remains unaffected when exposed to ribonuclease and
deoxyribonuclease. It means that annular material is protein in nature. The pore complex is a
rigid structure present in a fixed number according to cell type. In certain physiological
stages, however, they may change in number.
For example, they are reduced in number in maturing erythroblasts and spermatids
and it is due to low transcriptional activity of these cells. In some cases, the pore complex is
covered by a thin membrane. It has been suggested that the annulus may serve as a sphincter,
alternately decreasing and increasing the size of the pore with varying conditions.
Some evidence suggests the presence of myosin in the annulus area (Du Praw, 1970).
Annulus is supposed to be a hollow cylinder fitting into the nuclear pore (Witschnitzer,
1958). The lumen of the cylinder is 500 A in diameters representing the nuclear pore.
The wall of the cylinder consists of eight evenly placed microtubules or
microcylinders. Each microtubule is about 200 A in diameters. According to Viviers, a
central microtubule of 150 to 180 A diameters is present with in the lumen of annulus and is
attached to its inner wall by fibrous struts.
Amorphous annular material extends beyond the pore margins (Franke and coworkers, 1966-74). The materials exchanged between nucleus and cytoplasm must traverse
the nuclear pore complexes. Thus, annuli or pores control the passage of some molecules and
particles, even some ribosome components, between nucleus and cytoplasm. This exchange is
very selective and allows passage of only certain molecules of either low or very high
molecular weight. The nuclear envelope is a diffusion barrier for ions as small as K +, Na+ or
Сl– .
On the other hand, very large structures such as ribosomal subunits, which are
assembled in the nucleolus, are able to leave the nucleus through the nuclear pore complexes.
The unit membranes of karyotheca are composed of protein and lipid, like plasma membrane.
At the margins of these pores, the two unit membranes are continuous and at certain places
this nuclear membrane joins the membrane of endoplasmic reticulum.
2. Nuclear sap (karyolymph or nucleoplasm):
The nucleus contains a transparent, semi-solid, granular and homogeneous matrix
during interphase called as nuclear sap or karyolymph. This karyolymph is a fluid substance
containing many particles and network. Primarily it is composed of proteinous material and is
the main site for enzyme activity. This nuclear sap also shows variable appearance during
different stages of cell division. There is some evidence that karyolymph contributes to the
formation of the spindle apparatus in plants.
Nuclear constituents:
The nucleus contains RNA, DNA, proteins of two kinds, histone and nonhistone;
some lipids; various organic phosphorus compounds; and various inorganic compounds,
mostly salts (Davidson, 1976).
DNA:
Generally less than half the dry weight is DNA but the amount varies from species to
species. DNA remains constant and do not vary with nutrition or during starvation, while
proteins in nuclei vary, presistent (ultra-structure,)
RNAs:
Three kinds of RNA are present in cells: ribosomal (rRNA), transfer (tRNA) and
messenger (mRNA). RNA contains nucleotides of the purines, adenine and guanine and
pyrimidines, cytosine and uracil. In any of these RNAs the ratio of the bases to one another is
constant for a species. However, rRNA, tRNA and mRNA separated from one another
disclose different base ratio, indicating difference^ in their chemical composition.
The rRNA is synthesized and assembled in the nucleolus, tRNAs and mRNAs are
synthesized on the chromosomes, and all the RNAs enter the cytoplasm through the nuclear
pores.
Enzymes:
The nucleus contains a number of enzymes and performs metabolism, including
synthesis of DNA and various RNAs. Nucleus lacks the enzymes for aerobic metabolism that
are found in mitochondria, but it contains enzymes for anaerobic metabolism and for
formation of high-energy phosphates. It also contains enzymes for coenzyme synthesis
(nicotinamide adenine dinucleotide or NAD).
Proteins:
A variety of proteins is present in the nucleus: nucleoproteins, enzymes and structural
proteins.
The
nucleoproteins
form two
classes,
deoxyribonucleoproteins and
ribonucleoproteins.
Deoxyribonucleoproteins:
These largely form the chromosomes; consist primarily of histories and DNA in about
equal amounts. However, chromosomes also contain non-histone proteins in smaller
amounts.
Unlike histones, most of the non-histone proteins are acidic, and they vary
qualitatively in different cell types of the same organism.
Non-histone proteins are complexed to areas of DNA whose information is being
expressed. Hence it has been suggested that non-histone proteins, along with chromosomal
RNA which also binds to certain active portions of DNA, may somehow be involved in the
specific control of gene expression. However, if nonhistone proteins do regulate gene
expression, we do not know how this occurs.
Non-histone proteins contain the aromatic amino acid tryptophan. A considerable
amount of the contractile proteins actin, myosin, tropomyosin and tubulin are said to be
present. Nonhistone proteins have a more rapid turnover than histones do.
Both histones and non-histone proteins are synthesized in the cytoplasm and enter the
nucleus through the nuclear envelope. Histones are synthesized only when DNA is replicated,
whereas non-histone proteins are synthesized continuously. Histones induce a compact
structure in the chromosome.
Histones are also considered as stabilizers against heat damage (Tashiro and
Kurakawa, 1976) and against nucleases (Toczko et al., 1975). Activation and repression of
genic expression are thought to be carried out by nonhistone proteins. However, the
mechanism by which this is done in eukaryotic cells is less clear than it is in prokaryotic
cells. All the proteins are synthesized in the cytoplasm and then transported into the nucleus.
3. Chromatin:
DNA is the main genetic constituent of cells, carrying information in a coded form
from cell to cell and from organism to organism. Within cells, DNA is not free but is
complexed with proteins in a structure called chromatin.
Chromatin:
It appears as a viscous, gelatinous substance which contains DNA, RNA, basic
proteins called histones, and nonhistone (more acidic) proteins. The content of RNA and nonhistone protein is variable between different chromatin preparations, but histone and DNA
are always present in a fixed ratio about one to one by weight. The nonhistone proteins are
very heterogeneous; they vary in different tissues and include RNA and DNA polymerase.
Histones:
These are small proteins which are basic because they have a high content (10 to 20
percent) of the basic amino acids arginine and lysine. Being basic, histones bind tightly to
DNA which is acid. The four main histones, H2A, H2B and H4 are very similar in different
species.
Functions of histones:
It is quite likely that they aid in the packing of DNA within the nucleus, and the
binding of histone to DNA serves to prevent the expression of hereditary information.
Chromatin has a repeating structure of beads about 10 nm in diameter connected by a
string of DNA. The 10 nm fibre represents the first level of organization of chromatin within
cells.
The chromatin appears as thread-like, coiled and elongated structure which can be
stained with basic stains (e.g., basic fuchsin, orcein or Giemsa). These structures are termed
chromatin fibres or chromatin substance (Gr., chrome, colour). These are visible during
interphase stage.
In interphase, chromatin of the chromosomes spreads out as a fine threads of linin, but
at certain regions, the chromatin remains condensed in the form of darkly stained chromatin
mass. These condensed regions are heterochromatic regions or heterochromatin, and the
dispersed regions are euchromatin. Both regions are formed of DNA.
During cell division, chromatin fibres become thick ribbon-like structure known as
chromosomes. The chromatin fibres remain twisted or form a network in the nucleoplasm.
The chromatin material is of two types:
Heterochromatin:
Certain regions of chromosomes containing chromatin mass become more darkly
stained than other regions. These particular darkly-stained parts are called as heterochromatic
regions or simply heterochromatin. The darkly staining regions show numerous bead-like
bodies along the chromosomes, called as chromomeres.
There are evidences that heterochromatic regions have a higher ribonucleic acid
content than the euchromatic regions. The heterochromatin is supposed to be metabolically
and genetically inert since it possesses small amount of DNA and large amount of RNA.
Euchromatin:
The light stained region of chromatin is called the euchromatic part or euchromatin. It
contains relatively more DNA.
Chemical composition
[I] Nucleic acids:
These are the main constituents of nucleoproteins comprising about 15 to 30% of the
dry weight of nucleus, mostly as DNA. RNA is in very small amount as 1-2% of dry weight
occurring mainly in nucleolus.
These substances (DNA and RNA) are found associated with the proteins forming
deoxyribonucleoprotein or ribonucleoprotein. The nucleolus contains 8 to 10% of RNA by
dry weight and upto 90% acidic protein. The amount of DNA in the nucleus depends upon
the number of chromosomes; the higher the chromosome number, the greater the DNA
amount.
[II] Proteins:
These may be either acidic or basic proteins. Basic protein contents are comparatively
greater than acidic proteins and contain mainly histone and protamines, as nucleohistones and
nucleoprotamines. Histones are characterized by the preponderance of amino acids—
arginine, histidine and lysine, while protamines show mainly arginine.
The protamines are usually bounded with the DNA molecules by the salt linkage. The
histone proteins are associated with the DNA by the ionic bonds. Acidic proteins (nonhistone
proteins) in nuclei show the abundance of amino acids name у tryptophan and tyrosine.
[III] Enzymes:
These are the most important components of nucleus. Diphosphopyridine nucleotide
synthetase is an important nuclear enzyme necessary for the synthesis of diphosphopyridine
nucleotide (DPN) which is a coenzyme for protein synthesis. Besides, there are DNA
polymerase, RNA polymerase (forming m-RNA) necessary for the synthesis of DNA.
Other enzymes present in the nucleus are nucleotide phosphorylase, nucleotide
triphosphatase, NAD synthetase, adenosine diaminase, guanase, aldolase, enolase, 3hosphoglyceraldehyde, dehydrogenase, pyruvate kinase, and ribonuclease necessary for the
synthesis of RNA. The nucleus also contains ATP and acetyl Co-A, etc.
[IV] Inorganic contents:
These are present in minute quantities but are of great biological importance. The
inorganic materials include salts of calcium, potassium, sodium, magnesium, phosphorus,
iron and zinc. They may be both associated with proteins or with enzymes and related to the
organization of chromosomes as metallic ions.
4. Nucleolus
Embedded in the matrix of nucleus there is a dense rounded, oval and acidophilic
body called as nucleolus, first described by Fontana in 1781 (nucleolus meaning ‘small
nucluns’). Nucleolus has no membrane of its own and is denser than the surrounding
nucleoplasm and hence is distinctly visible.
Size:
The size of the nucleolus is related with the synthetic activity of the cell. Cells with
little or no synthetic activity (e.g., muscle cells, blastomeres, sperm cells, etc.) are found to
contain smaller or no nucleoli. On the other side, the secretory cells, neurons and oocytes
which synthesize proteins or other substances possess relatively large nucleoli. In the living
cell, nucleoli are highly refringent bodies due to large concentration of solid material.
Number:
The number of nucleoli in nucleus depends upon the number of chromosomes and
species. There may be only one nucleolus in many plants and animal cells for each haploid
set of chromosomes. In others, there may be two or more nucleoli for each haploid set of
chromosomes. In man, there are two pairs of nucleoli in each diploid nucleus.
Position:
There are certain heterochromatic regions of specific chromosomes found in
association with the nucleolus, constituting nucleolar organizing regions of chromosomes.
This indicates that although all of the chromosomes contribute to the formation of nucleolar
material but these certain organizing regions are responsible for its constitution. The
nucleolar organizers are now known to contain the genes coding for 18S, 28S and 5.8S and
RNAs.
The 18S, 5.8S and 28S RNAs are synthesized in the nucleolus, where as 5S RNA is
synthesized on the chromosomes outside the nucleolus and the 70 ribosomal proteins are
synthesized in the cytoplasm.
All these components migrate to the nucleolus, where they are assembled into
ribosomes and transported to the cytoplasm. The nucleolar organizer is usually located in a
secondary constriction on the chromosome, i. e., in a chromosomal site that becomes less
condensed during mitosis.
There may be more than one nucleolar organizer region among the chromatin strands
in a nucleus, and hence more than one nucleolus may be present in a single cell.
The nucleolus is known to be the cellular site for the synthesis of ribosomal RNA, the
RNA component of the ribosomes.
1. Chemical composition of nucleolus:
Maggio, Palade (1963) and Vincent (1952) have described the chemical composition
of nucleolus. In liver cells, nucleoli contain RNA as 3-5%, whereas in pea embryo, RNA
contents are 10% or 20% of total nuclear RNA. The protein components include mainly
phosphoproteins.
Histones have not been reported in the isolated nucleoli but Tandler (1962) has
described a high concentration of orthophosphates, which may serve as a precursor of the
RNA phosphorus. Besides, Sirlin, Jacob and Tandler (1963) have verified the presence of
acid phosphatase, nucleoside, phosphorylase, DPN synthetase and RNA methylase enzymes,
although some of them may be lacking in different nucleoli.
2. Types of nucleoli:
According to E.B. Wilson, there are two or three categories of nucleoli, namely
plasmosomes or true nucleoli and karyosomes (Ogata) or chromatin-nucleoli, etc.
(a) Plasmosomes:
The plasmosomes are of oxyphilic nature, i.e., get staind with acidic stains. Their
outer part is transparent, called as halo, and inner one is dense.
(b) Karyosomes (Ogata):
These are basophilic in nature, i.e., get stained with basic dyes. Montgomery calls
them as chromatin nucleoli. They are associated with the chromosomes formation during cell
division. The karyosomes are of 3 kinds: Firstly, they may be net-knot type (i.e., in the form
of nodes composing spireme threads).
Secondly, they include chromosome nucleoli which occur only in gametocytes and
are spheroidal bodies. They represent a group of chromosomes in a condensed form during
resting phase. The third category includes karyospheres which are called nucleolus—noyanx
by Carnoy. They are also spheroidal bodies having basic chromatin.
(c) Amphinucleoli:
Sometimes plasmosomes and karyosomes are closely associated to form a double
nucleolus or amphinucleolus. These are very common in the eggs of molluscs, annelids, etc.
3. Components of nucleolus:
During cell division the nucleolus generally disappears during the first stage or
prophase stage, but it reappears in the daughter cells. Morphologically, two components may
be defined in a nucleolus:
(a) Pars amorpha:
This is a component of nucleolus which first disappears but reappears at the end of
division. This is the amorphous part which begins to disappear just prior to the breakdown of
nuclear membrane during cell division and reappears in daughter nuclei as division
completes.
(b) Nucleolonema:
This is the second and permanent component which does not disappear but remains
persistent throughout the cell cycle. It is a filamentous structure having 80A fibrils with
which 150A particles of ribonucleoprotein are attached.
Functions of nucleolus:
(a) Role in mitosis:
The nucleolus plays a significant part in mitosis. In grasshopper neuroblasts, there are
two nucleoli in each nucleus. If any one of these nucleoli is injured by ultra-violet radiation
or by other source, the mitosis is permanently stopped. It gives clear evidence that both
nucleoli must be present for the initiation of mitosis.
(b) Help in protein synthesis:
The nucleoli help in protein synthesis by the formation of ribonucleic acid. This
ribonucleic acid (RNA) plays an important role in the formation of proteins. The cells with a
high rate of protein synthesis have large nucleoli with a high RNA content, whereas the cells
with a low rate of protein synthesis have small undeveloped nucleoli.
The nucleolus is a factory for ribosomes. The nucleolus is formed at the nucleolar
organizer, which is a chromosomal site that contains tandem (one behind another) repeats of
the genes coding for 18S and 28S rRNA.
This DNA becomes uncoiled and penetrates the nucleolus, where it is actively
transcribed. In addition, other ribosomal components such as 5S RNA and the ribosomal
proteins, which are synthesized in other parts of the cell, converge on the nucleolus, where
the assembly of ribosomal subunits starts.
(c) As intermediator in the transmission of genetic information:
The nucleolus serves as an intermediary for carrying the genetic information from
generation to generation. Evidences for this function are given by the structure of the salivary
gland cell of Bradysia mycorum (Sciaridae).
These gland cells contain large multiple nucleoli which arise from the primary
nucleoli associated with the chromosomes. These primary nucleoli become detached from
chromosomes and become congregated to form multiple nucleoli which are considered to be
the main site of genetic information.
Biogenesis of nucleolus cycle:
During mitosis the nucleoli undergo cyclic changes. Nucleoli are formed around the
DNA loop that extends from the nucleolar organizer. Thus, nucleoli are formed from loops of
one or more chromosomes combining with specific proteins. There may be several nucleoli
per cell, but frequently they tend to fuse into one or a few nucleoli at this stage.
During late prophase the DNA loop containing rRNA genes gradually retracts and
coils into the nucleolar organizer of the corresponding chromosome. Since this DNA is
highly extended as a result of intense RNA synthesis, the nucleolar organizer region is one of
the last to undergo condensation, thus producing a secondary constriction on the
chromosome.
The fibrillar and granular regions are gradually dispersed into the nucleoplasm. After
the cell divides, during telophase, the nucleolar organizer DNA uncoils and the nucleolus is
reassembled.
The chromatin material of the nucleolar organizer carries the information that directs
the formation of the nucleolus and of the ribosomal RNA. In the nucleolus, the newly
synthesized ribosomal RNA (probably the fibrillar material of nucleolus) combines with
proteins, which are apparently synthesized in the cytoplasm and transported to the nucleolus,
to form particles (nucleolar granules) that are precursors to cytoplasmic ribosomes.
Endosomes
These are rather smaller chromatin bodies present in the nucleoplasm of nucleus.
They are like nucleolus but smaller in size, showing changeable structure.