Cell Nucleus

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Cell Nucleus

• Organization of DNA

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Agenda

• Euchromatin and heterochromatin
• Organization of DNA as chromatin
• the histones
• a role for chromatin organization in gene
  expression
• Please read third ed. Fourth ed.
• DNA packaging 501-507 498-507
• Nuclear matrix 515-516 514-516 (fig 12.26)
• Gene structure 521-539 520-537

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Interphase chromatin

• DNA is an exercise in packing and organization
• the animal genome is written in 3 X 109
  nucleotide bases.
• DNA contains the 30,000 different genes
• in humans this is divided into 46 chromosomes,
  each an unbroken strand.
• A total of 2m in the nucleus of 10mm in diameter
• Although it cant be seen in interphase, each
  unwound chromosome does have an organized
  location in the nucleus. (see fig. 12.23 (third
  ed.) 12.21 second ed.)
• Held in place on the nuclear matrix and nuclear
  lamina. Protein frameworks

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Interphase chromatin
Chromatin structure is only visible as denser and
lighter regions
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• Euchromatin
• The regions of DNA containing active genes.
• Dispersed, active DNA
• Lighter appearance, located in interior of
  nucleus
• Autoradiography with 3H uridine demonstrates that
  this region is active in producing RNA
• Uridine is a raw material for new RNA, not DNA.

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Heterochromatin

• Inactive, condensed chromatin
• located at the edges of the nucleus
• visible by E.M. as a darker colour.
• Two reasons why they are condensed
• a) inactive genes, such as chromosome 18
• This will vary with cell type, depending if genes
  are active
• b) regions without genes. Non-coding DNA.

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• Centromeres
• the constricted central portion of a each
  chromosome,
• the DNA contains a-satellite DNA made up
  non-transcribed 171 base repetitive sequences.
• Repeated many thousands of times
• Attach to the kinetochores during M phase
• Telomeres
• non-coding regions at the ends of the
  chromosomes.
• Short repeated sequence repeated 500-5000X
• TTAGGGTTAGGGTTAGGG
• Serve to separate chromosomes from each other
• Anchor to the nuclear lamina.

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Heterochromatin

• Two types
• Constitutive heterochromatin is non-coding and so
  can never become active
• Facultative heterochromatin contains genes that
  are shut off and temporarily inactive. -depends
• In any cell only a portion of all the genes are
  active
• Example the Barr Body. Females have two X
  chromosomes, one of which is inactive (fig. 12.15)

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Facultative vs. Constitutive Heterochromatin
Facultative Heterochromatin
telomere
Both chromosome 18
Euchromatin in interior of nucleus
Facultative Heterochromatin Barr Body inactive
X. Euchromatin Active X-chromosome
Interphase nucleus
Constitutive Heterochromatin around centromere
and telomere
Telomere
Cytoplasm
11
DNA Packaging

• Structural and Functional Aspects

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Level 1 Histones

• DNA winds around nucleiosomes, which are a group
  of positively charged, highly conserved proteins

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Histone Varieties

• Nucleosome core particle made up of four types of
  histones, two of each
• Histones H2A, H2B, H3 and H4
• DNA wraps around the nucleosomes. 1.8 turns or
  146 nucleotide bases per nucleosome
• Histone H1 (fifth type)
• links adjacent nucleosome core particles.
• A total of 167 nucleotide bases per unit
• 71 packing ratio, the fiber is 10 nm thick

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H2A
H2B
H3
H4

naked DNA
nucleosome core particle
nucleosomes
Add H1
H1
- 168 bp in loop - 6 to 7 fold shortening - 10
nm fiber
H1
H1
H1
H1
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Level 2 30 nm Fibres

• Spontaneous assembly of adjacent nucleosomes
  results in a 401 condensation in length, with 30
  nm thick fibres

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H1
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Level 3 Looped Domains
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Looped Domains

• DNA strands associate with nuclear matrix
• Form loops of 20,000 to 100,000 bases
• AT-rich domains on DNA form MARS
• Matrix Associated Region
• Includes a satellite and other non-coding DNA
• proteins of the scaffold include 4 types of
  binding material

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Looped DNA domains
Nuclear matrix fig 12.26 (both eds.)
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Scaffold Proteins

• Nuclear matrix
• a protein fiber framework
• major organizing structure for RNA polymerase
  (makes messenger RNA), RNA processing, DNA
  replication.
• Topoisomerase II
• an un-tangling protein
• Insulator Protein
• keeps loops separate
• Nuclear lamina
• lamins bind telomeres and a satellite DNA of the
  centromere

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Level 4Mitotic Chromosomes
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Mitotic Chromosomes(a 10,000 fold decrease in
length)

• Also have looped domain structure
• No Nucleus is present
• DNA loops on Condensin protein
• SARs scaffold associated regions
• More compact than interphase chromatin
• Condensin is activated by phosphorylation via Cdk
  (cyclin dependent kinase)
• Centromere is a site of condensation
• contains a-satellite DNA
• binds to proteins, including the kinetochore

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The Histone Code

• DNA is being transcribed and duplicated
• Necessary machinery has to work around
  nucleosomes
• The cell makes DNA more or less accessible to
  other proteins by modifying histones
• Mechanisms of Histone modification
• Replacement with modified types of histone
• Chemical modification of the histones

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Modified Types of Histones

• H2AX
• DNA being repaired
• H2AZ and H3.3
• DNA transcribed by RNA polymerase II
• CNEP-A (a version of H3)
• on the centromere, kinetochore assembly
• macroH2A
• barr body
• keeps chromatin condensed and inactive.

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Chemical Modification of Histones

• Acetylated histones looser binding
• Phosphorylated histones
• Methylated histones
• Alter the accessibility of DNA to processing
  enzymes
• Alter binding to organizational structures such
  as nuclear matrix