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343 Cards in this Set
- Front
- Back
Cell
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fundamental units of life
|
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Three characteristics that define a cell
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1. Replaceable genetic information (DNA)
2. Limiting membrane (plasma membrane) 3. Metabolic machinery (catabolism and anabolism) |
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Prokaryotes
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have genetic material, but not in the nucleus
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Types of Prokaryotes
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1. Bacteria
2. Archaea |
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Eucaryotes
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Organization of cell in nucleus
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Types of Eucaryotes
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Protists
Plants Animals Fungi |
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Membrane bound organelles
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Plasma membrane
Nuclear Envelope Endoplasmic Reticulum (Smooth and Rough) Golgi Apparatus Mitochondria Chloroplasts Lysosome Peroxisome |
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Non membrane bound organelles
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Ribosomes
Cytoskeleton |
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Nucleus
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information storage, very complex structure: DNA, RNA, proteins
*appearance depends of devision phase |
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Nuclear lamina
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nuclear envelope
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Endoplasmic Reticulum
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the cells synthetic machinery
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Smooth ER
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Steroid hormone synthesis; detoxification
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Rough ER
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Proteins for export; the cells synthetic machinery
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Free ribosomes
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proteins for internal purposes
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Golgi apparatus
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protein modification, sorting and shipment
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Mitochondrion
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the cells power plant; have outer and inner membrane
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Chloroplast
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inner and outer membrane
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Lysosome
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digestion, some catabolic functions take place in the cytosol; most of the intracellular digestion is performed in the lysosomes
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Peroxisome
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oxidation; oxidized organic molecules
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Cytoskeleton
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polarity and cell movement
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Endoplasmic Reticulum
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the cells synthetic machinery
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Smooth ER
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Steroid hormone synthesis; detoxification
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Rough ER
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Proteins for export; the cells synthetic machinery
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What atoms make up 96.5% of an organisms weight?
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Carbon, Hydrogen, Oxygen, and Nitrogen
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Golgi apparatus
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protein modification, sorting and shipment
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Mitochondrion
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the cells power plant; have outer and inner membrane
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Chloroplast
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inner and outer membrane
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Lysosome
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digestion, some catabolic functions take place in the cytosol; most of the intracellular digestion is performed in the lysosomes
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Peroxisome
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oxidation; oxidized organic molecules
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Cytoskeleton
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polarity and cell movement
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Three factors that affect the Quality and Optical image
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Magnification
Resolution Contrast |
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Magnification
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increase in the relative size of an object
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Resolution
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the smallest objects that can be distinguished as a separate objects
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Contrast
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the degree to which the image of an object stands out from its background
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Types of Microscopes
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Light and Electron
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Light Microscope
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Illumination: Photons
Lenses: Glass Medium: Air Specimen: Hydrated Visualized: Eye or film |
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Electron Microscope
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Illumination: electrons
Lenses: Electromagnets Medium: Vacuum Specimen: Dried Visualized: Phosphorescent screen of film |
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Types of Contrast
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Amplitude of Contrast
Phase Contrast Interference Contrast Differential interference contrast Hoffman Modulation contrast Dark Field Fluorescence |
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Preparation of tissues for microscopy
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Fixation
Dehydration Sectioning Staining |
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Fixation
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Light: Formaldehyde
Electron: Glutarladehyde and osmium teroxide |
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Sectioning
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thin enough sections for the illumination graduation to pass through it
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Staining
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staining method largely determines what one sees in the microscope
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Electrostatic binding
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most dyes behave like acidic or basic compounds and form electrostatic bonds with ionized components of the tissues
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Acidophilia
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tissue components that stain with acid dyes are termed acidophilic (mostly proteins)
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Basophilia
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Tissue components that stain with a basic dyes are basophilic (nucleic acids and acidic sugars)
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Covalent binding
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Osmium tetroxide (OsO4)- binds covalently to lipids; black in the light microscope
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Double staining
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1. Nuclear Stain
2. Counterstain |
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Nuclear Stain
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stains the DNA of the nucleus
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Counterstain
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stains components of the cytoplasm and or ECM
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Polychrome Stains
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mixture of different stains
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Histochemical Staining
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detects and reveals the location of specific substances
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Light Microscope- Phase Contrast
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*phase contrast and differential interference microscope
-Powerful tool to observe living cells - Based on the principle that light changes its speed when passing through cellular and extacellular structures with different refractive indices |
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Light Microscope- Florescence microscope
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-Designed to illuminate the sample with light of the excitation wavelength and collect the emitted light while excluding exciting light from the image
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Fluorescence
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when substances are irradiated by light of a proper wavelength, the emit light with a longer wavelength
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Fluorescence Microscope
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-uses fluorescent dyes to provide contrast against a dark background
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Antibodies
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bind to specific antigens
|
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Ligands
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bind to receptors
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Enzymes
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bind to substrates
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Phalloidin
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bind to actin filaments
|
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lipids
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label membranes
|
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Electron Microscopy
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-uses electrons as the illuminating radiation
-required high vacuum |
|
Transmission Electron Microscope
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*analogous to bright field light microscope
*uses beam of electrons to penetrate the sample *Image formed on a phosphorescent screen from electrons that pass through the sample *used with ultra thin sections and metal |
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Scanning Electron Microscope
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*analogous to television
*uses narrow beam of electrons that scans over the surface of the sample *image formed on a video screen from electrons that are ejected from the surface of the sample and picked up by an electron detector that amplifies the signal *Used with intact objects |
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Contrant in transmission electron microscope
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-contrast is produced by the scattering of electrons from the beam by atoms in the sample (electron shattering)
-Bigger the atom, the more electrons it has and the more likely it is to repel and defect electrons from the beam -Image is formed by the electrons that are not deflected -Interactions between the electron beam and atoms in the sample produces X-rays and other types of radiation that provide information about the composition of the sample |
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Sources of Contrast in TEM
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Various methods used to enhance the ability of biological samples (composed mostly of atoms) to scatter electrons, thereby increasing contrast in the TEM image
|
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Positive staining
|
of sections with salt of heavy metals in solution (U, Pd, Os) reveals structures that bind the metals; structures appear dark compared to the background
|
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Negative Staining
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of structures with salts of heavy metals in solution (U, phosphotungstic acid) reveals structures that exclude the metal salts, producing a "negative" image; structures appear light compared to the background
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Shadowing
|
with evaporated metal atoms (Pt, W) reveals structures that accumulate a coating of atoms; structure appear dark against the background
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Ultra Thin sections
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Traditional method to visualize the internal structures of cells in the TEM
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Negative staining
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Used to visualize surface details of small samples; including macromolecules. supramolecular structures andy viruses in TEM
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Metal-Carbon Replicas
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Creates a shadow-cast replica of the surface of a sample that can be viewed in the TEM
|
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Freeze Fracture
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-Reveals the interior of biological membranes by splitting the lipid bilayer in half
-Exposes small particles (intra membrane particles) that are derived from transmembrane proteins |
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Contrast in Electron Scanning Microscope
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contrast in SEM is cause by ejecting of secondary electrons from the surface of the sample by high energy electrons in the beam
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How are atoms held together?
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Chemical bonds
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Chemical components of the cell
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properties of material from which living cells are made depend on which atoms they contain and the way these atoms are linked together to form molecules
|
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Types of Chemical bonds
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Covalent and non-covalent
|
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Covalent
|
involve sharing of electrons between atoms; strong bonds
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Non-Covalent
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attractions between atoms that do not involve sharing of electrons; weak bonds
|
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In covalent bonding...
|
multiple bonds have definite orientations in space relative to each other; specific bond angles, lengths and energies depend on atoms involved in formation
|
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Single covalent bond
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sharing of 2 electrons (one from each atom) allows the rotation around bond axis
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Double covalent bond
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sharing of more than 2 electrons; shorter and stronger than single bond; no oration around bond axis; 3D shape of molecules
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Polar covalent bond
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one atom attracts the shared electrons more than other. Positive charge concentrated toward one end of molecule and negative at the other
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Ionic bond
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caused by attraction between positively and negatively charged atoms (ions) formed by giving electrons to- or accepting electrons from- another atom
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Cation
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positively charged atom or molecule
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Anion
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negatively charged atom or molecule
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Hydrogen bond
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cause by attraction between positively charged hydrogen atom held in one molecule by a polar covalent bond and another atom (Typically N or O) that is partially negatively charged in another polar molecule
|
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Water
|
each molecule forms hydrogen bonds with 2 other water molecules -> network -> responsible for surface tension
|
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Van der Waals Attraction
|
attraction between atoms caused by fluctuating electrical charges
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Hydrophobic Interactions
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attraction between non-polar atoms and molecules in aqueous solution cause by their inability to form hydrogen bonds with water molecules
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Bond strength
|
measured by the amount of energy needed to break a bond
|
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Why is properties of water important to cells?
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Life evolved in an aqueous environment and cells are made up mostly of water.
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Properties of water
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Polar
Attracted by hydrogen bonds Cohesion- allows for surface tension and solubility properties |
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Solubility in Aqueous Solution
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molecules attracted to water are soluble
molecules that disrupt hydro bonding between water are insoluble |
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Hydrophlic
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water loving
|
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Ionic substances
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attract the polar end of water molecules with the opposite charge and surround themselves with a shell of water molecules
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Polar Substances
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form hydrogen bonds with water molecules and surround themselves with a shell of water molecules
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Hydrophobic
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Water fearing; insoluble molecules
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Hydrocarbons and non polar molecules
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Hydrophobic; break hydrogen bonds between water molecules
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Molecule
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cluster of atoms held together by covalent bonds
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Cell molecules
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organic- carbon compounds
|
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Four families of small organic molecules in cells
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Sugars
Fatty acids Amino Acids Nucleotides |
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Sugars/ Carbohydrates
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Monosaccaride, Disaccarides, Oligosaccharides, Polysaccarides, Glycoproteins, Glycolipids
|
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Monosaccarides
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single sugar molecule
(CH2O)n |
|
Types of Modnosaccarides
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Triose= 3 C
Tetrose= 4 C pentose= 5 C Hexose= 6 C Heptose= 7 C |
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Isomers
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Monosaccarides that differ only in spatial arrangement of atoms
|
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Glycosidic Bond
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Monosaccarides can be convalently linked to each other via a glycosidic bond to form chains
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Glycosidic Bond formation
|
can form between the oxygen associated with the 1-carbon and any carbon carrying a hydroxyl group
|
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Condensation (dehydration) reaction
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loss of water
|
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Hydrolysis
|
addition of water
|
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alpha position
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hydroxyl below the plane of the ring
|
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Beta position
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hydroxyl above the plane of the ring
|
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Glycosidic link
|
when glycosidic bond forms, the hydroxyl is fixed in either the alpha or beta position, forming an alpha or beta glycosidic link
|
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Complexity of Sugars
|
a small number of monosaccharides can form an extremely large number of chemically distinct molecules;
basis of chemical recognition |
|
Sources of Complexity
|
isomerization, multiple ways to link monosaccharides, branching, formation of chemical derivatives
|
|
Multiple linkage patterns
|
because each monosaccharides have several hydroxyl groups, two can be linked many different ways
|
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Branching
|
a monosaccharide can form more than one glycosidic bond, to produce a branched chain
|
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Chemical derivative of sugars
|
various chemical groups can be attached through dehydration reactions
+carboxylic acid= sugar acid +amino group= amino sugar +N-acetyl grp= N-acetyl sugar |
|
Lipids
|
are a family of hydrophobic molecules based on hydrocarbon chains
|
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Fatty acids
|
energy storage and building blocks of other lipids; unbranched hydrocarbon chains terminating in a carboxylic acid group
|
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Triacylglycerols
|
energy storage and building blocks of other lipids
|
|
Phospholipids
|
biological membranes; Glycerol + two fatty acids + phosphate group + hydrophilic compound
|
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Steroids
|
cell signaling
|
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Polyisoprenoids
|
membrane synthesis
|
|
Glycolipids (lipids - oligosaccarides)
|
cell signaling
|
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Phospholipid aggregates
|
in aqueous solution, phospholipids aggregate to form to form structures that remove the hydrophobic tails from contact with water
|
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Three forms of Aggregates
|
Micelles, Lipids monolayers, lipids bilayers
|
|
Micelles
|
form in aqueous solution when the concentration of lipids is relatively low spheres with hydrophobic tails in the center and hydrophilic heads on the surface
|
|
Lipid Monolayers
|
form at the air-surface of an aqueous solution file with hydrophobic tails in the air and hydrophilic head in the solution
|
|
Lipid Bilayers
|
form in solution at higher concentrations of lipids membrane composed of two-layers of lipid molecules with hydrophobic tails in the center and hydrophilic heads on the two surfaces
|
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Amino Acids
|
building blocks of proteins
|
|
General Amino Acid Structure
|
Central carbon atom, Amino group, Carboxyl acid group, Side group *R, hydrogen
|
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Amino Acid Charge
|
Can become positively charge on amino end, and negatively charge on carboxyl end
|
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Four families of Amino Acid Side chains
|
Acidic, Basic, Uncharged Polar, Non-polar
|
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Peptide Bond
|
two amino acids can be joined together via a condensation ration to form a covalent peptide bond
|
|
Covalent peptide bond
|
links the carboxyl end of one amino acid to the amino end of another C-N bond
|
|
Polypeptides
|
chains of amino acids linked by peptide bonds
*flexible *no branching *polarized molecule |
|
Amino acid naming:
|
Small= di, tri, tetra peptides
Medium= oligopeptides Large= polypeptide |
|
Glycine
|
does not have D and L forms like other amino acids
|
|
Nucleotides
|
molecule consistive of one or more nitrogen-containing rings (bases) covalently linked to a pentose sugar, and one or more phosphate groups attached to sugar
|
|
Nucleotide Functions
|
building blocks of nucleic acids
high energy compounds coenzymes Intercellular and signaling |
|
Nucleotide Structure
|
Pentose sugar, nitrogen base, phosphate
|
|
Nucleotide Nomenclature
|
Base + Sugar = nucleoside
Nucleoside + phosphate = nucleotide |
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Phosphodiester bond
|
between the phosphate group attached to the sugar of one nucleotide and a hydroxyl group on the sugar of the next molecule
|
|
Nucleic Acids
|
polymers of nucleotides linked by phosphodiester bonds
|
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Polarity of Nucleic Acids
|
are polarized molecules
3' end- bearing -OH group 5' end- bearing phosphate group |
|
Two types of Nucleic Acids
|
DNA: Deoxyribose A G C T
RNA: ribose A G C U |
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DNA
|
double stranded two polynucleotide chains running anti-paraellel, held together by hydrogen bonds between base chains
|
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RNA
|
single stranded chain
|
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Marcomolecules
|
polymers of small organic molecules linked by covalent bonds
|
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Metabolism
|
is the sum total of all the chemical reactions that occur in living cells
|
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Catabolism
|
reactions that break down complex molecules, release energy that can be used by cell, providers building blocks
|
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Anabolism
|
biosynthesis, reactions that synthesize new molecules using energy and molecules released by catabolic reactions
|
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Metabolism
|
Anabolism + Catabolism
|
|
2nd Law of Thermodynamics
|
states that the degree of disorder (entropy) in the universe can only increase
movement toward disorder is spontaneous and requires energy to reverse |
|
1st law of thermodynamics
|
energy can be converted from one form to another, but it can not be created or destroyed
|
|
Photosynthesis
|
light energy converted into chemical bonds energy in plant cells
|
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Respiration
|
in plants and animals, energy is extracted from food molecules by a process of gradual oxidation of organic molecules
|
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Oxidation
|
partial or complete loss of electrons
|
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Reduction
|
partial or complete acquisition of electrons
|
|
Oxidation of Carbon
|
as the carbon atom becomes increasing oxidized, the electrons in bonds spend decreasing amount of time with the carbon atom and increasing amount with the oxygen atom
|
|
Gibbs Free Energy (G)
|
the measure of energy potentially available in a molecule to do useful work
|
|
Change in Gibbs free energy
|
a change in G, measures the amount of energy released as heat (loss) when a reaction takes place. Its also measures the relative change in the order
|
|
Negative G
|
Decrease in order; reaction favorable
|
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Positive G
|
Increase in order; energetically unfavorable reaction
|
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Activation energy
|
the energy required to initiate an energetically favorable chemical retain
Sources: Heat and Enzymes |
|
Heat
|
increases molecular movement; increases the probability that potentially reactive molecules will collide with sufficient energy and orientation to react
|
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Enzymes
|
biological catalyst, responsible for carrying out the chemical reactions that make up metabolism; highly selective; have unique active bonding sites
|
|
Cells control the activities of enzymes
|
feedback inhibition, covalent modification, compartmentalization
|
|
Activated Carriers
|
the energy derived from oxidation of food molecules must be stored temporarily before use in production of small organic molecules and macromolecules
*ATP, NADH, NADPH |
|
ATP
|
most abundant and widely used activated carrier molecule in the cells; Captures chemical energy released from a n energetically favorable reaction and uses it to produce energy to drive an energetically unfavorable reaction
|
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ATP hydrolysis
|
often involves the transfer of the terminal phosphate to another molecule
|
|
NADPH and NADH
|
differ by phosphate group; serve as carriers for electrons and protons, in oxidation-reduction reactions
|
|
NADH
|
functions primarily in catabolic reactions- serves as an intermediate in the catabolic system of reactions in the oxidation of food molecules that generate ATP
|
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NADPH
|
functions primarily in anabolic reactions- supplies the high energy electrons needed to synthesize energy rich biological molecules
|
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Biosynthesis
|
macromolecules are made from subunits that are linked together in condensation reaction
require energy input |
|
Digestion
|
occurs in digestive tract/organelles; provide small molecules
*enzymatic hydrolysis of macromolecules into their subunits |
|
Glycolysis
|
occurs in cytosol; does not require oxygen; basis of anaerobic metabolism
|
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Citric Acids Cycle
|
occurs in matrix of mitochondria; requires oxygen
|
|
Oxidative phosphorylation
|
driven by electron transport across the inner mitochondrial membrane; generate ATP, requires oxygen
|
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Breakdown of marcromolecules
|
Fats-- >> Fatty acids
Polysaccharides-->> monosac Polypeptides-->> Amino Acids Nucleic Acids-->> Nucleotides |
|
Synthesis of nucleic acids, proteins and polysaccharides
|
produced by the repeated addition of a subunit onto one end of a growing chain
|
|
Energy from ATP
|
Mechanism used to link ATP hydrolysis to monomer addition in condensation reactions in polymer synthesis in cells is very complex
|
|
Compartmentalization of Metabolism
|
Cells use various strategies to organize enzymes to increase efficiency:
*speed up reactions by spatially rearraging *Control runs by separating enzymes from potential substrates |
|
Proteins
|
building blocks of cells, execute nearly all of cells functions
|
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Protein functions
|
Enzymes, structural proteins, transport proteins, motor proteins, storage, signal proteins, receptors, gene regulatory, special-purpose proteins
|
|
structural proteins
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mechanical support in cells and tissues
|
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Transport proteins
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carry small molecules and ions
|
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Motor proteins
|
generate movement in cells and tissues
|
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Storage proteins
|
store small molecules in cells and tissues
|
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Signal proteins
|
carry signals from cells to cells
|
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receptor proteins
|
detect signals and transmit them
|
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gene regulatory proteins
|
bind to DNA to switch genes on/off
|
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Proteins
|
macromolecules composed of one or more flexible chains of amino acids held together by peptide bonds
|
|
Chaperones
|
bind to partly folded chains and help to fold in crowded cell environment prevent association with other molecules until folding is complete, recognize product of mutated genes
|
|
Hsp 70
|
acts early during initial folding of polypeptide
|
|
Hsp 60
|
forms a barrel-like cage into which misfiled proteins are placed and the folding corrected
|
|
Prions
|
misfiled forms of proteins that can covert properly folded proteins into the abnormal configuration
|
|
Protein shapes
|
globular, fibrilar, filaments, sheets, rings, spheres
|
|
Common folding patterns of proteins
|
Alpha helix
Beta sheet |
|
Alpha Helix
|
hydrogen bonds formed between every 4th peptide bond
|
|
Beta Sheet
|
two or more beta sheets can occur together in one of two different configurations
|
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Parallel
|
adjacent chains have the same polarity
|
|
Antiparallel
|
adjacent chains have opposite polarity
|
|
Coiled-Coil
|
2 a helixes; very stable, hydrophobic side in the middle
|
|
Protein level of structural organization
|
Primary, Secondary, Tertiary, Quaternary
|
|
Primary
|
amino acid sequence- determines the pattern of folding of a polypeptide
|
|
Secondary
|
folding of the polypeptide into stable configurations some stable folding patterns occur repeatedly in polypeptides:a-helix, b-sheet, coiled-coil
|
|
Tertiary
|
full 3D conformation formed by entire polypeptide chain
|
|
Quaternary
|
association of two or more polypeptides into functional proteins
|
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Domains
|
regions of 100-150 Amino acids that fold independently of the rest of a polypeptide to form stable, compact structure
|
|
Domain
|
the modular unit for construction of larger proteins
|
|
Evolution of proteins
|
new proteins form by altering existing proteins
|
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Mutation
|
random changes in amino acid sequence
|
|
Natural selection
|
elimination of cells with deleterious mutations and selection of cells with advantageous mutations
|
|
Conservation of domains
|
once developed, functionally useful domains tend to be conserved
|
|
Genetic recombination
|
combine old domains in new ways to form new proteins
|
|
Protein families
|
have the same amino acid sequences and functional domains
Serine proteases, Calcium-binding proteins, and ATPases |
|
Mechanism of Assembly for proteins
|
polypeptides can assemble into large structures
|
|
Globular
|
polypeptide folded into a compact shape (ball)
|
|
Fibrous
|
long, relatively simple 3-D structure and cytoskeletal
|
|
Protein turnover
|
have a finite life span, and eventually are broken down by cells
highly controlled, not random |
|
Proteasome
|
large protein complexes in the cytosol in which individual proteins are degraded
|
|
Binding properties of proteins
|
biological properties depends on their physical interactions with other molecules
|
|
Ligand
|
molecule to which a protein binds
|
|
Binding site
|
region in the 3D conformation of a protein to which a ligand binds
|
|
Antibodies
|
Immunoglobulins- produced by immune system
each binds to particular target molecule very tightly |
|
Allostery
|
most proteins change their conformation as a result of binding to other molecules
|
|
Allosteric protein
|
a protein that can adopt two or more stable conformations
|
|
GTP-binding proteins
|
can be changed as a result of cyclic gain and loss of a phosphate group
|
|
Motor proteins
|
the conformational changes of some proteins can be coupled to do mechanical work
aid in movement |
|
ATPases
|
proteins that bind and hydrolyze ATP, some are GTP-binding proteins
|
|
Methods of studying proteins
|
*Centrifugation
*Column Chromatography *Gel Electrophoresis *Antibody methods *Amino acid sequencing *X-ray crystallography *Nuclear Magnetic Resonance (NMR) spectroscopy |
|
Cell fractionation
|
As a first step in preparing cells for study by many different procedures it is necessary to break the cells open and separate their major components
|
|
Methods to disrupt cells
|
*Homogenization
*Osmotic shock *Ultrasonication *Mechanical shear *Detergent extraction |
|
Centrifugation
|
separates cellular components on the basis of size, density or buoyancy using centrifugal force from a centrifuge
|
|
differential centrifugation
|
separates on the basis of size; The faster the speed and the longer the time, the smaller the components that will be pelleted at the bottom of the tube
|
|
velocity sedimentation
|
separates on the basis of size and shape; Cellular components separate into bands on the basis of their density
|
|
bouyant density or equilibrium sedimentation
|
separates on the basis of buoyancy
|
|
pellet
|
material that collects at the bottom of the centrifuge tube
|
|
supernatant
|
fluid above the pellet
|
|
Sedimentation Coefficient - S
|
*Characterizes the rate at which a component sediments during velocity centrifugation =
function of size and shape *Provides a useful measure of the relative size of large subcellular components and macromolecules *Determined by measuring the distance a component migrates in a density gradient over time |
|
Column Chromatography
|
Used to separate macromolecules, especially proteins; eparates macromolecules on the basis of how they interact with a solid support as they flow under gravity through a glass or metal column
|
|
Types of chromatography
|
Gel filtration, Ion-exchange, and Affinity
|
|
Gel Filtration
|
separates on the basis of size
|
|
ion-exchange
|
separates on the basis of electrical charge
|
|
affinity
|
separates on the basis of specific binding = affinity
|
|
Electrophoresis
|
Separates macromolecules (proteins, nucleic acids) on the basis of their ability to move through a gel, either in a slab or in tubes, driven by an electrical current;Direction of movement determined by the net charge of the molecule (+ or -)
|
|
Types of Electrophoresis
|
SDS-polyacrylamide gel electrophoresis (SDS-PAGE )
Isoelectric focusing and 2-dimensional gel electrophoresis |
|
SDS-polyacrylamide gel electrophoresis (SDS-PAGE )
|
separates on the basis of relative size
|
|
Isoelectric focusing
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separates on the basis of isoelectric point
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2-dimensional gel electrophoresis
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eparates on the basis of both size and isoelectric point
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Proteins - Antibody methods
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Uses antibodies to label or purify cellular components; Because of their high specificity of binding to antigens, antibodies have become a principle tool for studying cells
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Uses of Antibodies
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*as specific labels for light and electron microscopy
*to purify molecules by immunoprecipitation *to purify molecules by immuno-affinity chromatography *to identify proteins on electrophoretic gels |
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Structure of antibodies:
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proteins with paired binding sites that recognize and bind with high affinity to specific molecular sequences (epitopes) on other molecules (antigens)
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Types of Antibodies:
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Polyclonal and Monoclonal
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Polyclonal
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Made by injections of purified antigen into an animal
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Monoclonal
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Made by fusing B cells (from an animal injected with purified antigen) with a tumor cell in cell culture Þ immortal hybrid cell secreting monoclonal antibody
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Monoclonal and Polyclonal antibodies used in a variety of methods
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*Immunoaffinity column chromatography
*Immunocytochemistry Light, fluorescence and electron microscopy *Identification of molecules separated by electrophoresis |
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Amino acid sequencing
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provides an analysis of the amino acid sequence of polypeptides
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X-ray crystallography
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reveals the three-dimensional structure of macromolecules
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Nuclear Magnetic Resonance (NMR) spectroscopy
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reveals the structure of small molecules and parts of large molecules
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DNA structure
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consists of two long polynucleotide chains - DNA strands; Each strand made of 4 types of nucleotide subunits
(linked by phosphodiester bonds Þ sugar-phosphate backbone with N-bases sticking out) |
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purine
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adenine, guanine
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pyrimidines
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cytosine, thymine
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Complementary base pairing
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*enables the base pairs to be packed in the energetically most favorable arrangement (same width, 1-ring base pairs with 2-ring base Þ same distance between sugar-phosphate backbones along the molecule)
*provides the basis for replication of nucleic acids |
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double helix
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2 sugar-phosphate back-bones twist around one another Þ form a double helix with 10 bases per helical turn
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Genome
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a complete set of genetic information in a cell
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Genes
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ragments of DNA molecule coding for proteins + many non-coding sequences Þ extremely long sequence of nucleotides (message written in 4 letter code)
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Eucaryotic Cell Nucleus
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Provides a compartment in which the DNA and DNA- dependent functions are sequestered
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Nuclear Pores
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Nuclear envelope is penetrated by numerous nuclear pores
*allow passage of molecules and large particles: from the nucleus to the cytosol and from the cytosol to the nucleus *movement through the pores is regulated |
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Functions of the nucleus
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*DNA replication
*DNA packing - chromosomes *DNA transcription Þ mRNA, rRNA and tRNA *processing of mRNA *mRNA transport *ribosome assembly *dissolution and reformation of the nuclear envelope during mitosis and meiosis |
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Chromosomes
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Composed of DNA + proteins = chromatin
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ploidy
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number of sets of chromosomes per cell
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haploid
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one set of chromosomes per cell
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diploid
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two sets of chromosomes per cell
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tetraploid
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four sets of chromosomes per cell
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Karyotype
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display of the full set of mitotic chromosomes
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Giemsa dye
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stains regions rich in A-T in DNA
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Chromosome functions
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carrying genes
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Interphase chromosomes
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tangled treats - can not be distinguished in light microscope
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Metaphase Chomosomes
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highly condensed - easy to identify in light microscope
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Replication Origin
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start site for DNA replication
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Centromere
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site where daughter chromosomes remain attached during division, marks site where the kinetochore will form to attach microtubules during division
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Telomere
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epeated nucleotide sequences at the ends of chromosome, solves "end-replication" problem in eukaryotes
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Problem: extremely long DNA molecules in the cell must be packed into a very small volume and in such a way as to:
*prevent tangling *be reversible *allow rapid, localized, on-demand access to DNA |
Solution: DNA is packed into a series of higher order structures by specialized proteins
that coil and fold the DNA |
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Chromosomal proteins
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Histone and nonhistone chromosomal proteins
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Histones
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*responsible for the first level of chromatin packing
·nucleosomal histones = H2A, H2B, H3 & H4 small, highly conserved proteins responsible for coiling of DNA into nucleosomes ·H1 histones - pack the DNA+nucleosomes into a coil *positively charged *the most highly conserved of all known eucaryotic proteins |
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Nucleosomes
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*fundamental packing units of DNA
*made of: ·protein core - complex of 8 histone proteins (histone octamer - 2 of each H2A, H2B, H3 & H4) ·double stranded DNA - ~146 nucleotide pairs wrapped twice around octamer ·linker DNA - up to 80 nucleotides |
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Formation of Nucleosomes
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*1st level of packing
*converts a DNA molecule into a chromatin tread - ~ 1/3 of its initial length *“beads on a string” form of chromatin |
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Formation of 30 nm fiber
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*2nd level of packing
*native form of DNA *nucleosomes bundled together by H1 histones *unclear how nucleosomes are packed in a fiber - most probable zigzag model or solenoid structure |
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Levels of DNA packing
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1. Nucleosomes formed
2. Formation of 30nm fiber 3. Looped domain - current model Loops of 30 nm fiber attached to proteins that form the chromosomal axis 20,000 - 100,000 bp per loop 4. Metaphase chromosome - final level of packing |
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Interphase Cells
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Chromatin in an interphase chromosome is not in the same packing state throughout the chromosome:
*regions with genes that are being expressed are more extended, regions with quiescent genes - more compact Þ chromosome structure can differ from cell to cell and during cell lif |
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Forms of chromatin in interphase cell
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Heterochromatin, Active euchromatin, inactive euchromatin
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Heterochromatin
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10% of chromatin)
·highly condensed ·transcriptionally inactive ·most of the heterochromatin does not contain genes ·concentrated around centromere and telomeres |
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Active euchromatin
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(10% of chromatin)
·least condensed ·histone H1 less tightly bound ·nucleosomal histones chemically modified |
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Inactive euchromatin
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(80% of chromatin)
·more condensed than active euchromatin ·can become active euchromatin |
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Mitotic Cells
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*Metaphase chromosomes - very condensed chromatin
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Sperm Cells
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*Sperm head - most condensed form of chromatin
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Nucleosome Replication and Assembly
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*Nucleosomes must be moved out of the way to permit DNA to replicate or be translated
*New nucleosomes must be assembled as DNA is replicated *New histones are synthesized at the same time as DNA replication *New nucleosomes assemble on the daughter DNA helices shortly after the DNA is replicated |
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Nucleosome-binding to DNA affected by
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Base sequence (AT-rich regions easier to bend)
Binding of other proteins - may displace nucleosomes |
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Eucaryotic cells have mechanisms to adjust the local structure of chromatin
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*chromatin remodeling complexes
*reversible modification of histone tails Chromatin remodeling complexes and histone tails modifying enzymes may work in concert allowing rapid changes in chromatin structure according to cell needs |
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chromatin remodeling complexes
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·protein machines
·use ATP to change nucleosome structure ·make DNA more accessible to specialized proteins (e.g. these involved in replication, gene expression and DNA repair) ·inactivated during mitosis - helps maintain tightly packed chromosome structure |
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reversible modification of histone tails
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·N-terminal tails function in regulating chromatin structure
·undergo covalent modifications after nucleosome assembly ·modified tails bind and attract specific proteins to different chromatin regions (some facilitate further chromatin condensation, some facilitate access to DNA) ·histone modifying enzymes are strongly regulated ·different combinations of tail modifications and different sets of histone-binding proteins give different signals (e.g. for gene expression, replication) |
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DNA - Replication
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Must occur before cell division.
Cell has to copy its genome with great accuracy - Mistakesin replication cause mutations |
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template
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Each strand of a DNA double helix can serve as a template for the replication of the other (complementary)strand
-Complementary base pairing with the template strand determines which new nucleoside is added tothe new (daughter) strand |
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DNA replication in cells is semiconservative
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*both of the two parent strands are conserved, one ineach
of the two daughter molecules *daughter strands are complementary to the respective parent strands *therefore, the two daughter molecules are identical to the originalparent molecule |
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Initiator proteins
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bind to the DNA and open double helix
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Replication Origin
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Site on the DNA double helix where replication is initiated
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replication bubble
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Site where the double helix first open.
*consist of specific nucleotide sequences recognized byinitiator proteins *A-T rich (easier to separate) |
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Replication origins and Cell type
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Prokaryotes
*1 replication origin per chromosome Eucaryotes *multiple replication sites on each chromosome |
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Replication Fork
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-Replication of DNA occurs at replication forks
-Each replication origin generates 2 replication forks -Y-shaped structure resulting from the separation of the DNAdouble helix into two strands during replication -Move away from origin in both directions |
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DNA Polymerase
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-Enzyme responsible for DNA synthesis
-can catalyze DNA synthesis in only one direction: 5' to 3' |
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Leading strand
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synthesis in the same direction that the replication fork is opening, allows continuous DNA synthesis
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Lagging Strand
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synthesis in the opposite direction in which thereplication fork is opening (Okazaki fragments)
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Proofreading Ability of DNA Polymerase
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DNA polymerase is very accurate (~1 error in 107copied nucleotides), but if the wrong nucleotide is inserted, the polymerasecan correct its mistakes
Contains both polymerizing and editing sites |
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Proofreading
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1. Before adding a new nucleotide polymerase checks if theprevious nucleotide is correctly base-paired to the template strand
2. If the complementary base pairing is inaccurate, DNA polymerase recognizes the mismatch and hydrolyzes the phosphodiester bond (exonucleaseactivity), removing the nucleotide 3. DNA polymerase inserts a new nucleotide and moves forward |
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Removal of RNA primers and Closing of the DNA-DNA gaps
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1. Nuclease beaks down and removes the RNA primer
2. Repair polymerase replaces primer with a DNA usingadjacent Okazaki fragment as a primer 3. DNA ligase joins 5'-phospate end of one new DNA fragment to 3'-hydroxyl end of the next fragment (there is no high energy phosphate bondto supply energy, DNA ligase uses energy from ATP to catalyze the formation ofa phosphodiester bond) |
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Meeting of replication bubbles
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When adjacent replication bubbles meet, the daughter DNAstrands separate and DNA replication ceases
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DNA helicase
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separates the bases of the DNA doublehelix at the replication fork
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Single-strand binding protein
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monomers bind tosingle stranded DNA in the replication bubble to stabilize it
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Sliding clamp protein
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keeps the DNA polymerasefirmly attached to the DNA template, allows it to slide along DNA
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DNA topoisomerase
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transiently cuts one or bothstrands of the DNA double helix ahead of the replication fork to allow thereplication bubble to spin as the DNA untwists
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Telomerase
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Adds multiple copies of telomere DNA sequence to the ends ofchromosomes Þproduces template for lagging strand completion
Daughter strand replicated by DNA polymerase (with anassociated DNA primase) Without Telomere replication and Telomerase activity Þshortening of chromosomes Þ loss of important information |
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DNA - Repair
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Maintenance of the accuracy of the DNA genetic code iscritical for the long- and short-term survival of cells and species
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Errors in DNA
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Errors in DNA = mutations
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DNA Mismatch Repair System
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Removes replication rare errors that escape the replication machine
mutations in genes coding for mismatch repair proteins -molecular base of inherited predisposition to certain cancers: Deficiency in mismatch repair Þ higher rate ofaccumulating mutations Þ high chance of neoplastic transformation |
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Depurination
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spontaneous hydrolysis of a purine base produces adepurinated sugar, affects 5000 bases per cell per day
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Deamination
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spontaneous hydrolysis of an amine group, converts cytosineto uracil, affects 100 bases per cell per day
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Pyrimidine dimerization
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Ultraviolet light promotes covalent bonds formation betweenadjacent thymine or cytosine bases in the same DNA strand = pyridinedimerization reactions
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3 common steps in DNA repair:
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Excision, Resynthesis, and Ligation
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Excision
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enzymatic removal of the damaged nucleotide, enzyme -various nucleases
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Resynthesis
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replaces the missing nucleotide, replacement - complementaryto base of opposing strand
enzyme - DNA repair polymerase |
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Ligation
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seals the nick in the sugar-phosphate backbone, enzyme - DNAligase
|
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DNA repair processes:
|
*operate continuously during the life of a cell to repairspontaneous damage
*operate during replication to repair mistakes *extremely important for health and survival of individualorganisms and species *cells make a large investment in DNA repair systems *single cell organisms have more than 50 different proteinsinvolved in DNA repair *DNA repair pathways are more complicated in eukaryotes |
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DNA Recombination
|
Although a stable genome is essential for the existence ofboth the individual and the species, the origin of the differences betweenindividuals and the origin of species depends on the ability of the genome toundergo change
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Homologous recombination
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Caused by unequal crossing over between homologouschromosomes ÞChromosome Rearrangement
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Crossing Over:
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Duplicated homologous chromosomes pair up to form tetrads inwhich similar DNA sequences align and are in register
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Site-specific recombination
|
Allows DNA exchange between helices with different nucleotidesequences
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Transposable Elements
|
Cause mutations directly by duplication, addition, ordeletion of nucleotide sequences
2 types: Transposons & Retrotransposons |
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Transposons
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Pieces of DNA that move from place to place within thegenome by a cut-and-paste mechanism. Process mediated by enzymes = transposasesresiding within the transposable element. Most move rarely
|
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Retrotransposons
|
Move via an RNA intermediate:
RNA copied from DNA by RNA polymerase ÞDNAcopied from the RNA by reverse transcriptase Þ DNA reintegratesinto another site in the genome |
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Viruses
|
fully mobile genetic elements that can move fromcell to cell - genes enclosed by a protein coat
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Retroviruses
|
use single-stranded RNA as their genetic material and as atemplate to make DNA
Enzyme responsible - reverse transcriptase |