reading –alberts chapter 8 p. 335-385--primary reference for nuclear architecture –alberts...
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• Reading– Alberts Chapter 8 p. 335-385--Primary
reference for nuclear architecture– Alberts Chapter 16 p. 800-801--Nuclear
Lamins– Alberts Chapter 18 p.921-924--Centromeres– Alberts Chapter 4 p.139-148--Microscopy– Alberts Chapter 4 p. 186-188--Antibody
labeling
Alberts 4-20
•Nucleus–Storage of genetic
information
–Replication of genetic information
–Transcription of genetic information into “functional forms”
–Control of gene expression
Alberts 1-18
• Prokaryotic cells– No nucleus
– DNA localizes to nucleiod body
– Lack of compartments
Alberts 1-12
• Replication, Transcription and Translation occur in the cytoplasm of prokaryotic cells– Mechanisms of control
are different than in eukaryotic cells.
Alberts 3-15
• Active transcription and translation in E. coli– Coupled events
– Ribosomes attach to transcript while it is still being synthesized
Voet 29-18
• Eukaryotic cells– Compartmentalized
– Replication, transcription and processing occur in the nucleus
– Translation occurs in the cytoplasm
• Mitochondrial protein synthesis
Alberts 3-15
•Nuclear organization– Nuclear envelope
– Nuclear lamina
– Nuclear pore
– Nucleolus
– Heterochromatin
– Euchromatin
Alberts 8-1
• Electron micrograph of nucleus
Alberts 8-71
•Nuclear envelope–Lipid bilayer
• Permeable to small nonpolar substances
–Inner membrane• Continuous with outer
membrane
• Associates with nuclear lamins
–Intermembrane space• Closely resembles ER
–Outer membrane• Continuous with lumen of
ER
Voet 11-13
Nucleus
Cytoplasm
Alberts 16-18
–Nuclear pores• Major connection between
nucleus and cytoplasm
• Active and passive transport
–Nuclear Lamina• Meshwork of intermediate
filament protein lamin
• Major structural support for nucleus
• Nuclear Lamina– Grid-like structure
– Composed of lamins A, B, C
– Assembly dependent on phosphorylation
– Lamin B attaches to inner membrane
Alberts 16-18
Alberts 12-18
• Nucleolus• Euchromatin• Heterochromatin
– Constitutive
– Facultative
Cooper 8-15
• Metabolic labeling with tritiated uridine to identify areas of active transcription
• Euchromatin
• Heterochromatin
Alberts 8-26
Other Nuclear Structures• Spliceosomes
– May function as storage centers for splicing factors
– Or may be sites of splicing
– Contain splicing factors
– Speckles
• PML bodies– Disrupted in acute
premyelocytic leukemia
– Associated with nuclear matrix
– Function unknown
• Coiled bodies– Cajal bodies or Nucleolar
accessory bodies• Ramon y Cajal described
in 1903
• May be involved in snRNP production
• Gems– Associated with coiled
bodies (Gemini of coiled body
– Similar in structure to CB, but contains different proteins
• Antibodies are important tools for identification and localization of proteins in cells– Primary antibody
– Secondary antibody
– Fluorescent, gold or enzymatic label detects antibody labeling indirectly
Alberts 4-64
• Fluorescence Microscopy– Path
– Confocal
– Two photon
– Deconvolution
• Excitation• Emission
Alberts 4-7
• What is a fluorophore?–Heterocyclic
compounds
–Absorption of light raises energy to an excited state
–Molecule decays to its ground state by emitting photons
Alberts 4-8
ExcitationEmission
S 1
S0
S1‘
Step 1
Step 2
Step 3
• Other fluorochromes– Dapi and Hoechst stain nucleic acids– Mito-trackers designed to stain specific
organelles (mitochondria, lysosomes, etc).
• GFP--jellyfish green fluorescent protein– cDNA isolated in 80’s by Bill Ward– Protein folds into a fluorochrome– Make chimeric genes with GFP cDNA and your
cDNA– Fluorescent tag that can be visualized in live
cells
• Fluorescent staining of a fibroblast nucleus– Blue: DNA stained with Dapi
– Red: RNA stained with rhodamine labeled poly dT
– Green: Fluorescein labeled antibody to a protein involved in splicing
Lodish 11-21
• GFP fusion proteins expressed in the nucleus
• Phair and Misteli (2000) Nature 404: 604-609 Figure 1
•FRAP– Fluorescence recovery
after photobleaching
– High intensity pulse of laser
– Fluorochrome hit by laser is dead
– Monitor the fluorescence over time
– Recovery of fluorescence due to movement of new molecules into the area
– Measures movement
•FRET– Fluorescence resonance
energy transfer
– Requires 2 fluorochromes
– Emission of one must overlap excitation of the second
– Close proximity of labeled components allows energy transfer
– Measures proximity of components
Voet 11-16
• We have 2 m of DNA in every nucleus in our body.
• Each nucleus is only 10 µm in diameter.
• How does all this DNA get packed into such a small space?
• Answer: dense packing of the DNA into chromatin.
Alberts 18-14
• A. Native structure of condensed chromatin– 30 nm fiber
• B. Structure of “decondensed” chromatin– Beads on a string
• Kornberg 1974
Alberts 8-9
• Kornberg’s experiment
– Limited digestion of DNA with nuclease release DNA fragments 200 bp long (string).
– Further digestion released nucleosome beads.
– Dissociation of beads revealed 148 bp of DNA and histones
Alberts 8-10
• Histones– Small, highly basic
proteins (20-30% lys or arg)
– Highly conserved
• Core histones– Nucleosomal histones
– H2A, H2B, H3, H4
– Two copies per nucleosome.
• H1 histones– 6 highly related species
Alberts 8-10
• Model for the structure of the nucleosome.– DNA is sharply bent.
– Spool like structure 11 nm in diameter.
– Core histones possess similar folds.
Alberts 8-10
• H1 Histones– Associates with the
nucleosome and additional DNA
– Chromatosome
– Interacts with H1 histone of adjacent chromatosome
Alberts 8-15
• Interaction of H1 histones with adjacent H1 histones produces the 30 nm structure known as the solenoid.
Cooper 4-10
• Radial Loops– EM of isolated
chromosomes display loops of chromatin extending from a backbone
– Lampbrush chromosomes also display loops
• More extensive folding
• Scaffold for chromosome structure
Alberts 8-29
• Double helix
• Nucleosomes
• Solenoids
• Radial loops
• Condensed loops
• Metaphase chromosome
• Heterochromatin may closely resemble metaphase chromosome
• Euchromatin may be structurally similar to 10 nm/30 nm structures
Alberts 8-30
• Scaffolds– Extensive radial loops
appear to extend from a backbone
– Removal of histones allows observation of what appears to be a fibrous core or scaffold
• Real or Artifact?– Harsh treatment may
cause deposition of protein in the sticky nucleic acid
Voet 33-14
How does chromatin structure effect replication and
transcription?
• Model for dealing with histones during replication– Nucleosome splits in
half as the replication fork approaches
– Histones remain associated with one strand
– Nucleosome re-forms after replication passes
– New histones are added to other pair
Alberts 8-40
• Activation– Acetylation of histones correlates with actively transcribed genes
– Acetylation reduces the net positive charge of the histones
– HMG14 or 17 compete with H1 histones for binding to the nucleosome
• De-acetylation is involved in turning transcription off
Cooper 6-32
Chromosome organization
• Metaphase chromosome– Chromatids
– Telomeres
– Centromere• Holds sister chromatids
together
• Attachment of kinetochore during mitosis
Alberts 18-15
• Origins of replication-sites where DNA replication begins.
• Telomeres are specialized sequences at the end of linear chromosomes that ensure that genetic material is not lost during replication.
• Centromeres hold chromatids together prior to cell division and associate with kinetochores.
Alberts 8-4
• Kinetochore– Attachment site for
spindle microtubules
– DNA/ protein structure
– Required for proper chromosome segregation
Alberts 18-16
• Yeast centromere has been characterized.• Simple DNA sequence that binds to microtuble
Alberts 18-17
• Chromosome Banding• Chromomeres• Distinctive banding
pattern for each chromosome– Hoechst (G bands--AT
rich sequence)
– Olivomycin (R bands--GC rich sequence)
– Giemsa
– Feulgen reagent
Alberts 8-31
• Domains of replication– Metabolically label
cells with bromodeoxyuridine
– Substitutes for thymidine
– Alters staining of G bands or can use antibodies to detect
• Synthesis occurs in distinct domains during S phase
Alberts 8-37
• In situ hybridization
– Examine position of a gene on a chromosome
– Examine position of chromosome in nucleus
– Examine distribution of transcript in cytoplasm
• Basic hybridization technique
– Label probe
– Hybridize via base pairing to target
Alberts 7-17
• Result of in situ hybridization– Gene specific probes
– Unique labels for each gene
– Metaphase chromosome 5
• Duplicate spots for each probe
• Positions on each pair similar
Alberts 7-19
Alberts 6-23
Alberts 6-22