Tutorial 3 - Interphase Nucleus

Unit 3: Interphase Nucleus
Interphase Nucleus
Heterochromatin
Nucleolus:
Heterochromatin:
Nuclear
Euchromatin
Euchromatin:
site
dark, ofcondensed
ribosomal
Envelope:
lighter,
assembly
DNA aand
that is
Contains
transcriptionally
rRNA
transcriptionally
double
active
inactive
membraneDNA
transcription
during
Nuclear Envelope
interphase.
surrounding the
nucleus.
Contains nulear
Nucleolus pores
throughout.
Nucleolus
Nucleoli are not bounded by a membrane.
They are modified chromosomal loops that are
transcriptionally active and involved in synthesis
of rRNA.
Three types of components:

1. DNA that is not actively synthesizing r-RNA.
2. RNA molecules in the process of rRNA synthesis
3. maturing ribosomal subunits.
The Nuclear Pore
• There are lots of

nuclear pores
• They have an
elaborate structure
• They are involved
in the transport of
RNAs and
proteins.
What goes In/Out
In: Histone molecules
Polymerases and other enzymes required
for replication and regulation of replication
Ribosomal proteins and other proteins
(including snRNPs) that complex with
newly formed transcripts.
Out: Ribosomal subunits

m-RNA Protein complexes.
Only fully processed and spliced
transcripts are allowed out of the nucleus.
Transport into
Nucleus
Nucleoplasmin is a large protein

with distinct head and tail
regions. The tails can be
separated from the heads by
limited proteolysis. Colloidal
gold is microscopic gold
particles that can be made to a
desired size and seen in the
TEM.
Steps of Import:
1. NLS protein combines with nuclear import receptor
2. The complex binds to a fibrils attached to the
cytosol surface of the nucelar pore complex.
3. The fibril then bends
4. The protein is transferred to the central component
of the nuclear pore complex.
5. The central component of the nuclear pore complex
undergoes a conformational change that results in
the protein being transferred to the nuclear side of
the pore complex.
CHROMATIN and
CHROMOSOMES
Experimental studies
Short region of DNA
Chromatin
double helix
Beads on a string
form of chromatin This schematic drawing
30 nm chromatin shows the orders of
fiber of packed
nucleosomes chromatin packing that give
Section of rise to the highly
chromosome in an
extended form condensed mitotic
chromosome.
Condensed section
of chromosome
Entire mitotic
chromosome
Each DNA molecule has been packaged into a mitotic

chromosome that is 50,000 X shorter than its extended length
Nucleosomes
Histone molecules:
2 - H2A
2 - H2B
2 - H3
2 - H4
• The nucleosome consists of the core histone

octamer surrounded by DNA wrapped around the
outside of the proteins in approximately two turns per
nucleosome.
• This interaction forms a 'beads on a string structure'
What are Nucleases?
• Enzymes that cleave nucleic acids

(RNA or DNA).
• Nucleases can be very specific,cutting
the DNA or RNA between specific base
pairs (targeting a specific nucleotide
sequence).
• A mixture of different nucleases can be
used to create random cuts.
Step 1 of Digestion
• Chromatin is exposed to nuclease digestion.

• The timing of the digestion is such that not
every stretch of linking DNA is digested.
Step 2
• The incomplete digestion of chromatin results in

fragments of varying length, but in multiples of the
length of one DNA/histone “bead on a string”.
• For example, if 100 bp was involved in one “bead on
a string”, this digestion will produce fragments of
roughly 100, 200, 300 bp etc.
Step 3
• Protein is removed to leave sections of

naked DNA of varying length.
Step 4
• The DNA fragments are separated via

electrophoresis on an agarose gel.
• The fragment lengths will be roughly multiples
of the amount of DNA involved in one segment
of the “beads on a string” model of histone-
DNA packaging.
Characterization of Nucleosomes
• A mixture of different nucleases will cleave
any exposed section of DNA indiscriminately.
• Thus, a section of DNA that is protected by
histones will not be digested.
• Nucleases can be used to determine the
length of DNA involved in one nucleosome,
because there is a repeating pattern of
histone protection.
• Naked DNA, however, would produce a
smear of many sized pieces of DNA until it is
digested down to 1 base pair fragments.
Example:
Lane 1: Nuclease digestion of naked DNA.

Lane 2: No nuclease digestion.
Lane 3: Nuclease digestion of DNA associated
with histones.

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Unit 3: Interphase Nucleus

Three types of components:

• There are lots of

Out: Ribosomal subunits

Nucleoplasmin is a large protein

Each DNA molecule has been packaged into a mitotic

• The nucleosome consists of the core histone

• Enzymes that cleave nucleic acids

• Chromatin is exposed to nuclease digestion.

• The incomplete digestion of chromatin results in

• Protein is removed to leave sections of

• The DNA fragments are separated via

Lane 1: Nuclease digestion of naked DNA.

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