The Nucleus: DNA, Chromatin And Chromosomes

Professor Alfred Cuschieri

Department of Anatomy, University of Malta.

Objectives
By the end of this unit the student should be able to:
1. List the major structural components of the nucleus
2. Name the fundamental properties of DNA
3. Explain the role of proteins in the organization of DNA, chromatin and
chromosomes
4. Outline the structure and functions of nuclear pores
5. Name the types of molecules that pass between the nucleus and the
cytoplasm
6. Identify the components of the nucleolus, their composition and
major functions

Practical skills objectives

The student should be able to:
Identify the ultrastructural components of the nucleus and nucleolus in
electron micrographs
Distinguish between eu- and hetero- chromatin
Identify variations in shape, size and texture of nuclei in histological
sections
Interpret the significance of prominent nucleoli in cells.

Recommended Reading
The World of the Cell Becker WM, Kleinsmith LJ, Hardin J
Chapter 16: The structural basis of cellular information: DNA,
chromosomes and the nucleus
Components and functions of the nucleus.

The nuclear envelope consists of two

membranes enclosing a space, surrounds the
nucleus, and is traversed by nuclear pores
Controls movement of molecules
between nucleus and cytoplasm

Chromatin and nuclear matrix

Formed of the genetic material (DNA)
and associated proteins

The nucleolus
Produces ribosomal RNA

The nuclear envelope consists of two membranes enclosing a

space
The cytoplasmic surface of the outer membrane has
numerous ribosomes and is continuous with RER

The nuclear surface of the inner membrane is

associated with a nuclear lamina for the attachment of
chromatin

At the nuclear pore (P) the two membranes are fused

Nuclear pores:
Are membrane-bound channels
60-90 nm diameter
Regulate passage of substances between nucleus and cytoplasm
Vary in number according to the cells function and stage of development
Consist of proteins forming a distinct complex as shown in the diagram:
 an annulus of 8 proteins

radial spokes

a central plug

Nuclear pore complexes can be isolated by chemical treatment

1. Differential centrifugation separates the nuclei
2. NaHCO3 makes the chromatin soluble
- the remaining nuclear envelopes are termed nuclear ghosts
3. Differential centrifugation separate the nuclear ghosts
4. Detergents are used to dissolve the lipid membranes
the isolated pore complexes (protein) remain
5. Centrifugation isolates the pore complexes
The pore complexes have a distinct structure consisting of
a rosette of 8 granules.

Passage of Substances Through Nuclear Pores

The molecules that pass from the cytoplasm into the nucleus are mainly:
(a) Ions;
(b) Nucleotides for DNA synthesis;
(c) Proteins for binding to DNA
The molecules that pass from the nucleus into the cytoplasm are m-RNA and
r-RNA bound to protein as RNA-protein complexes.
Protein molecules require a special mechanism to pass through
the nuclear pores:

- a signal at the -COOH end of the protein

Protein
- a signal receptor at the pore boundary

- membrane-bound ATPase to releases energy for the process

A fibrous lamina is situated on the nuclear surface of the

nuclear envelope:
- It consists of proteins.
- It has a woven fibrillar structure
- It serves to anchor the chromatin to the nuclear envelope

The main proteins of nuclear envelope are:

1. Pore proteins:
Large pore proteins (known as gp210)
- Anchor the pore complex to membrane
Small pore protein (known as p62)
- Regulate transport through the pore
2. Lamins:
Consist of 3 types: Lamins A, B, C
Form a meshwork of filaments (similar to intermediate filaments) in
the nuclear lamina
Bind chromatin to receptor proteins in the inner membrane of the
nuclear envelope. They are attached in the following sequence:
Receptor protein

Lamin B

Lamins A & C

Chromatin
The nucleus consists of fibrillar material termed chromatin

Chromatin in nuclei exists as:

a. heterochromatin - closely packed or condensed
fibrils, where DNA is inactive.
b. euchromation - loosely packed fibrils, where active
transcription occurs
The amount of euchromatin reflects the state of
activity of the nucleus. Nuclei of cells that are
actively synthesising proteins have a large amount of
euchromatin (e.g. neurons renewing their organelles;
plasma cells producing antibodies). Cells that are not
actively synthesizing protein have heterochromatic
nuclei (e.g. circulating lymphocytes are inactive cells in transit; sperm cells
have very closely packed heterochromatin)

The nuclear material consists of

DNA
Histone protein
Non-histone chromosome protein
RNA

DNA and proteins are organised in a highly regular pattern to form

nucleosomes and chromatin.

The proteins regulate the packing of DNA. Proteins constitute over 80% of
the total mass of nuclear material, while DNA forms 15 to 20%.

There are 5 histone proteins associated with DNA

Two molecules each of

histones H2A, H2B, H3
and H4 form a histone
core.
DNA is wound around the histone cores - two turns of DNA around each
core form a nucleosome. The DNA of each nucleosome contains 146
nucleotide pairs. The stretch of DNA between one nucleosome and the next
is called linker DNA. H1 histone is associated with the linker DNA.
Nucleosomes can be isolated from chromatin by treatment with a hypotonic
solution in an aqueous medium.

H1 histones are responsible for the primary coiling of DNA

When chromatin is exposed to dextran sulphate, it is depleted of histones.
The histone-depleted chromatin forms free loops of DNA emanating from
the chromatin mass.

Further coiling of chromatin fibres is responsible for different

levels and types of packaging.
This supercoiling is dependent on the non-histone chromosome proteins.
(NHCP)
Among the large variety of NHCP are two enzymes which control the
supercoiling are topoisomerase II and topoisimerase I.

Complex coiling of nucleosome chains form chromatin fibrils.

20nm
Chromatin fibres have a diameter of 20-30 nm diameter.

Note that the chromatin in electron micrographs has a fibrillar

structure. Each fibril is a chromatin fiber with a diameterof 20-
30nm.
The nucleolus consists of:

A dense fibrillar part, consisting of DNA, carries

the message for synthesis of r-RNA

A pale granular part, consisting of r-RNA is

combined with protein to form ribonucleoprotein
particles

The presence of a prominent nucleolus indicates that the cell is actively

synthesizing protein. A prominent nucleolus is present in protein-
secreting cells such as those of the pancreas, plasma cells, developing
blood cell precursors and others
The nuclear matrix is a residual network of protein fibrils remaining
after:
Removal of nuclear envelope (using detergent)
Removal of histones (using high salt concentration)
Removal of DNA (using nuclease)

The DNA Molecule Consists of a Long Nucleotide Chain.

Each nucleotide consists of 3 elements:
1. A pentose sugar molecule (deoxyribose)
2. A phosphate group that links one
nucleotide to another
3. A base that could be one of the following:
Adenine
Cytosine
Thymine
Guanine

Some Features of the DNA Molecule

DNA is a very long molecule consisting of two strands
of nucleotides.
The two polynucleotide strands run in opposite
directions
Each strand has a polarity indicated by 3 and 5 ends
Specific base pairing links the two strands:
Adenine always pairs with Thymine;
Cytosine always pairs with Guanine
Characteristics Of DNA - Shape and size:

The DNA molecule:

* has the form of a double helix
* has a diameter of 2 nm.
* one spiral turn = 3.4 nm length
contains 10 base pairs.

Note: 1 nanometre (nm) = 10-9m.

DNA has 3 basic properties

1. DNA REPLICATES ITSELF
DNA makes an identical copy of itself .
This function is possible because of specific
base pairing.

The two DNA strands separate by breaking

open the H-bonds linking the bases. Each DNA
strand serves as a template for the synthesis of
a complementary chain of nucleotides. Specific
base pairing ensures that the sequence of bases
is preserved on both molecules of DNA.
DNA replication is the basis of propagation of
life.

DNA replication involves two main steps:

1. The two strands of DNA separate:
2. Each strand serves as a template for synthesis of a complementary
strand.
The two resulting daughter molecules of DNA are identical to the original
DNA molecule.

2. DNA CARRIES THE GENETIC MESSAGE.

A stretch of DNA coding for one particular protein constitutes a gene.
Each gene is specific for one specific protein.
Each protein is responsible for a particular function in a cell

In a gene the coded message is contained in exons (expressed sequences),

which are usually interrupted by introns (intervening sequences) that are not
parts of the gene message.

The number of introns varies from 0 to 80 in different genes, but is the

same for a particular gene. Introns are like blank spaces separating
paragraphs in a written message. They intervene but do not interrupt the
message. They also serve a purpose.

The Genetic Message Is First Transcribed and Then Translated

Transcription is the process of synthesis of messenger RNA using the
coded message in a stretch of DNA (a gene) as template. It occurs in the
nucleus.
Translation is the process of synthesis of a specific protein using m-RNA as
a template. It occurs in the ribosomes in the cytoplasm.

Note that both exons and introns are transcribed. The introns are then
spliced off the m-RNA. Only the exons are translated.
Translation Requires 3 Types of RNA
i. Messenger-RNA (m-RNA)
- Serves as a template for the formation of a specific protein.
- Is specific - There is one m-RNA for each protein.
ii. Ribosomal-RNA (r-RNA)
- Is required for the assembly of ribosomes.
- Ribosomes are the machines that synthesize proteins based on the
m-RNA template. Ribosomes are non-specific i.e. they are used in the
synthesis of all proteins.
iii. Transfer-RNA (t-RNA)
- Transfers the appropriate amino-acid to a growing protein chain
- There is one t-RNA for each amino-acid

3. DNA IS UNIVERSAL
Whatever the living organism - bacteria, plants, animals or man - the
basic structure of DNA is the same.
Only the sequence of bases differs and consequently the genetic
message; the resulting proteins and their specific functions are also
different.
Because DNA is universal, DNA from one organism can be grafted on
to the DNA of another organism. This is the basis of genetic
engineering.

Note the main processes that occur within the nucleus:

Replication - the process of synthesis of DNA, which occurs prior to
mitosis of meiosis
Transcription - the process of synthesis of m-RNA, a copy (transcript) of
the genetic message in one gene
Synthesis of r-RNA - important components of the ribosomes
Synthesis of t-RNA - to be used in the cytoplasm for translation of
proteins
DNA and RNA are always coupled with protein in the nucleus

The proteins that form part the nucleus are synthesized in the cytoplasm
and imported into the nucleus.

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