Prokaryotic vs. Eukaryotic Cells

Organelles

PROKARYOTES EUKARYOTES

Cell (plasma) membrane Cell (plasma) membraneCell wall Cell wall (plant cells only)Cytoplasm Cytoplasm Nucleus Nucleus

Rough Endoplasmic reticulumSmooth Endoplasmic reticulum Golgi complexLysosome (animal cells only)Vacuole (plant cells only)Chloroplast (plant cells only)MitochondriaCytoskeletonPeroxisome

Ribosomes RibosomesFlagella Flagella (animal cells only)Pili

Prokaryotic (bacterial) Cell

Eukaryotic Cell

Animal cell

Plant cell

Nuclear materialProkaryotes - circular DNA:

Nuclear materialEukaryotes - 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 CellWe have 2 meters of DNA in all our somatic cellsHOW 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 chargedHistones proteins contain many basic amino acids (Lys, Arg), positively charged

Beads on a String(in Colorado)

Methylation - yellow hexagonAcetylation - green flagPhosphorylation - grey circle Other modifications??

The Histone Code

Methylation - yellow hexagonAcetylation - green flagPhosphorylation - grey circle Other modifications??

The Histone Code

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

Methylation - yellow hexagonAcetylation - green flagPhosphorylation - grey circle Other modifications??

The Histone Code

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 antibody-tagged 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

Secretory cells of pancreas

Skeletal muscle cell

Sperm cells

Red blood cells

Human embryo at 2-cell stage

Cell breakageAKA cell disruption, cell disintegration, lysis

Goal - destroy outer cell membrane without destroying organelle membranes

Cells broken open (plasma membrane dissolved) by:

Mechanical Chemical• freeze-thaw • solubilize with detergents• grinding • organic solvents• shearing (homogenizer) • alkali treatment• shearing (french press) • 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 wallpump 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 cellsdetergents destroy cell lipid membrane

• organic solventsdissolves cell membrane

• alkali treatmentuses 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 digestiondissolves cell membrane, “gentle” technique since enzyme attack specific components of cell membraneEX: 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

Low speed

Tissue homogenization

DIFFERENTIAL CENTRIFUGATION

Supe to medium speed

Tissue homogenate

Supe to 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

Supe to very high speed

Supernatant contains soluble proteins

Cell fractionation

Centrifugation

Separate proteins by size or densityDifferential centrifugation - separates large from small particlesIsopycnic (sucrose-density) centrifugation - separates particles of different densities

Low speed

Tissue homogenization

DIFFERENTIAL CENTRIFUGATION

Supe to medium speed

Tissue homogenate

Supe to 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

Supe to very high speed

Supernatant contains soluble proteins

ISOPYCNIC (SUCROSE-DENSITY)CENTRIFUGATION

centrifugation

Sample

Sucrose gradient

Less dense

More dense

Fractionation

Object of lab:

Isolate DNA from nuclei of eukaryotic cell (calf thymus)

1. Isolate nuclei (centrifugation after cell disruption)thymus tissue + buffercell membrane breakage using Waring blenderfilter homogenate through cheese clothcentrifuge homogenateresuspend pellet (contains nuclei) w/ buffer filter through cheese clothNUCLEAR SUSPENSION

2. Compare isolated nuclei to intact nucleiuse 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 solutionuse glass rod to pull DNA fibers into alcohol layer on top