reading –alberts chapter 8 p. 335-385--primary reference for nuclear architecture –alberts...

• 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