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Prokaryotic vs. Eukaryotic Cells
Organelles
PROKARYOTES
EUKARYOTES
Cell (plasma) membrane
Cell wall
Cytoplasm
Nucleus
Cell (plasma) membrane
Cell wall (plant cells only)
Cytoplasm
Nucleus
Rough Endoplasmic reticulum
Smooth Endoplasmic reticulum
Golgi complex
Lysosome (animal cells only)
Vacuole (plant cells only)
Chloroplast (plant cells only)
Mitochondria
Cytoskeleton
Peroxisome
Ribosomes
Flagella (animal cells only)
Ribosomes
Flagella
Pili
Prokaryotic (bacterial) Cell
Eukaryotic Cell
Animal cell
Plant cell
Nuclear material
Prokaryotes - circular DNA:
Nuclear material
Eukaryotes - linear DNA:
Eukaryotic Cell
Most cells (somatic cells) have 2 copies of each chromosome
(diploid)
Gametes, germline cells (egg and sperm) have only 1 copy of
each chromosome (haploid)
Eukaryotic Cell
We have 2 meters of DNA in all
our somatic cells
HOW DOES IT ALL FIT??
Condensation of DNA by
proteins!!
The nucleosome
• 8 histones form a complex
• 140-150 bp DNA wound twice around it. 50-70 bp DNA
between each nucleosome
• + Linker histones
DNA - acidic, negatively charged
Histones proteins contain many basic amino
acids (Lys, Arg), positively charged
Beads on a String
(in Colorado)
The Histone Code
Methylation - yellow hexagon
Acetylation - green flag
Phosphorylation - grey circle
Other modifications??
The Histone Code
Methylation - yellow hexagon
Acetylation - green flag
Phosphorylation - grey circle
Other modifications??
Chromatin types
• Euchromatin: open chromatin - associated with gene
activity
• Heterochromatin: densely packed chromatin - indicates
little or no gene activity
Chromatin types
• Euchromatin: open chromatin - associated with gene
activity
• Heterochromatin: densely packed chromatin - indicates
little or no gene activity
Euchromatin
Heterochromatin
The Histone Code
Methylation - yellow hexagon
Acetylation - green flag
Phosphorylation - grey circle
Other modifications??
Packing and the cell cycle
• Between cell divisions (interphase) - euchromatin
dominates, so open chromatin & gene activity
• When the cell is about to divide (metaphase) the
chromsome is densely packed
In mitosis, the chromosomes appear as the thick rod-shaped bodies which
can be stained and visualized under light microscopy.
The modern way to visualize condensed chromosomes is by FISH -fluorescence in situ hybridization. In this method, fluorescent antibodytagged DNA probes hybridize to their complementary sequences in the
chromosomes. By using FISH probes with different colored fluorophores,
one can color each human chromosome independently, and thus identify all
23 chromosomes. This is called chromosome painting.
M-FISH/SKY (multifluor-FISH/spectral karyotyping)
Isolate cell nuclei
Red blood cells
Skeletal muscle cell
Secretory cells
of pancreas
Sperm cells
Human embryo at
2-cell stage
Cell breakage
AKA cell disruption, cell disintegration, lysis
Goal - destroy outer cell membrane without destroying organelle
membranes
Cells broken open (plasma membrane dissolved) by:
Mechanical
• freeze-thaw
• grinding
• shearing (homogenizer)
• shearing (french press)
Chemical
• solubilize with detergents
• organic solvents
• alkali treatment
• enzymatic digestion
Cell breakage
Mechanical
• freeze-thaw
ice crystals form and disrupt cell after slow freezing and
thawing
• grinding (mortar & pestle or blender)
use force to grind and smash cells
• shearing (homogenizer)
use pressure to induce a shear force on cell wall
pump cell slurry through a restricted orifice valve
• shearing (french press)
use pressure to induce a shear force on cell wall
uses HIGH pressure & rapid decompression to disrupt cell
Cell breakage
Chemical
• solubilize with detergents
mostly used to disrupt animal cells
detergents destroy cell lipid membrane
• organic solvents
dissolves cell membrane
• alkali treatment
uses NaOH/SDS (sodium hydroxide/sodium dodecyl sulfate)
to solubilize the phospholipid and protein components of cell
membrane, fast & reliable, most commonly used for plasmid
DNA isolation out of prokaryotic cells
• enzymatic digestion
dissolves cell membrane, “gentle” technique since enzyme
attack specific components of cell membrane
EX: lysozyme digests peptidoglycan layer of bacterial cell
wall
Cell fractionation
If done correctly, disruption reduces cells to EXTRACT (homogenate)
with soluble components, intact organelles and plasma membrane
fragments
DIFFERENTIAL
CENTRIFUGATION
Tissue
homogenization
Low speed
Supe to medium speed
Supe to high speed
Tissue
homogenate
Supe to very high speed
Pellet of whole cells,
nuclei, cytoskeleton,
plasma membrane
Pellet of mitochondria,
lysosomes,
peroxisomes
Pellet of micorsomes
(fragments of ER),
small vesicles
Pellet of ribosomes,
large macromolecules
Supernatant contains soluble
proteins
Cell fractionation
Centrifugation
Separate proteins by size or density
Differential centrifugation - separates large from small particles
Isopycnic (sucrose-density) centrifugation - separates particles of
different densities
DIFFERENTIAL
CENTRIFUGATION
ISOPYCNIC (SUCROSE-DENSITY)
CENTRIFUGATION
Tissue
homogenization
centrifugation
Low speed
Supe to medium speed
Supe to high speed
Tissue
homogenate
Sample
Supe to very high speed
Pellet of whole cells,
nuclei, cytoskeleton,
plasma membrane
Sucrose
gradient
Less dense
Pellet of mitochondria,
lysosomes,
peroxisomes
Pellet of micorsomes
(fragments of ER),
small vesicles
Pellet of ribosomes,
large macromolecules
More dense
Supernatant contains soluble
proteins
Fractionation
Object of lab:
Isolate DNA from nuclei of eukaryotic cell (calf thymus)
1. Isolate nuclei (centrifugation after cell disruption)
thymus tissue + buffer
cell membrane breakage using Waring blender
filter homogenate through cheese cloth
centrifuge homogenate
resuspend pellet (contains nuclei) w/ buffer
filter through cheese cloth
NUCLEAR SUSPENSION
2. Compare isolated nuclei to intact nuclei
use microscope
3. Isolate DNA from nuclei (nuclear membrane disruption, dissociation
of protein (histones) from DNA, alcohol-insoluble DNA isolated)
add SDS to dissolve nuclear membrane and dissociate protein
from DNA
add alcohol to top of solution
use glass rod to pull DNA fibers into alcohol layer on top