Nucleus and

chromosomes
Xu Zhaoyang
Department of cell biology
Basic Medical college
Zhengzhou university
 A brief summary to nucleus

The cell nucleus is a remarkable organelle

becauseit forms the package for our genes
and their controlling factors.
Cell's control centre (usually only 1)
Often spherical, 5-10 microns diameter
large and pale in active cells
Organization of the nucleus

Nuclear envelope

Nuclear matrix

Nucleolus

Chromatin

nulceoplasm
 The nuclear envelope

The nuclear envelope is shown in

an electron micrograph in the
figure to the right. The filaments
outside the envelope are not
visualized with these protocols.
Also, the nuclear lamina just
inside the nuclear envelope is not
shown well. However, one can see
ribosomes on the outer
membrane and the sac enclosed
by the two membranes.
 The envelope of nucleus

The space between the outer and

inner membranes is also continuous
with rough endoplasmic reticulum
space.

The inner nuclear membrane

(above)defines the nucleus
itself.
The outer membrane (right)
is continuous with the rough
endoplasmic reticulum and has
ribosomes attached.
 Nuclear envelope
The nuclear envelope has
two membranes, each
with the typical unit
membrane structure.
They enclose a flattened
sac and are connected at
the nuclear pore sites.
The nuclear envelope is
enmeshed in a network of
nuclear lamina for
stability.
 Selective transport materials
to and from the nucleus
Diffusion
5,000 MW are freely diffusable
17,000 MW-- take 2 min to establish equilibrium
44,000 MW--take 30 min to establish equilibrium
60,000 MW--cannot move in by diffusion
Active Transport
This form of transport is assumed when molecules larger
than the pore diameter (10 nm) get into the nucleus. Studies
with gold markers show that the pore can actually dilate up
to 26 nm when it gets the appropriate signal.
 The nucleolus

The nucleolus is the most obvious

structure seen in the nucleus when
viewed in the light microscope.
The nucleolus is organized from
the "nucleolar organizing regions"
on different chromosomes. A
number of chromosomes get
together and transcribe ribosomal
RNA at this site.
 The structure of nucleolus
The nuclear organizing (NO) regions are
seen as circular areas (pale) surrounded by a
rim of electron dense filaments. These
filaments collectively are called the pars
fibrosa (PF). This is formed from newly
transcribed ribosomal RNA.
After the ribosomal RNA is transcribed, it is
linked to proteins and one can see
accumulation of ribonucleoprotein particles
in the pars granulosa (PG). These particles
form the two types of ribosomal subunits
(large and small) which are then transported
out of the Nuclear Pores separately.
 The nucleolus
A preparation of nucleoli is dissociated and spread on a liquid
surface. Then, the synthesis of ribosomal RNA is stimulated
and after a period of time, the DNA from the nucleolar
organizing region begins to look like a Christmas tree. The top
of the tree is the start site.
As you move down the
"tree", the branches appear
longer. Each branch is a
growing strand of ribosomal
RNA. The DNA code is
being transcribed and the
nucleotides added to the
growing RNA strand.
 The function of nucleolus
rDNA

45S rRNA

18S rRNA 32S rRNA

protein
5S rRNA5.8S rRNA 28S rRNA

Small subunit
protein
Transcribe
from the large subunit
DNA out of
the nucleolus
 The nuclear matrix

The interphase nucleus

is thought to contain a
3-dimensional
filamentous protein
network referred to as
nuclear matrix. It can
be isolated by
dissolving nuclei in
lipid detergents and
solubilizing most of the
DNA with DNases.
 The nuclear matrix
Transmission
electron
micrograph
(X47,000) of a
portion of
nuclear matrix
(left) and
surrounding
cytoplasm.
Cytoskeletal
filaments are
clearly visible.
 The function of matrix

It provides a framework to maintain the overall size and

shape of the nucleus.
The matrix acts as a structural attachment site for the DNA
loops during the interphase: evolutionary highly conserved
300-1000 bp long DNA sequences, referred to as MAR
(Matric-attached Region), have been identified that define the
base of DNA loops, anchoring them to specific proteins. By
means of such chromosomal attachment sites, the matrix
might help to organize chromosomes, localize genes, and
regulate DNA transcription and replication within the
nucleus.
Speculative model
of interphase
chromatin
organization,
envisioning the
nuclear matrix as a
series in internal
channels .
Chromosomal
DNA loops
attached to the
nuclear matrix
through
replication origins.
 Chromatin and chromosome

Chromatin and chromosome are the same material

with different structure in the different stage of cell
division.
The chromatin is composed of DNA, histone(H1 H2A
H2B H3 H4 ) ,non-histone (help to coiling chromatin
into chromosome, ravel the DNA suppressed by
histone) and RNA.
 Two kinds of chromatin

Heterochromatin

Euchromatin
 Heterochromatin

Heterochromatin is the condensed form of chromatin

organization. It is seen as dense patches of chromatin.
Sometimes it lines the nuclear membrane, however, it
is broken by clear areas at the pores so that transport
is allowed. Sometimes, the heterochromatin forms a
"cartwheel" pattern. Abundant heterochromatin is
seen in resting, or reserve cells such as small
lymphocytes (memory cells) waiting for exposure to a
foreign antigen. Heterochromatin is considered
transcriptionally inactive.
 Euchromatin

Euchromatin is threadlike, delicate. It is most

abundant in active, transcribing cells. Thus,
the presence of euchromatin is significant
because the regions of DNA to be transcribed
or duplicated must uncoil before the genetic
code can be read.
 Nucleosomes: the lowest level of
chromosome organization

The nucleus is only 6 micrometers in diameter. The

total length of DNA in the human genome is 1.8 meters.
Thus, in order to pack the DNA into the nucleus as in
the photograph of the metaphase chromosome , there
must be several levels of coiling and supercoiling. There
is nearly a 10,000-fold reduction in length in an
interphase nucleus. Each chromosome contains 1 long
molecule of DNA plus associated histones (basic
proteins) which help in the condensation and regulation
processes.
 Nuclearsome

histone octomer + DNA =

nucleosome

146 nuceotides + 2H2a +

2H2b + 2H3 + 2H4 (histone
octomer)

nucleosomes joined by linker

DNA and histone H1 to form
chromatin fiber .
 The nuclearsomes
in Electron microscope

A length of chromatin
that has been
experimentally
unpacked ,or
decondensed,after
isolation to show
nucleosomes
 Solenoid Model
of Chromosome Structure

first level : nuclearsome (packing ratio -7)

2nd level: 6 nulearsomes coiling into a helical array - 30nm

solenoid (packaging ratio -6)

3rd level : Solenoid coiling into a 400nm supersolenoid

(packaging ratio -40)

4th level: chromotids (packaging ratio -5)

Scaffold-radial
loop structure
model
 The function of nuclear

Store genes on chromosomes;

Organize chromatin into chromosomes to allow
cell division;
Transport gene products via nuclear pores;
Produce messages (mRNA) that code for
proteins;
Produce ribosomes in the nucleolus;
Organize the uncoiling of DNA to replicate key
genes;
 Nuclear pore complex

An electron micrograph of a
nuclear pore. It appears as if
the two membranes are
pinched at that site, leaving a
space filled with filamentous
material. Sometimes a thin
diaphragm may be seen
running horizontally through
the pore
 The nuclear pore

This electron micrograph shown in

the figure to the right depicts a
nuclear pore complex seen with the
transmission electron microscope. As
is obvious, little detail can be seen.
The inner and outer membranes of
the nuclear envelope are joined and
there appears to be a diaphragm-like
structure in the center. However, the
intricate detail pictured in the
foregoing figure cannot be
appreciated.
 The structure
of the pore complex
The right figure shows a view of the nuclear pore from the top. It
contains 8 subunits that "clamp" over region of the inner and outer
membrane where they join. Actually, they form a ring of subunits 15-20
nm in diameter. Each subunit projects a spoke-like unit into the center
so that the pore looks like a wheel with 8 spokes from the top. Inside is a
central "plug". The next (left) figure shows a cross section of the pore
with the clamp-like complex adjacent to the membranes. The projected
spoke is directed towards the central "plug' or granule.
 The method to visualize
the nuclear pore complex
Negative staining
This protocol deposits heavy metal stains around structures and
delineates their surface structure. When placed in an electron
microscope, the heavy metal around the structure retards the electron
beam. The structure itself allows the electron beam to pass and this
activates the photographic emulsion. Thus, a "negative" image is created
in the photograph.
Freeze-fracture/freeze-etch
This protocol involves the rapid freezing of structures followed by
fracturing. The membranes are cleaved along their lipid bilayer and
either the face next to the cytoplasm (protoplasmic or P face) or the
extracellular (E) face of the membrane is shown. Then, a replica is made
of the membrane by evaporating heavy metal over the surface. This
replica is what is viewed in the transmission electron microscope
 Negative stainning
The figure to the right
illustrates a preparation of
nuclear pore complexes that
were isolated from an oocyte
and spread on plastic. Then,
the heavy metal stain was
applied to delineate their
structure. Note that one can
visualize the 8 subunits, the
spokes of the wheel and the
central granule.
 Freeze-fracture/freeze-etch

The right figure shows a

surface view of nuclear pores
scattered in the inner nuclear
envelope membrane. The
subunits cannot be appreciated
with this preparation.
However, it can be used to
study formation of nuclear
pore complexes. This varies
with the physiological state of
the cell.
 Another preparation

The subunits forming the rings

and their spokes. Note that one of
the pores appears to be open in
the center, forming a channel.

The subunits also project fibrils

from either side. At the nuclear
side, these fibrils join to form a
"nuclear cage" The fibrillar
structures cannot be appreciated
in this micrographs.
 Nuclear lamina

An electron micrograph of
a portion of the lamina in a
Xenopus oocyte prepared
by freeze-drying and metal
shadowing. The lamina is
formed by a regular lattice
of specialized intermediate
filaments.
 Structural characteristics
of nuclear lamina

1. Consists of "intermediate filaments", 30-100 nm

thick.
2. These intermediate filaments are polymers of
lamin, ranging from 60-75 kD
3. A-type lamins are inside, next to nucleoplasm; B-
type lamins are near the nuclear membrane (inner).
They may bind to integral proteins inside that
membrane.
 Function of nuclear lamina

Play a role in assemble and disassemble of

envelope before and after mitosis. After they are
phosphorylated, this triggers the disassembly of
the lamina and causes the nuclear envelope to
break up into vesicles. Dephosphorylation
reverses this and allows the nucleus to reform.

If antibodies to lamins are injected into cells,

the nuclei cannot reform after cell division.
Therefore, these lamins are vital to reassembly.
 Function of nuclear lamina

Provide a dock place for chromatin .Arrange the

chromatin on the inner side of lamina to stop the
coiling process. In prophase, accompanied by the
disassemble of the laminar, the chromatin lost the
anchor, coiling into chromosome.
 Function of nuclear lamina

Play a role in assemble of the nuclear. Removed

of the laminar will suppress the assemble of
envelope and the pore complex.
 Signal

The signal is in the peptide sequences. These are

recognition sequences rich in lysine, arginine, and
proline.
Signal may control direction of transport: Gold-
labeled tRNA or 5S RNA may leave the nucleus, but
may not come back.
Also, transport of RNA is inhibited by alteration of
the 3' end or the 5' cap structure.
 The evidence for the signal existance
A peptide sequence, called
nucleoplasmin, was isolated and
linked to colloidal gold. It was then
injected into an oocyte and traced
with electron microscopy. As
shown in the figure to the right, the
gold particles mark the site of
transport of the nucleoplasmin and
the studies showed that it was
transported into the nucleus. Small
gold markers are evident inside the
nucleus.
 The evidence that
transport requires energy

Transport of mRNA can be inhibited by cooling

the cells (placing them at 4 C).
ATP Hydrolysis is required to import a protein
into the nucleus. In the absence of ATP, the proteins
bind specifically at the cytosolic face of isolated frog
oocyte nuclei. When ATP is added, the proteins are
allowed to enter. This can be traced with colloidal
gold labeling of the proteins. Studies show that ATP
is needed for entry, but not for binding to specific
receptors.