Unit 3: Interphase Nucleus

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Unit 3 Interphase Nucleus
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Interphase Nucleus
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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.

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The Nuclear Pore

• There are lots of nuclear pores
• They have an elaborate structure
• They are involved in the transport of RNAs and
  proteins.

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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.

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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.
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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.

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CHROMATIN and CHROMOSOMES Experimental studies
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Chromatin
Short region of DNA double helix
Beads on a string form of chromatin
This schematic drawing shows the orders of
chromatin packing that give rise to the highly
condensed mitotic chromosome.
30 nm chromatin fiber of packed nucleosomes
Section of chromosome in an extended form
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
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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'

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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.

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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.

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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.

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Step 3

• Protein is removed to leave sections of naked DNA
  of varying length.

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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.

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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.

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Example

• Lane 1 Nuclease digestion of naked DNA.
• Lane 2 No nuclease digestion.
• Lane 3 Nuclease digestion of DNA associated with
  histones.