Prokaryotic vs. Eukaryotic Cells

Organelles PROKARYOTESEUKARYOTESCell (plasma) membrane Cell wall 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 PeroxisomeRibosomes FlagellaFlagella (animal cells only) 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 bp DNA wound twice around it 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)

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?? 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 EuchromatinHeterochromatin

Methylation - yellow hexagon Acetylation - green flag Phosphorylation - 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 breakage AKA cell disruption, cell disintegration, lysis Goal - destroy outer cell membrane without destroying organelle membranes Cells broken open (plasma membrane dissolved) by: MechanicalChemical 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 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 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 density Differential centrifugation - separates large from small particles Isopycnic (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 + 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