FA13 Cell Biology Final

Molecular & Cell Biology Fall 2013 Semester
List of Objectives, Key Concepts and Diseases covered in Block 1

Presentation # 1: Molecules of the cell
Must read Chapter 2 of The Cell, A Molecular Approach
Understand the important properties of water
o Most abundant molecule in cells. Polar, cohesive properties, high capacity to stabilize, and excellent
solvent (not everything can dissolve in water such as non-polar and hydrophobics)
o Know the importance of polarity in the molecule
First, since water is polar, it has an unequal distribution of charge. This means that water
molecules are slightly positive and negative. This quality is important because that makes water a
good solvent (it can dissolve many things). Other polar compounds and ions can easily be
dissolved in water because polar + polar = even distribution of charge, and ions have a charge, so
it's attracted to the opposite charge on the water molecule.
Second, the polarity of water is important in repelling nonpolar compounds. Nonpolar
compounds don't dissolve well in water (like how oil, a nonpolar solution, forms "beads" in
water). This is important to cell membranes in the body. The shape and function of cell
membranes depend on the interaction of polar water with nonpolar membrane molecules.
o Know the importance of hydrogen bonding between water molecules
Hydrogen bonding is important in many chemical processes. Hydrogen bonding is responsible for
waters unique solvent capabilities.
The 104.5 degree angle of the two hydrogens relative to the oxygen very important in making
water molecules polar
Hydrogen bonds involve weak, attractive interactions between a proton, and two electronegative
atoms.
Water molecules can form up to 4 hydrogen bonds
Know the forces responsible for the hydrophobic effect
o Hydrophobic interactions occur as a consequence of water trying to maintain max number of hydrogen
bonds with itself, thus driving hydrophobic molecules together. Another way to look at it: the association
of molecules unable to interact with water minimizes holes in the water structure.
Know the terms Transcription and Translation and where they occur in the cell
o Transcription is producing an RNA copy from a DNA template (occurs in the nucleus and
mitochondrial matrix) and this occurs in the nucleus. Translation is producing a polypeptide using an
mRNA as a template (most occurs in the cytosol and also found in the mitochondrial matrix what
structure is critical for translation? Ribosomes).
Understand the structures and functions of the major classes of macromolecules
o Proteins
Function: catalysts, structural elements, chemical messengers, transport agents, protective
barriers.
Understand the basic structure of amino acids and peptide bond formation
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Know the following amino acids and their structures:

Uncharged amino acids
Glycine -Simplest amino acid, found every 3rd position in collagen
Cysteine forms disulfide bonds
Proline - Hydroxylated in mature collagen, disrupts secondary structure
Polar uncharged amino acids

Serine - Phosphorylation, O-linked glycosylation (major site of glycosylation is the
golgi)
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Threonine - Phosphorylation, O-linked glycosylation
Tyrosine Phosphorylation
Asparagine N-linked glycosylation (initially takes place in the lumen of the rough
ER)
Positively charged amino acids

Lysine - Hydroxylated in mature collagen
Glutamic acid Vitamin K dependent carboxylation, excitatory neurotransmitter in

CNS
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Understand the levels of organization for protein structure

Primary, secondary, tertiary, quaternary
o Must know the bond(s) important for each level
Level of Basis of structure Kinds of bonds
structure
Primary Amino acid sequence Covalent peptide bonds
Secondary Folding into alpha helix or beta pleated sheet, or Hydrogen bonds
random coil
Tertiary 3D folding of a single polypeptide chain Disulfide bonds, hydrogen bonds, ionic
bonds, van der waals, hydrophobic
interactions
Quaternary Association of two or more folded polypeptides to Same as for tertiary structure!
form a multimeric protein
Alpha helix every 4th AA forms hydrogen bonds, which are parallel to the helix axis. Carbonyl (coo-) binds with
the amino (NH) of another.
Beta sheet hydrogen bonding is perpendicular to the plane of the sheet and either parallel or antiparallel.
Structural motifs units of secondary structure that consist of small segments of alpha helix and beta sheets.
A domain is a locally folded unit of the tertiary structure (containing alpha helices and beta sheets) and is
associated with particular functions.
Understand clinical consequences of certain misfolded proteins and polypeptides

(Alzheimer, Huntington and prion disorders)
Misfolding of proteins and peptides results in spontaneous assembly of insoluble amyloid
fibrils, toxic to the cells they are found in.
Huntingtons disease (HD) - polyglutamine expansion disease expanded
polygutamine repeat in the protein huntingtin (Htt) causes protein to aggregate and
damage neurons. HD is an autosomal dominant condition caused by an expansion of
CAG (glutamine) trinucleotides. People without HD have less than 26 CAG repeats;
WITH HD >40 repeats.
o Symptoms: early stage: minor chorea, difficulty in mental planning, and often
depressed or irritable state. Next stage: more pronounced chorea, poor
articulation (dysarthria) and dysphagia (difficulty in swallowing). Late
stage: behavioral problems are lessened; severe motor disability becomes severe,
and individual often becomes dependent, mute, and incontinent.
Alzheimers disease (AD) - common form of dementia; caused by the abnormal
cleavage of the amyloid precursor protein (APP encoded on chromosome 21) that
creates a neurotoxic peptide. Secretases cleave APP
o Symptoms: confusion, poor judgment, language disturbance, agitation,
withdrawal, hallucinations, parkinsonian features, mutism, increased muscle
tone, myoclonus, and poorly recognized failure of memory; forgetfulness;
o Clinical duration: 8-10 years with a range of 1-25 years. About 25% of all AD is
familial.
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Prion diseases (transmissible spongiform encephalopathies) affect both humans and
animals family of rare, progressively neurodegenerative disorders.
o Long incubation periods, neuronal loss, and failure to induce an inflammatory
response.
o Prion: abnormal transmissible agent capable of inducing abnormal folding of
cellular prion proteins (leads to brain damage)
o Normal PrP(c) protein is mostly alpha helix and SOLUBLE
o Abnormal PrP(sc) protein has 45% beta sheets, insoluble, and protease is
insensitive, produces cell surface aggregates that kills cell.
Usually rapidly progressive and always fatal
o Nucleic Acids (RNA & DNA)
Know the common types of RNA
mRNA: translates information for the production of polypeptides.
tRNA: carry amino acids and transfer them to growing chain during protein synthesis
rRNA: integral components of ribosomes
snRNA: components of splicosomes involved with mRNA splicing
miRNA: regulatory RNAs involved with control of gene expression
other types include regulatory RNA, antisense RNA, ribo-switch RNA, and small
nucleolar RNA
Know the basic structures of nucleotides (both NTPs and dNTPs)
Must know the structure of the bases!!!
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Know which bases are complementary with each other

Know which bases are used in transcription and which used during DNA replication
In DNA, A/T and G/C
In RNA, A/U and G/C
o Carbohydrates/polysaccharides
Be familiar with the difference between alpha and beta glycosidic linkages
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Know the key monosaccharides: glucose, mannose, galactose and fructose
Know the key disaccharides:
Lactose: composed of galactose and glucose
Sucrose: composed of glucose and fructose
Maltose: composed of two glucoses
Understand the basic structural and functional differences between Glycogen vs Amylose vs
Amylopectin vs Cellulose including types of glycosidic bonds
Cellulose- found in plants; beta 1-4 bond as in cellobiose and is unbranched
Amylose- found in plants (starch); alpha 1-4 bonds and is unbranched
Amylopectin- found in plants (starch); alpha 1-4 bonds; branched every 12-25 glucose
units via alpha 1-6 bond; side chain about 20-25 glucose long
Glycogen- found in animal cells and some bacteria; alpha 1-4 bonds; highly branched
every 8-10 glucose units via alpha 1-6 bonds; side chains about 8-12 glucose units. Will
have a high concentration in your liver.
Know that chitin is a linear homo-polymer of N-acetylglucosamine GlcNAc
Carbohydrate
Other important structural carbohydrates n-acetyalmuramic acid chitin in cell walls;
o Lipids
Know the basic structure of the six categories of lipids
Fatty acids, - Saturated and unsaturated fatty acids
o Saturated: no double bonds (linear)
o Unsaturated: has double bond. Kinked shape if cis double bonds.
Triacylglycerols (3 fatty acids attached to a glycerol backbone).
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o found in lipid droplets and adipocytes
Phospholipids (glycerol based and sphingosine based)
arachidoic acid
Glycolipids
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- ceramide is composed of a sphingosine and a fatty acid.

Steroids
o Cholesterol is an important steroid found in the body.
Terpenes (examples: coenzyme Q- electron transport, dolichol- important carrier and

vitamin A eye sight)
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Understand that many proteins and other macromolecules can self-assemble into functional 3-D
conformations
o The information required to specify the folding of macromolecules and their interactions to form more
complicated structures with specific biological functions is inherent in the polymers themselves.
o Anfinsens Dogma: postulate in molecular biology that, at least for small globular proteins, the native
structure is determined only by the protein's amino acid sequence. Demonstrated using ribonuclease
(RNase)
o Molecular Chaperones assist the assembly of some proteins
Hsp60 & Hsp70
Presentation # 2: Cell and Organelles

Read Chapter 1 of The Cell, A Molecular Approach
Learn about key differences between Eukaryotic and Prokaryotic cells
o Eukaryotes have membrane bound organelles and nuclei whereas prokaryotes do not
o Eukaryotes often have linear chromosomes, whereas prokaryotes have circular chromosomes
o Both eukaryotes and prokaryotes have ribosomes
Understand key facts about structure and function of the following cellular organelles:
o Plasma membrane
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Structure: lipid bilayer with proteins imbedded in it.
Function: define boundaries; permeability barrier; regulates transport of solutes; mediate cell-
cell communication
o Nucleus*
Structure: nuclear envelope is composed of two membranes. Double membrane bound organelle
Function: Site of DNA storage and transcription; nuclear pores regulate transport
o Nucleolus*
Site of ribosomal RNA synthesis and ribosomal subunit assembly
o Mitochondria*
Double membrane bound organelle
Four key structures
Outer membrane
Intermembrane space acidified by protons localized here and used for the ATP synthase
of the inner membrane.
Innermembrane
o Cristae folds and the site of electron transport and ATP synthesis.
Matrix
o Semi-fluid inside that mitochondria site of krebs cycle
Know the structure and function for each compartment
o Rough endoplasmic reticulum (rER)*

A region of ER associated with ribosomes,
often appearing as flattened sheets.
Function: site of protein synthesis and
modification of synthesized proteins
o Smooth endoplasmic reticululm (sER)*
Structure: often consists of short
anastomosing tubules that are not
associated with ribosomes.
Function: lipid and steroid metabolism;
membrane formation and recycling;
principal organelle involved in detoxification and conjugation of noxious substances; glycogen
metabolism; calcium storage
o Golgi complex*
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Structure: stacks of flattened membrane, often next to one side of the nucleus. Flattened
cisternae close to rER is cis-golgi network, and the cistaernae facing away is trans-golgi network.
Between the two is the medial golgi
Function: chemical modifications of proteins and glycolipids; site of glycosylation. Sorting and
packaging of molecules for secretion or transport
o Recycling, early and late endosomes

Early endosome
Structure: membrane enclosed tubulovesicle located near the plasma membrane. The
lumen is sub-divided into cisternae by invaginations. [pH- 6.2-6.5]
Function: sort and recycle proteins internalized by endocytotic pathways
Recycling endosome
function: store and recycle membrane components
Late endosome
Structure: complex structures that often exhibit onion like internal membranes. pH ~ 5.5
Function: mature into lysosome
o Lysosomes*
Structure: single membrane bound organelle and contains acid hydrolases.
Function: digestion of macromolecules
o Peroxisomes
Structure: single membrane bound and contains catalase (destroys hydrogen peroxide)
Function: breakdown of long chain FA; generation and degradation of hydrogen peroxide;
detoxification of harmful compounds
o Ribosomes*
Responsible for translating proteins. Composed of two sub-units and each is made up of rRNA
and ribosomal proteins. There are size differences between eukaryotes and prokaryotes
You MUST know the sizes of prokaryotic and eukaryotic ribosomal subunits
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Proarkyotes
o Complete: 70S
o Large subunit: 50S
o Small subunit: 30S
Eukaryotes
o Complete: 80S
o Large subunit: 60S
o Small subunit: 40S
o Cytoskeletal elements
Microtubules (composed of alpha- and beta- tubulin)
25 nm
Microfilaments (composed of actin)
7 nm
Intermediate filaments(composition varies)
8-12 nm
Function: cytoskeleton is responsible for 1) cell locomotion, 2) intracellular movement, 3)
framework for anchoring cell components, 4) and to provide cell structure.
o Centrosomes and Centrioles*
Centrosomes organize microtubule spindles; microtubule organizing centers (MTOC). Often
associated with the nucleus and golgi complex. The dominant feature of centrioles is the
cylindrical array of triplet microtubules with associated proteins.
Centrioles are involved in the organization of the mitotic spindle and in the completion of
cytokinesis
o Cilia, Flagella and Microvilli (know main cytoskeletal components)
Cilia and flagella are microtubule based structures. Important for movement of extracellular
fluid or cell locomotion.
Microvilli- actin microfilament based structure. Increases surface area of plasma membrane and
is important for absorption.
o Vesicles
o Glycogen granules*
Usually found in the cytoplasm. Highly branched polymers of glucose. Its function is the storage
of glucose.
*Students should be able to recognize the above structures with asterisks in an electron microscope
o
picture
Understand what is the endomembrane system and basic process of secretion
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o Endomembrane system is interconnected system of cytoplasmic organelles in eukaryotes. Consists of
rough and smooth ER, nuclear envelope, golgi complex, endosomes, lysosomes, and vesicles.
o Process of secretion: 1) synthesized on the rough ER; 2) passed to the golgi complex for processing; 3)
compartmentalized into secretory vesicles; 4) vesicles then make their way to the plasma membrane, fuse,
and release to the exterior of the cell.
Know that there is an extracellular matrix
o Provides mechanical and structural support for tissues and influences communication and cell migration.
Consists of: proteogylcans, multiadhesive glycoproteins, glycosoaminoglycans (GAGs), collagen an d
elastin.
Know the cellular junctions
o Tight junctions- prevent the free passage of molecules between epithelial cells and separate the apical
and basolateral domains of epithelial cell plasma membranes
o Gap junctions- direct connections between the cytoplasm of adjacent cells. Open channels through the
plasma membrane allowing ions and small molecules
o desmosomes- localized spot-like junctions between cells
o hemidesmosomes- anchors cells to basement membrane
o adhesive junctions- connect cells into sheets
Presentation # 3: Membrane Structure and Dynamics

Be able to describe membrane function and structure
o Membranes define boundaries and serve as permeability barrier. Membranes are also sites of specific
activity. Regulate transport of solute; detect and transmit electrical and chemical signals. Also useful for
cell-to-cell communication
o Structure: composed of lipids, proteins, and carbohydrates. The most abundant lipid in membranes is the
phospholipids (amphipathic both hydrophilic and hydrophobic regions)
Know the structure and function of the following components of a membrane:
o Lipids
Phospholipids
Phosphatidylcholine
o Found in outer monolayer
Phosphatidylethanolamine
o Found on monolayer facing cytoplasm
Phosphatidylserine
o Found on monolayer facing cytoplasm
o Negatively charged
Sphingomyelin,
o Sphingosine back bone
o Ceramide and phosphocholine sphingomyelin
o Found in outer monolayer
Cholesterol (Sterol)
Found in both layers. A single cholesterol molecule doesnt span both layers.
o Proteins
Integral membrane proteins
o Have hydrophobic transmembrane segments. Some are lipid anchored proteins
(eg. GPI). To disrupt integral membrane proteins, use detergents to isolate
Single-pass, multipass, and monotopic
o Single pass- single alpha helix transmembrane domain. Extracellular:
oligosaccharide chains and disulfide bonds. Cytoplasmic: sulfhydryl groups do
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not usually form disulfide bonds because of the reducing environment and
maintain their SH bond.
Know that most single-pass proteins have an alpha-helical
transmembrane domain
o Multipass- multipass proteins having the same features as single pass except for
more than one alpha helix in the transmembrane.
o Monotopic- proteins have only one on the extracellular or cytoplasmic side.
Peripheral membrane proteins
Associated through weak electrostatic and H-bonding; lack discreet hydrophobic
segments; disassociated from membrane by change in pH or high salt concentration
Lipid-anchored proteins
Covalently linked to lipids in the membrane
Cytoplasmic facing: linked to either a fatty acid or isoprene derivative (prenyl groups
such as farnesyl)
Extracellular facing: glycosylphosphatidylinositol (GPI)
o GPI made in ER as single pass, cleaved, then linked to GPI. Released by
phospholipase C.
Know freeze-fracture technique
Frozen sample is fractured by a sharp blow along the plane between the bilayer, giving
rise to a P (protoplasmic or cytoplasmic) and an E (extracellular layer) by the electron
microscope.
o Carbohydrates
Glycolipids- no phosphate
Other neutral glycolipids (eg. A, B, O blog group antigens) contain many sugar moieties
Cerebrosides
o Neutral- single uncharged sugar.
Gangliosides (example: GM2 tay sachs disease is inability to degrade GM2)
o Gangliosides have one or more negatively charged sialic acid
Glycoproteins
Both glycolipids and glycoproteins found facing on the extracellular side of the bilayer
Know about the production of eicosanoids from the precursor arachidonic acid
o Lipid component of the membrane releases arachidonic acid in response to physical injury or
inflammatory response. Enzymes catalyze the breakdown of membrane lipid to arachidonic acid, and
converted to:
Straight chain eiconsanoids (leukotrienes) by lipoxygenase
Cyclic eiconsanoids (prostaglandins, prostacyclin, and thromboxane) by cyclooxygenase
Inappropriate blood clotting, increased temperature etc. sometimes you want to block the
production of prostaglandins.
o COX and prostaglandin production
COX1 produces eiconsanoids used in many normal physiological process
COX2 produces eiconsanoids used in the inflammatory response
Know the Fluid Mosaic Model of membranes
o Now accepted view of membrane structure. This model depicts proteins as either embedded (integral) or
associated (peripheral) with the membrane.
o The mosaic part comes from a mosaic of proteins and lipid rafts. Evidence from freeze fracture
microscopy abundance of proteins on the cytoplasmic side, but may be a result of the cytoskeleton
pulling extracellular layer proteins inside.
Know about membrane asymmetry
o Membranes are asymmetric inside and outside faces. Synthesis of new membranes begins in the ER on
the cytosolic side of the membrane, further modified in the ER and golgi determines their distribution
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Two lipid bilayers differ in their lipid composition. Carbohydrates are restricted to the
membranes exterior. Glycolipid (found on the surface of all plasma membranes) and
glycoproteins are also found in the outer-membrane.
o P.choline and sphingomyelin are more abundant in the OUTER-layer
o P.serine, p.ethanolamine, p.inositol are more abundant in the INNER-layer
o The role of Flippases asymmetry, in part, caused by phospholipid translocators such as flippases. Move
p.ethanolamine and p.serine from the outer-layer (using ATP) and transports them into the inner
membrane
Know about the effects of lipids & sterols on membrane fluidity
o Evidence from the FRAP (fluorescence recovery after photobleaching) test showed the fluidity of the
membrane. In a few seconds, the bleached area was filled back up.
o Fatty acids are essential to regulation of membrane fluidity. Most are 12-20 FA in length and the thickness
of the membrane is dictated by these lengths. Unsaturated FA do not tightly pack because of kinks (double
bonds disrupt van der waals interactions). Long chain FA increase melting temperature more than short
chain. Membranes with UNsaturated FA decrease melting temperature and increases fluidity.
o Effect of sterols: makes membranes temperature dependent less fluid at higher temperatures and
increases membrane fluidity at lower temperatures. Prevents FA from getting too close. Sterols also
decrease permeability of a lipid bilayer to ions and small molecules (better barrier and insulator)
Know about the lipid rafts
o Localized regions of membrane that sequester proteins involved in cell signaling (microdomain). Outer-
membrane rafts have elevated cholesterol and glycosphingolipids. The lipids tend to be saturated, thicker,
and less fluid than the rest. Inner-membrane rafts may contain kinases.
Know about the Glycocalyx
o It is a barrier comprised of glycolipids, integral membrane glycoproteins (proteoglycans). Carbohydrates
on the cell surface are important recognition sites of membrane receptors. Glycoproteins have shorter
chains attached, and proteoglycans have longer chains attached to the integral protein.
Must know the role of spectrin and ankyrin in the red blood cells
o Ankyrin: anchors band 3 to spectrin (band 3 is an anion exchange protein of Cl- and bicarbonate)
o Spectrin: provides scaffold for linking proteins on membrane.
o Glycophorin: provides and external negative charge that repels other cells.
o Band 4.1: links glycophorin to actin and spectrin
o Must know the images showing the spectrin-based cytoskeleton
Clinical Correlations:
o Inhibition of COX by NSAIDs
Aspirin is a non-steroidal anti-inflammatory drug that irreversibly inhibits cyclooxygenase and
is used to reduce effects of MI, inhibit platelet aggregation, and reduce pain and fever, and
general anti-inflammatory agent.
Ibuprofen will reversibly bind to cyclooxygenase
o Hereditary Spherocytosis
Presentation # 4: Transport Across the Membrane

Be familiar with mechanisms of transporting material across the membrane and key examples
o Know the differences between passive and active transport
Passive transport is the movement of particles down a concentration gradient and do not require
energy. Examples of passive transport are channel-mediated and transporter-mediated.
Active transport requires the expenditure of energy in order to move particles against the
concentration gradient.
o Recognize examples of different mechanisms of transport across the membrane
Simple diffusion vs facilitative diffusion vs direct active transport vs indirect active transport
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Simple diffusion- move solutes towards equilibrium and proceeds from a region of
higher to lower energy (equilibrium being the lowest energy state).
o Example: water and oxygen entering and leaving the membrane of erythrocytes.
Facilitated diffusion- protein mediated movement down the gradient BUT DOES NOT
REQUIRE input of energy.
o Example: aquaporin to transport water into and out of the erythrocyte
Uniport vs Antiport vs Symport
Uniport- single solute
o GLUT1-5 provides the transport of glucose across the cell membrane into the
cytoplasm of the cell.
1 found in RBC, glia, and endothelial cells of the BBB, where it
functions in basal transport of glucose into the cell.
2 found in hepatocytes where it functions in bidirectional glucose
transport.
3 found in neurons.
4 found in adipocytes, skeletal muscle cells, and cardiac myocytes
where it functions in insulin-stimulated transporter of glucose into the
cell.
5 found on surface absorptive cells of the small intestine mucosa where
it functions in the transport of fructose into the cell.
antiport- solutes transported in opposite directions of the same protein transporter
o example: erthryocyte anion exchange protein: an antiport carrier
Chloride shift Cl- across the membrane by counter transport with
bicarbonate. This is important in the transport of carbon dioxide in the
body and for helping to regulate pH
symport- both solutes in the same direction of the same protein transporter
Know that carrier-mediated transport exhibits saturation kinetics whereas simple diffusion
does not
Simple diffusion rate is a linear function of the concentration. There is no plateau as there
is in the transporter-mediated diffusion. [solute]= [transporter]. A plateau indicates
saturation. For transporter mediated diffusion, a plateau is reached. 1/2 Vmax is called
the Km. this concentration gives you max obtainable velocity. It does NOT say that
the transporters are occupied.
o Osmosis
Know and recognize examples of the terms hypertonic, hypotonic, and isotonic
Osmolarity- concentration of osmotically active particles in solution, which may be
quantitatively expressed in osmoles of solute per liter of solution.
Hypertonic- a solution with a higher solute concentration than inside the cell
Isotonic- a solution with an equal solute concentration compared to the inside of the cell
Hypotonic- a solution with a lower solute concentration than inside the cell
Know how ouabain effects cells
An inhibitor of sodium/potassium pump. Exposure to ouabain causes cell swelling. At
low concentrations, ouabain will stimulate the Na/K pump. These pumps maintain
gradients.
o Diffusion across membranes
Know that gases such as oxygen, nitric oxide and carbon dioxide cross the membrane by
simple diffusion
Effect of size, polarity and charge
Membranes are generally more permeable to smaller molecules than larger ones.
Membranes are also permeable to nonpolar, and less permeable to polar. Membranes are
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relatively impermeable to ions. Small nonpolar molecules such as oxygen, carbon
dioxide, and ethanol can easily diffuse across membranes.
Nitrogen is more hydrophobic and can get into the fats. This is how divers may get the
bends. Swimmers would not be affected as much as couch potatoes.
o Facilitated diffusion (channels and carriers)
Two main classes of proteins involved with facilitated diffusion: carriers and channels.
o Carriers
Carriers bind one or more solutes on one side of the membrane and undergo a conformational
change to bring solutes in.
Uniporter (Examples: Glut permeases)
Co-transporters
Antiporter (Example: Anion exchange protein)
o The erythrocyte anion exchange protein aka band 3 protein or
chloride/bicarbonate exchanger
Solute binding sites of the anion exchange protein interact with different
ions on opposite sides of the membrane. The anion exchanger transports
chloride across the membrane by counter-transport with bicarbonate.
This is important in the transport of carbon dioxide in the body and for
helping to regulate pH.
Symporter (Example: Sodium- glucose symporter)
Channels
channel proteins form hydrophilic channels (often transport ions ion channels, porins,
and aquaporins).
o Porins
Rapid passage of various solutes.
Aquaporins (transport water specifically)
o Ion channels
Ligand-Gated- usually require two or more ligand binding of the ligand
to open the channel.
Mechanosensitive- open in response to stimuli or stretching.
Voltage-Gated- open in response to some electrical charge change.
Only involved with passive transport
o Know what is the Patch Clamp technique
A micropipette is used to isolate a small patch of membrane, allowing the flow of ions through a
single channel to be analyzed. When the patched area is isolated, you can determine certain
characteristics about the channel including its baseline conductance.
o Know the four types of ATPases
P-type (P for phosphorylation; examples: Na/K pump and proton pump in stomach)
Reversibly phosphorylated by ATP as part of the transport mechanism. They are
responsible for maintaining ion gradients across the membrane.
F-type (example: ATP synthases)
Proton transporter found in bacteria, chloroplasts, and mitochondria. Not only can ATP be
used as energy source to generate and maintain electrochemical gradients, but such
gradients are used as an energy source to synthesize ATP
V-type
Controlling pH (proton gradients) in organelles of the endomembrane system
Pumps protons into vesicles, vacuoles, lysosomes, endosomes, and the golgi complex in a
phosphorylation independent manner. ATP is hydrolyzed.
ABC-type (ATP binding cassette)
Handles a wide variety of solutes. Flippase is another example. (removes p.ethanolamine
and p.inositol from the outer leflet to the inner by hydrolyzing an ATP).
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o Example: CFTR and Cystic Fibrosis
Know tissue-specific directionality of chloride transport for skin and
respiratory tract
Chloride normally is transported into epithelial cells. With a
dysfunctional CFTR, the chloride cannot enter and leaves
through the duct.
The CFTR gene encodes for a chloride transporter. Mutation in this
gene causes CF.
o Example: Chloroquine transporter and Malaria
Expressed by plasmodium falciparum (causes malaria) and confers
resistance to the antimalarial drug chloroquine by transporting the drug
out of p. falciparum.
o Example: MDR1/2 and cancer
MDR1 and MDR2 are uniporter carriers in hepatocytes that transport
cholesterol (MDR1) and phospholipids (MDR2) into the bile canaliculus.
MDR1/2 is expressed by cancer cells and confers resistance to cancer
chemotherapeutics by transporting the drug out of p. falciparum.
o Indirect and direct active transport
Direct active transport requires energy (usually ATP) to be expended in the process. The
accumulation of solute molecules or ions on one side of the membrane is coupled directly to an
exergonic chemical reaction (ATP hydrolysis).
Example of direct active transport: Na+/K+ Pump
o Know how cardiac glycosides digoxin and digitoxin work
Indirect active transport depends on the co-transport of two solutes with the movement of one
solute DOWN its gradient, driving the movement of the other solute UP its gradient.
Example of indirect active transport: Na+/glucose symporter
o Know how glucose is transported from the lumen of the intestine, through intestinal epithelial and
into the extracellular fluid on the basal-lateral side of the epithelium
o a symporter protein most commonly found in surface absorptive cells of the
small intestine mucosa and the simple columnar epithelium of the proximial
convoluted tubule of the kidney that transport Na across the luminal membrane
into the cytoplasm by contransport with glucose. Glucose is pumped into the cell
through the apical domain with the Na/Glucose symporter. Glucose passes out of
the cell down its concentration gradient (GLUT-2), and cellular concentration of
Na is kept low by the Na/K pump on the basal side. (3 Na out and 2 K in).
o Endocytosis- uptake of macromolecules from the extracellular surroundings by localized regions of
plasma membrane. The material is surrounded by localized regions of plasma membrane, which buds off
inside the cell to form a vesicle containing ingested material.
Phagocytosis- endocytosis of large particulate substances including bacteria.
Pinocytosis- endocytosis of fluid and dissolved solutes.
Receptor mediated- binding of ligands to receptors triggers vesicle formation
o Exocytosis- secretion of macromolecules by transport vesicles.
Presentation # 5: Biotechnology & Human Diseases

Please note that students are required to know the biotechniques and must know how to interpret
biotechnique results for every exam this semester!!!!
Learn about restriction enzymes and their importance for genetic research
o Restriction enzymes are naturally occurring in bacterial enzymes and cut double stranded DNA.
Restriction enzymes cut at DNA palindromic sequences. Can produce sticky or blunt ends.
o Recognize and complete palindromic restriction enzyme recognition sequences
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A DNA palindrome is when the sequence is identical 5 3 on both ends. For example,
5 3 GAATTC
3 5 CTTAAG
Know how to identify a palindromic sequence embedded in a longer sequence
Know how to calculate the frequency of DNA cleavage by a restriction endonuclease
(assuming equal % A, G, T and C)
There are 4 different bases, so the probability of finding a particular base at one location
on a DNA strand =
The probability of finding the require base at each of n = locations is (1/4)^n (n= size of
the palindrome sequence).
The smaller the palindromic sequence, the more frequently the cut will occur. The longer
the sequence, the less frequently the cut will occur.
o Know how restriction enzymes cleave DNA; Blunt vs Sticky ends
If a restriction enzyme cuts a palindromic sequence down the middle blunt ends
If a restriction enzyme cuts a palindromic sequence in separate locations sticky ends
o Know how to interpret a restriction enzyme digestion gel electrophoresis pattern or a restriction
enzyme map
On a gel, the smallest fragment will travel the furthest because the apparatus uses a charge to
separate fragments. The DNA back bone is negative because of the phosphates. Larger fragments
will not travel as far because of their size.
Learn about the key steps in molecular cloning
o 1) Insertion of DNA into a cloning vector; 2) introduction of the recombinant vector into bacterial cells;
3) amplification of the recombinant vector in bacteria; 4) selection of cells containing recombinant DNA;
5) identification of clones containing the DNA of interest.
o When to uses various vectors
Plasmids- circular double stranded DNA molecules that replicate extrachromasomally in bacteria
or yeast.
Bacteriophage lambda- bacterial virus with a relatively large double stranded DNA. Can accept
DNA up to 20 kb
Cosmids- plasmids that use the ability of bacteriophage infection particles to package large
linear pieces of DNA into protein shells that can infect bacteria. Can accept foreign DNA up to
45 kb
Bacterial artificial chromosomes- huge plasmids. Can accept foreign DNA up to 300 kb
Yeast artificial chromosomes- contains all the necessary chromosomal elements to allow the
DNA to be accepted, replicated, and maintained in yeast cells. Can allow foreign DNA up to 400
kb.
o Use of Restriction enzymes and DNA ligase
Restriction endonucleases cleave their recognition sequences as staggered sties, leaving
overhanging single stranded tails that can associate with each other by complementary base
pairing. DNA ligase can then seal the ends permanently.
o Genomic vs cDNA libraries
Genomic library- all the various genomic fragments (after partial RE digestion) cloned into
separate vector molecules to create a pool for further isolation. You can use this library when you
want to observe the entire genome before splicing and intron removal.
Complementary libraries- cDNA copies of mRNA present in the cell cloned into vectors. More
useful for cloning direct representation of the coding sequence and gene expression. Nucleic acid
probes for library screening include: nucleic acid hybridization, DNA fragments, RNA probes,
and oligonucleotide probes (short polymers).
Know the role of reverse transcriptase in synthesizing cDNA
Reverse transcriptase uses an mRNA to make DNA.
Learn about expression vectors
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o An expression vector, otherwise known as an expression construct, is usually a plasmid or virus
designed for protein expression in cells. The vector is used to introduce a specific gene into a target cell,
and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the
gene. The plasmid is engineered to contain regulatory sequences that act as enhancer and promoter
regions and lead to efficient transcription of the gene carried on the expression vector
Expression in eukaryotic cells may be needed if posttranslational modification is required. Cloned
genes are inserted in virus vectors that direct high level gene expression.
o Use of GFP expression vectors to determine cellular location of proteins
Constructed and express green fluorescent protein (GFP) tagged fusion proteins. The GFP
expression vector is then introduced into cells and express. The subcellular localization of the
GFP tagged protein could then be determined by fluorescence microscopy.
Learn how DNA can be introduced into an animal cell
o Cloned DNA can also be introduced into plant and animal cells through gene transfer. The first
methodology used infectious viral DNAs called transfection.
o Other methods include: direct microinjection into the nucleus; coprecipitation of DNA with calcium
phosphate to form small particles that are taken up by the cells; Incorporation of DNA into liposomes that
fuse with the plasma membrane; exposure to cells to a brief electric pulse that opens pores in the plasma
membrane (electroporation).
The synthesis of cDNA
o Hybridize the mRNA strand with primer. Make complementary DNA copy with reverse transcriptase.
Degrade the RNA with RNase H. synthesize a second cDNA strand using DNA polymerase; incubate
with terminal transferase to add single stranded tails. Double stranded DNA is ready for insertion into
vector
Random fact: if there is an RNA probe in a DNA solution, it will bind and form an RNA/DNA hybrid. The base
pairs must match up.
Learn how to amplify DNA targets by PCR
o Must know how to identify proper primers used to amplify a given target
o Must know key reagents and the 3 key steps in the reaction (melt, anneal , polymerization)
Learn about the following techniques for analyzing nucleic acids and their application(s) (For each
technique you should know the key reagents- what type of probe is used, and key targets). You should also
be able to interpret results from these techniques as taught in class
o Gel electrophoresis
Separates fragments by size. A current is run through the apparatus. Is a method for separation
and analysis of macromolecules (DNA, RNA, and protein) and their fragments, based on their
size and charge.
o Techniques NOT requiring gel electrophoresis
These techniques look for the PRESENCE and ABUNDANCE of nucleic acid targets (CANNOT
determine size)
Dot/slot blots
Biomolecules to be detected are not first separated by chromatography. Instead, the
mixture containing the molecule to be detected is applied directly on a membrane as a
dot. Followed by detection by either nucleotide probes (for RNA or DNA) or antibodies
(for proteins). DNA or RNA is bounded directly to a solid support filter. This method is
ideal for multiple samples and quantitative measurements.
Analyzing results: look for the darkest spots (blots) has the most hybridization.
Lighter regions have less hybridization. CANNOT DETERMINE SIZE
ASO (allele-specific oligonucleotide)
This method is preferred when there is a common mutation in a relatively frequent
disease such as cystic fibrosis and sickle cell anemia. Oligonucleotides will only
hybridize to perfect matches and not to imperfect complementary sequences.
21
This method is able to detect point mutations, and takes advantage of the fact that the
mismatch between the oligoprobe and the template due to point mutation lowers Tm of
the probe. The probe will fall apart if there is a single mismatch. This is a rapid method to
detect mutations. NOT useful for disorders with allelic and/or locus heterogeneity.
Comparative genome hybridization
Measures the differences in copy number or dosage of a particular chromosomal segment
between two different DNA samples. Also able to study chromosomal aberrations in
cancer cells.
Comparing ratio of green to red fluorescence
o Tumor DNA (green), control DNA (red) the two samples are mixed together
and hybridized competitively to normal metaphase chromosomes. If the tumor
sample contains more DNA than the control, there will be an INCREASE in
green. Deletions result in reduction of ratio
Monosomy ratio: 0.5:1
Trisomy: 1.5:1
Euploidy: 1:1
o If there are 4 times the number of chromosomes for a particular chromosome,
there will be twice the amount of binding and increase in the ratio.
o Southern Blot
Target: DNA; probe: complementary nucleic acids
Restriction endonuclease fragments of DNA are separated by gel electrophoresis. Specific DNA
fragments are then identified by hybridization with an appropriate probe.
Used to detect DNA mutations such as insertion or deletion of nucleotides. Can also be used to
detect point mutations that cause the loss or gain of restriction enzyme cleavage (RFLP)
To remove the DNA/RNA, you use a filter to immobilize (trap) it onto a filter paper. Run aqueous
solution through it.
o Northern Blot (Know how to interpret a Northern blot)
Target: RNA; probe: complementary nucleic acids
o FISH (fluorescence in-situ hybridization)
Can be used to detect homologus DNA or RNA sequences in cell extracts, chromosomes, or intact
cells. Hybridization of fluorescent probes to specific cells or subcellular structures is analyzed by
microscopic examination.
Targets: Examine presence or absence of a particular DNA sequence. Evaluate number or
organization of a chromosome or chromosomal region. Uses DNA probes with high specificity
and detected by a fluorescent dye. Can analyze multiple targets simultaneously utilizing different
fluorochromes.
o RFLP (restriction fragment length polymorphism)
Must know how to interpret RFLP blot and predict fragment sizes
Principle: when two alleles differ due to a point mutation within a restriction endonuclease
recognition sequence so that the cleavage of the site by the enzyme is abolished in one of the
alleles, they can be identified on the basis of the lengths of their restriction fragments.
o DNA microarrays
Allow the expression of thousands of genes to be monitored simultaneously. One application of
DNA microarrays is in studies of gene expression; for example, a comparison of the genes,
expressed by two different types of cells.
DNA microarrays consist of thousands of individual sequences bound to closely spaced regions
on the surface of a glass microscope slide. Also allows for the simultaneous analysis of the
expression of thousands of genes.
o RT-PCR
A template cDNA is first synthesized from mRNA, using viral reverse transcriptase, prior to PCR
amplification.
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o DNA fingerprinting
No two individuals, except identical twins, can have the same DNA fragment. When you compare
the ladders of southern blots, you will find different patterns in each person. In babies, you need
to amplify DNA before testing blood because you do not get very much from collecting their
blood.
o DNA sequencing
Know the importance of dye-labeled dideoxyribonucleotides in modern DNA sequencing
ddNTPs lack both a 2 and 3 oxygen.
o One method is based on premature termination of DNA synthesis. DNA synthesis is initiated with a
synthetic primer. ddNTP are included along with the normal nucleotides. They are labeled with different
fluorescent dyes. The ddNTPs stop DNA synthesis because no 3 OH group is available for addition of the
next nucleotide. The fragments are separated by gel electrophoresis, and a laser beam excites the
fluorescent dyes and data is analyzed by a computer.
o You need the 3 OH to carry out the dehydration reaction. By knowing the color associated with each of
the ddNTPs you can determine which nucleotide was used. Another important technique is gel
electrophoresis to order the fragments by size and then determine the proper sequence DNA sequence
analysis
Know how to interpret DNA sequencing pattern
Know the terms carrier, homozygous (homozygotic) and heterozygous (heterozygotic)
o If there is a disease trait that is recessive, a carrier for the disease will not be affected because the person
will have both a good gene and a bad gene. To be homozygotic is to have two alleles of the same genes.
To be heterozygotic is to have both a good gene and bad gene (carrier).
Learn about protein analysis by SDS-PAGE, Western Blot , Immunoprecipitation and ELISA
o ELISA (enzyme linked immunosorbent assays)
These assays are performed in the wells of a plastic microtiter dish. The antigen (protein) is
bound to the plastic of the dish. The probe used consists of an antibody specific for the particular
protein to be measured.
The antibody is covalently bound to an enzyme which will produce a colored product
when exposed to its substrate.
The amount of color produced can be used to determine the amount of protein (or antibody) in the
sample to be tested
A rapid immunologic technique for testing for antigen-antibody reactivity.
A patients blood sample can be probed with EITHER:
Test antigen to see if immune system recognizes it. (ex. Testing a patient to see if they
have anti-HIV antibodies in their blood)
Test antibody- to see if a certain antigen is present
Know how to interpret an immunofluorescence image
o Antibodies can be used to visualize proteins in intact cells. Cells can be stained with antibodies labeled
with fluorescent dyes, or tags visible by electron microscopy.
Understand what is proteomics and what the commonly used techniques to identify proteins, their cellular
location and their protein interactions
o The systematic analysis of cell genomes (genomics) is one example. The proteome is all the proteins
expressed in a given cell. The systematic analysis of cell proteins (proteomics) aims to identify all
proteins expressed in a cell, where they are expressed, and their interactions.
o 2-D gel electrophoresis
Can be used for large scale separation of cell proteins. Proteins are separated based on charge
(isoeletric focusing) AND size (SDS page). This technique is biased towards the most abundant
proteins.
o Mass spectrometry
Proteins can be excised from the gels and identified by mass spectrometry.
23
A protease cleaves the protein into small peptides. These are ionized and analyzed in a mass
spectrometer, which determines the mass-to-charge ratio of each peptide. The mass spectrum is
compared to a data base of known spectra.
o GFP
Know the importance of antibodies in detecting specific proteins
o Antibodies are proteins produced by B lymphocytes that recognize specific molecules called antigens.
Antibodies can be generated by inoculation of an animal with any foreign protein.
The animal usually produces a mixture of antibodies that react against multiple sites on the
protein
o Monoclonal antibodies can be produced by the culturing of clonal lines of B lymphocytes.
Each B lymphocyte is programmed to produce only a single type of antibody. A clonal line of
lymphocytes produces a monoclonal antibody that recognizes only a single antigen.
o Polyclonal antibodies- antibodies that can recognize multiple antigens.
Learn about production of transgenic mice
o Cloned genes can also be introduced into the germ line of multicellular organisms. Mice that carry foreign
genes (transgemic mice) are produced by microinjection of cloned DNA into the pronucleus of a fertilized
egg.
Introduction of genes into ES cells
o Embryonic stem cells (ES) are another way to introduce cloned genes into mice. Cloned DNA is
introduced into ES cells in culture, then stably transformed cells are introduced back into mouse embryos.
Learn about gene inactivation by homologous recombination
o ES cell carrying normal copies of gene on both chromosomes. The inactive copy of gene is introduced
into the cells, and the ES cell will carry one mutated one and one normal. Inject into blastocyst and
transfer to foster mother. After mating, some offspring are produced that carry mutated copies of the gene
on both chromosomes.
Learn how antisense RNA targets messenger RNA and prevents its translation into protein
o Other approaches are used to interfere with gene expression or function. Antisense nucleic acids are RNA
or single-stranded DNA complementary to the mRNA of the gene of interest (antisense). They hybridize
with the mRNA and block its translation into proteins.
Learn about RNA interference
o 1998 Experiment with c. elegans found that injection of double stranded RNA inhibited expression of a
gene with a complementary mRNA sequence. Double stranded RNA resulted in extensive degradation of
the target mRNA, whereas single-stranded antisense RNA had only a minimal effect. This unexpected
observation demonstrated a role for double stranded RNAs in gene regulation. This has been developed
into a powerful experimental tool for inhibiting expression of target genes.
o When double stranded RNAs are introduced into cells, they are cleaved into short double stranded
molecules (siRNAs) by an enzyme called Dicer. SiRNAs with a complex of proteins known as the RISC
where mRNA is cleaved.
Presentation # 1: Cytoskeleton
Read Chapter 12 of The Cell
Know the three major elements of the cytoskeleton, what they look like, how they are assembled
o Microtubules (25 nm)
Hollow tube with a wall consisting of 13 protofilaments
Treadmilling can also occur with microtubules. Incorporation at plus end equals disassociation at
minus end.
o Microfilaments (7 nm)
Two intertwined chains of F-actin (filamentous actin); composed from G-actin (globular actin)
Dense network of microfilaments beneath the plasma membrane are critical components of the
cell cortex. Also are involved in locomotion and cytokinesis.
24
Different actins localize to different regions of the cell and appear to have varying interactions
with actin-binding proteins.
G-actin monomers polymerize into f-actin microfilaments and can perform treadmilling.
o Intermediate filaments (8-12 nm)
8 protofilaments joined end-to-end with staggered overlaps.
o Microfilaments
Know Wiskott-Aldrich syndrome
Know key features and function of microvillus
Key features and components: electron-dense plaque; actin bundling proteins: fimbrin
and villin; terminal web (anchors microvilli) proteins: myosin and spectrin; plasma
membrane; lateral cross links composed of myosin 1 and calmodulin.
Are fingerlike extensions that are abundant on cells involved in absorption. The
microvilli of epithelial cells lining the intestine form a layer on the apical surface (a brush
border) of about 1000 microvilli per cell. They increase the surface area for absorption by
ten to twenty fold. The filaments are cross linked in part by fimbrin, but the main protein
is villin.
The actin bundles are attached to the plasma membrane by the calcium binding
calmodulin in association with myosin I.
Know the role of monomeric GTPases Rac, Rho and CDC42 in controlling microfilament
structures

Know that the compounds cytochalasin B and Phalloidin target microfilaments
Cytochalasin binds to barbed ends and blocks elongation. This can inhibit movements
such as cell division.
Phalloidin binds to actin filaments and prevents dissociation. It can be visualized with
fluorescent dye to allow visualization of actin filaments.
Awareness of actin binding proteins and the types of microfilament structures
25
Diverse actin-binding proteins and functions. Some actin-binding proteins bind along
the length of actin filaments, stabilizing them or cross-linking them to one another.
Others promote dissociation with others regulate the exchange of ATP for ADP.
CapZ is a capping protein that helps to stabilize the ends of microfilaments. Actin-
binding proteins regulate interactions between microfilaments.
ADF/cofilin enhance the rate of dissociation of actin/ADP monomers from the pointed
end, and remain bound the monomers, preventing their reincorporation. Profilin reverse
this by stimulating exchange of bound ADP for ATP and dissociating the actin/ATP
monomers from ADF/cofilin, so that they are available for reassembly. ADF/cofilin can
also bind to and sever actin filaments.
Formin and arp2/3 complex determine where filaments are formed by facilitating
nucleation.
Actin bundles versus networks
Bundles actin filaments are cross linked into closely packed parallel arrays and can
closely align with other bundles. Networks cross linked in arrays that form 3D
meshworks with the properties of semi-solid gels and tend to be large and cross-link with
perpendicular filaments.
Two types of actin bundling proteins
Parallel bundles are closed spaced actin filaments aligned in parallel, all with the same
polarity and barbed ends adjacent to the plasma membrane. Fimbrin is one protein in
parallel budles.
Contractile bundles are more widely spaced and cross linked by alpha actinin
increased spacing between filaments allows motor protein myosin to interact with actin
filaments.
Actin networks and filamin
In actin networks, proteins such as filamin (v-shaped molecule with actin binding
domains at the end of each arm) form flexible cross links.
Actin filaments are concentrated at the junction that forms the 3D network beneath the
plasma membrane. This network and associated proteins (cell cortex) determines the cell
shape and is involved in activities such as movement.
o An example of the cell cortex and its importance is in red blood cells. RBC does
not have any organelles, so it was easily studied by isolation. (To determine
interior, place RBC into hypotonic solution, and the cell will swell and lyse).
Spectrin
26
Major structural protein and is a tetramer of two polypeptide chains (alpha and beta). The
ends of the spectrin tetramers associate with short actin filaments, resulting in the
spectrin-actin network.
Spectrin based cytoskeleton:
Protein 4.1 is another link that binds spectrin actin junctions and the transmembrane
protein glycophorin (provides negative charge to repel other cells).
Other types of cells have linking proteins
Fodrin, a non-erythroid spectrin (provides scaffolding for membrane proteins)
ERM proteins, related to protein 4.1 (binds glycophorin to the spectrin-actin network)
Dystrophin links actin filaments to transmembrane proteins of muscle cell plasma
membranes. Transmembrane proteins link to the ECM, which helps maintain cell stability
during contraction.
Know Duchenne and Becker Muscular Dystrophies

Muscular dystrophy, an X-linked inherited disease, results in progressive degeneration of
skeletal muscle. Dystrophin is absent in patients with Duchennes muscular dystrophy.
Dystrophin is abnormal (usually truncated) in patients with beckers muscular dystrophy.
DMD
o Difficulty rising from the groun using gower maneuver (rising by pushing on the
ground with hands, and supporting the knee and the thigh). Affected boys are
wheel chair bound by age 11 (due to severe proximal muscle weakness).
Subsequent deterioration leads to lumbar lordosis and joint contracture. Death by
age 18 and due to cardiorespiratory failure. There is also moderate intellectual
compromise. There is an increase in serum creatine kinase (CK) level.
o Age of onset: male children at 3-5 years (1/3500); slowly progressive muscle
weakness, resulting in awkward gait. Inability to run quickly, and inability to
climb stairs. There is psudohypertrophy of calf (increase in the size of the calf
and replaced by fat and fibrous connective tissue).
Becker muscular dystrophy
o Clinical features similar to DMD, but is less aggressive. The mean age of onset is
11 years, many patients remain ambulant until well into adult life, overall life
expectancy slightly reduced, some form of dystrophin (usually truncated) is
produced.
Know the normal cellular role of dystrophin at the sarcolemma
o Dystrophin is a membrane associated intracellular protein expressed
predominately in skeletal, smooth, and cardiac muscle; also in some brain
neurons (explains the decrease in IQ). Dystrophin is part of a large complex of
sarcolemma associated proteins that among others include ECM protein and
other proteins. Dystrophin attaches the cytoskeleton to the sarcoplasmic
membrane.
Know basic structure and function of focal adhesions, stress fibers and adhesion belt
27
Cultured fibroblasts secrete extracellular matrix proteins that stick to the surface of the
dish. The fibroblasts attach to the matrix via binding of transmembrane proteins
(integrins). The sites of attachments (focal adhesions) are also attachment sites for
large actin bundles called stress fibers.
o Stress fibers are contractile bundles, cross-linked by alpha actinin and stabilized
by tropomyosin. Two other proteins, talin and vinculin, are involved in binding
stress fibers.
In sheets of epithelial cells, cell-cell contacts (adherens junctions) form a continuous
belt like structure (adhesion belt) around each cell. Contact is mediated by
transmembrane proteins called cadherins. They bind to cytoplasmic catenins, which
anchor actin filaments to the plasma membrane.
Know the structures of pseudopodia, lamellipodia and filopodia
Pseudopodia are extensions of moderate width, responsible for phagocytosis and the
movement of amoebas.
Lemellipodia are broad, sheetlike extensions at the lead edge of fibroblasts.
Many cells also extend microspikes or filopodia, thin projections of the plasma
membrane supported by actin bundles.
Know the structure of the skeletal muscle sarcomere, the process of muscle contraction and
the proteins involved.
Structure of the sarcomere- sarcomeres consist of several regions discernible by
electron microscopy. These bands correspond to the absence of myosin filaments. A
bands are dark, I bands are light. The actin filaments are attached at their barbed ends to
the Z disc, which includes the cross linking protein apha-actinin.
Each myofibril is organized as a chain of contractile units called sarcomeres, which are
responsible for the striated appearance of skeletal and cardiac muscle.
Know that the I band, H zone and distance between z-lines gets smaller, but the A band
size does not change.
Actin is attached to the Z bands, which are protein plates. The I band is only filaments of
actin. The A band is overlapping regions of actin and myosin. The H zone is only myosin
fibers. Myosin consists of cross links that bind to specific sites on the actin. The cross links
pull the myosin along the actin. The cross links attach independently, which is good because
if they all let go at the same time, they would let go of the actin.
Sarcomeres contain ordered arrays of actin, myosin, and accessory proteins
o Thick filaments composed of myosin organized in staggered array.
o Thin filaments components of actin, tropomyosin, and troponin
28
o Organization of filament proteins capZ; alpha-actinin; myomesin; titin; nebulin;
tropomodulin
Process of muscle contraction
o Electrical impulse (AP) travels down axon of neuron; signal reaches neuromuscular
junction; axon terminals secrete acetylcholine; open channels allow Na to enter and
induce electrical impulse to be transmitted away from the sarcolemma at motor
endplate; the electrical impulse/AP travels over the sarcolemma via the T tubules and
into the interior of the muscle cell; depolarization activates L type voltage dependent
Ca channels in the T tubule membrane; voltage gated Ca channels cause the release
of Ca from the sarcoplasmic reticulum; Ca binds to troponin C and induces
conformational changes of the tropomyosin, and makes actin sites available to
myosin; cross-bridges form between actin and myosin contraction (when nerve
impulses to the muscle cell cease, Ca levels decline quickly and the muscle relaxes).
Know the process of smooth muscle contraction, regulation, and the proteins involved
including calmodulin, myosin light chain kinase, myosin and actin
Smooth muscle is more similar to nonmuscle cells than to skeletal muscle. Smooth
muscle is responsible for involuntary contractions. The structure of smooth muscle is
dense bodies containing intermediate filaments and is connected to actin microfilaments.
Regulation of contraction in smooth muscle cells: myosin light-chain kinase
calmodulin
o Role of Ca ions
In non-muscle cells and smooth muscle, contraction is regulated primarily by
phosphorylation of one the myosin light chains. This is catalyzed by myosin light chain
kinase which is a regulator of the Ca binding protein calmodulin.
Nerve impulse or hormonal signal influx of Ca ions Ca calmodulin complex binds
to Ca calmodulin binding sites on MLCK Active MLCK (myosin light chain kinase)
phosphorylates myosin II in its curled or uncurled form Phosphorylated myosin can
bind to actin.
Know the roles of non-muscle myosins in intracellular transport
Non muscle myosin is important for transport of cargo in the cell. For example, sending
processed mRNA requires motor proteins to carry it. Non-muscle myosin will help with
this movement.
Over 12 classes of nonmuscle myosins have been identified. Myosin I proteins are much
smaller than myosin II; but contain a globular head group that acts as a molecular motor.
The short tains bind to other structures. Movement of myosin I along an actin filament
can then transport its attached cargo, such as a vesicle. Myosin V is a two headed myosin
that transports organelles and other cargo toward the barbed ends of actin filaments.
Know the basic step of cell migration
Extending protrusions
o Retrograde flow; actin assembly; rearward translocation
Cell attachment
o Integrins; focal contacts
Translocation and detachment
o Contraction of rear and breaking adhesive contacts
Polymerization of the microfilaments drives extension.
Actin organized in lamellipodia is greater than cell cortex, but less than microvilli.
Branching facilitated by a complex of ARPs. The Arp2/3 complex helps branches form
by nucleating new branches on existing microfilaments.
o Monomeric G proteins regulate microfilament organization
Actin polymerization is regulated by small GTP binding proteins (Rac, Rho, Cdc42)
29
These proteins regulate kinases that phosphorylate inositol phospholipids, causing
increase in polyphosphoinostiol production. The polyphosphoinositol bind regulatory
proteins.
o Intermediate filaments
General information: Intermediate filaments have diameters intermediate between actin and
microtubules. There are not directly involved in cell movements, but provide mechanical strength
and a scaffold for localization of cellular processes. Intermediate filament proteins are tissue
specific! (can be used a diagnostic tool for cancer detection) all the intermediate filaments
proteins have a central alpha-helical rod domain of about 310 AA, which plays a central role in
filament assembly. The head and tail domains determine the specific functions.
More general information: filament assembly starts when the central rod domains of two
polypeptides form a coiled coil. These dimers then associate in a staggered antiparallel fashion to
form tetramers, which assemble end to end to form protofilaments. 8 protofilaments filament;
intermediate filaments DO NOT have distinct ends; they are MORE stable and DO NOT exhibit
the dynamic behavior of actin or microtubules (e.g. treadmilling). Phosphorylation CAN regulate
assembly and disassembly.
More general information: in addition to forming bundles themselves, intermediate filaments
are sometimes held together by accessory proteins. For example, plectin an important cross-
linking protein which bundles IF and links IF to MT and MF. Also, IF confer mechanical strength
on tissues. A defect here will result in epidermolysis bullosa simplex (EBS).
Know the different classes of IF and the tissues they are found
Type I and II are keratins, in epithelial cells.
Type III includes vimentin, which forms a network extending out from the nucleus
toward the cell periphery.
Desmin is expressed in muscle cells where it connects the Z discs of individual
contractile elements.
Type IV includes neurofilament (NF) proteins, the major intermediate filaments of
many mature neurons, especially long motor neuron axons.
Type V are the nuclear lamins.
Nestins (type VI) are expressed during embryonic development in several types of stem
cells.
Tonofilaments Epithelium Keratinizing and

(cytokeratins) non-keratinizing epithelia
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Vimentin Mesenchymal Fibroblasts, chondroblasts,

cells osteoblasts, macrophages,
endothelial cells, vascular
smooth muscle cells
Desmin Muscle Striated and smooth muscle
Neurofilaments Neurons Most neurons
Glial filaments Glial cells Astrocytes (GFAP)
Know the basic structure of desmosomes and hemidesmosomes

Desmosomes- junctions between adjacent cells. Keratin filaments attach to dense protein
plaques on the cytoplasmic side. The attachments are mediated by plakins; cadherins
link adjoining cells.
Hemidesmosomes- junctions between
epithelial cells and underlying

connective tissue. Keratin filaments are attached to different
plakins. Transmembrane integrins link to the ECM.
Know Epidermolysis bullosa simplex

below
o Microtubules
Know of microtubule disrupting drugs:
Colchicine (anti-gout); Vinblastine/Vincristine/Nocodazol (antineoplastic) anti-
cancer drugs; Albendazole (antihelminthic) used on parasites and disrupts normal
function of microtubules.
o INHIBIT MICROTUBULE GROWTH
Taxol and griseofulvin
o STABILIZE MICRTUBULES
Know how microtubules are assembled in vivo and the role of centrosomes and gamma
tubulin
Microtubules originate from microtubule-organizing centers (MTOC) within the cell.
Centrosomes are composed of two centrioles.
o Centrosomes of most animal cells contain a pair of centrioles, oriented
perpendicular to each other and surrounded by amorphous periecentrolar
material. Centrioles are cylindrical, based on nine triplets of microtubules and are
31
necessary to form basal bodies, cilia, and flagella. They are not found in plant
cells, many unicellular eukaryotes, and most meiotic animal cells.
o MTOCs organize and polarize the microtubules within cells. (-) ends are
anchored in MTOCs. Each MTOC has a limited number of nucleation and
anchorage sites. Fluctuation of pericentrin during cell cycle is highest at prophase
and metaphase of mitosis.
o Functions of the MTOCs
Control # of microtubules, polarity, # protofilaments, timing of new
microtubule biosynthesis, place of new microtubule biosynthesis.
Microtubules stability within cells is highly regulated kinetochores stabilize MTs.
The role or gamma tubulin in initiation
Know what are Microtubule Associated Proteins (MAPs)
Microtubules are regulated by microtubule associated proteins (MAPs); motor MAPs are
kinesin and dynein.
Non-motor MAPs
o Normal function of Tau is to help organize and stabilize microtubules in axons of
nerve cells; causes MT to form tight bundles found in axons. MAP2 causes MT
to form looser bundle found in dendrites. Tau protein is involved in Alzhemiers,
but is not mutated. Instead, Tau is hyperphophorylated, and results in
neurofibular tangles.
o Random fact: frontal temporal dementia is also associated with dementia, but
is the result of an inherited mutation of Tau.
Know how and in which direction dynein & kinesin move cargo on microtubule
Dynein will transport towards the (-) end. Kinesin will transport toward the (+) end.
Kinesin and dynein are responsible for powering the movements in which microtubules
participate.
Kinesin moves along microtubules by hydrolyzing ATP and have varying structures and
functions. At the start of each step, one of the two kinesin heads, the rear or lagging head,
is tightly bound to the microtubule and to ATP, while the front or leading head is loosely
bound to the microtubule with ADP in the front motor domain. The rear motor domain is
then pulled forward once it has detached from the microtubule with ADP bound.
Must know: axoneme 9+2 structure and function in cilia and flagella
Cilia and flagella consist of an axoneme connected to a basal body. Axonome has
characteristic 9+2 pattern with nine outer doublets of tubules and 2 additional
microtubules in the center. Each doublet has a complete A tubule and a B tubule with 10-
11 protofilaments, fused to the A tubule. Nexin links the microtubules, and two arms of
dynein are attached to each tubule.
Microtubule motors and movement

Microtubules are usually oriented with their minus end anchored in the centrosome and
their plus end extending toward the cell periphery.
The role of axonemal dynein
Know the role of actin and myosin in chemotaxis and cell migration
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Chemotaxis is an example of actin-based motility. Cell movement toward a chemical
attractant or away from a chemical repellant.
Know what are primary cilia and 9+0 cilia
Usually found on cells with only one cilium. Some primary cilia might be motile and be
involved in embryonic development and involved with generating left-right asymmetry of
internal organs. Some primary cilia function as mechanoreceptors sensing a flow of fluid
in developing kidneys.
Two important disorders associated with mutations that disrupt the function of primary
cilium (primary ciliary dyskinesia)
o adult polycystic kidney disease
results from mutations in genes encoding polycystin-1 or 2. Polycystin 1
or 2 are essential in the formation of calcium channels associated with
primary cilium in the developing kidney, thus disrupting the normal
mechanoreceptor function of these cilium.
Patients usually have multiple large cysts in both kidneys. You will also
see problems with the liver, and may present with pain in the lower back.
o Kartageners syndrome
Also known as primary ciliary dyskinesia (PCD) or immotile ciliary
syndrome. The main symptom is reduced or absent mucus clearance
from the lungs and thus increased susceptibility to chronic recurrent
respiratory infections.
Many patients also experience hearing loss. Infertility is common in both
male and female. For females, problems with ciliary action in the
fallopian tubes. For males, problems with sperm motility.
About 50% have reversal of organs (situs inversum)
Most cases result from a mutation in the axonomal dynein arms of
cilia/flagella.
Know the four types of microtubule that make up the mitotic spindle
Kinetochore microtubules
o Attach to the condensed chromosomes at the centromeres, which stabilize them.
Chromosomal microtubules
o Connect to the ends of the chromosomes via chromokinesin.
Polar microtubules
o Are not attached to chromosomes but are stabilized by overlapping with each
other in the center of the cell.
Astral microtubules
o Extend outward from the centrosomes and have freely exposed plus ends.
List of diseases you must know for block 1

1) Alzheimers disease
a. Most common neurodegenerative disorder. Twice as common in women and costly to society. 25% of AD
is familial.
i. Symptoms: failure of memory, confusion, poor judgment, increased muscle tone, incontinence
etc. death usually results from general inanition, malnutrition, and pneumonia. Caused by the
abnormal cleavage of the amyloid precursor protein (APP) creates neurotoxic peptide.
2) Cystic Fibrosis
a. Malfunction of the CFTR gene resulting in the defect of the chloride transporter. Caused by an autosomal
recessive mutation in the cystic fibrosis transmembrane conductance regulator (member of the ABC
transporter family). The most common CFTR mutation in Caucasian is the delta F508 deletion of the
508th amino acid or 3 codons.
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b. Mechanism: the mutation results in the inability of cells to transport chloride and water into the skin. A
result, the chloride will go to the skin and will have a salty taste. Results in increased frequency of
respiratory infections. Also, causes pancreatic insufficiency. Males are often sterile. End stage,
progressive lung disease is the principle cause of death.
3) Hereditary Spherocytosis
a. Most commonly caused by autosomal dominant mutations in the ankyrin-1 or the spectrin genes (50%
of cases are caused by mutations in the ankyrin gene). Can also result from inherited defects in the
ankyrin band 3 or protein 4.2 genes.
b. Mechanism: the mutation results in uncoupling of the lipid bilayer from the underlying cytoskeleton.
This causes the sphereocyte formation. These abnormal cells are more rigid and cannot traverse the
spleen. The cells ultimately succumb to extravascular hemolysis.
c. There is a decreased ability to transport oxygen and results in fatigue. Another observation is jaundice.
Look at blood smear and you would see small spherical blood cells.
4) Huntingtons disease
a. Caused by an expanded polyglutamine repeat in the protein huntingtin (Htt), which causes the protein
to aggregate and damage neurons. HD is an autosomal dominant condition caused by an expansion of
CAG (glutamine) trinucleotide repeats in Htt. People without HD have < 26 CAG repeats; WITH > 40
CAG repeats. Early, mid, and late stage symptoms. Early subtle changes in coordination, minor chorea,
difficult in mental planning, often depressed mood. Mid more prominent chorea, dysphagia. Late
behavioral problems and person is often mute, dependent, and incontinent.
5) Transmissible spongiform encephalopathies (prions): Creutzfeldt-Jakob
a. above
6) Wiskott-aldrich syndrome
a. Cannot produce functional WASP and therefore their platelets have difficulties undergoing changes in
shape and so have difficulties in forming blood clots. WAS usually presents in infancy (males most
commonly affected- X-linked).
b. Symptoms and signs
i. Bloody diarrhea; intermittent or chronic petechiae and purpura; eczema; recurrent bacterial and
viral, particularly recurrent ear infections. At least 40% of those who survive the early
complications develop one or more autoimmune conditions some as hemolytic anemia etc.
individuals previously exposed to EBV have an increased risk of developing lymphomas, which
often occur in unusual, extranodal locations such as the brain, lung, or GI.
7) Kartageners syndrome
a.
8) Duchenne and becker muscular dystrophies
a. above
9) Adult polycystic kidney disease
a.
10) Epidermolysis bullosa simplex
a. Blistering skin disease that can be caused by mutant keratins. Intermediate filaments confer mechanical
strength on tissues through tonofilaments, desmosomes, and hemidesmosomes, and attach to the basal
lamina.
List of drugs/poisons you must know for block 1
Drug function
1) Aspirin (acetylsalicylic acid) Aspirin is a non-steroidal anti-inflammatory drug that
irreversibly inhibits cyclooxygenase and is used to reduce
effects of MI, inhibit platelet aggregation, and reduce pain
and fever, and general anti-inflammatory agent.
2) Ibuprofen Ibuprofen and naproxen are NSAIDs that reversibly

inhibit cyclooxygenase and are used to reduce pain, treat
rheumatoid arthritis, and to treat osteoarthritis.
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Na and K ATPase blockers that elevate intracellular Na

3) Cardiac glycosides: Digitoxin and Digoxin levels within cardiac myocytes. The elevated Na
overwhelms the Na and Ca exchanger so that more Ca can
be re-accumulated by the sarcoplasmic reticulum. During
the next contraction, more Ca is released from the sER,
which increases the force of contraction. They are used in
congestive heart failure.
4) Oubain An inhibitor of sodium/potassium pump. Exposure to

ouabain causes cell swelling. At low concentrations,
ouabain will stimulate the Na/K pump. These pumps
maintain gradients.
5) Colchicine (anti-gout); INHIBIT MICROTUBULE GROWTH

Vinblastine/Vincristine/Nocodazol (antineoplastic)
anti-cancer drugs; Albendazole (antihelminthic)
used on parasites and disrupts normal function
of microtubules.
6) Taxol and griseofulvin STABILIZE MICRTUBULES
7) Cytochalasin B Binds to barbed ends and blocks elongation. This can

inhibit movements such as cell division.
8) Phalloidin Binds to actin filaments and prevents dissociation. It can be

visualized with fluorescent dye to allow visualization of
actin filaments.
Presentations # 3: Signal Transduction

Different types of chemical signals can be received by cells. The type of chemical messenger varies and can be
anything from amino acid and derivatives to steroids. It is not exclusive or a certain type.
o Ligand, primary messenger, and second messenger.
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Certain type of signaled responses such as increased cell growth and gene expression occur more slowly, on the
order of an hour or more. Other responses such as changes in cell movement and gene transcription occur much
more quickly on the order of seconds to minutes. A fast response suggests the machinery is likely already
available. A slow response has to alter chromatin before making favor able changes. Signals are not exclusive
and can alter both states.
Signal transduction pathways can amplify the cellular response to an external signal. Different type of cells
responds differently to the same extracellular signal molecule. Each cell is programmed to respond to specific
combinations of extracellular signal molecules.
o For example, if deprived of appropriate survival signals, a cell will undergo apoptosis.
The fate of some developing cells depends on their position in morphogen gradients.
Adjusting cellular response to stimuli: receptor sequestration, receptor down regulation, receptor inactivation (for
example).
Know where receptors for hydrophobic and hydrophilic ligand are usually found Know the terms:
Endocrines, Paracrines and Autocrines
o Most signal molecules are hydrophilic and are therefore unable to cross the target cells plasma
membrane directly; instead, they bind to cell-surface receptors, which in turn generate signal inside the
target cell.
o Some small signal molecules, by contrast, diffuse across the plasma membrane and bind to receptor
proteins inside the target cell either in the cytosol or in the nucleus.
Many of these small molecules are hydrophobic and nearly insoluble in aqueous solutions; they
are therefore transported in the blood stream and other extracellular fluids bound to carrier
proteins from which they dissociate before entering the target cell.
o Endocrine- each endocrine hormone is secreted by a specific cell type, enters the blood, and exerts its
actions on specific target cells, which may be some distance away.
Endocrine cells secrete hormones into the blood, and these act only on those target cells that
carry the appropriate receptors: the receptors bind the specific hormone, which the target cells
thereby pull from the extracellular fluid.
o Paracrine- paracrine actions are those performed on nearby cells, and the location of the cells plays a
role in the specificity of the response. It is very important in limiting immune response to a specific
location in the body.
o Autocrine- autocrine actions involve a messenger that acts on the cell from which is secreted, or on
nearby cells that is the same type as the secreting cells.
Understand the basic characteristics of signal transduction
o Detection of specific signals at the cell surface and the mechanism by which such signals are transmitted
into the cells interior, resulting in changes, in cell behavior, and or gene expression.
Must know both nuclear and membrane-initiated steroid hormone receptor signaling,
o Nuclear initiated steroid signaling
(classical signaling) Slower and involves changes in gene expression.
Lipophilic hormones use intracellular gene-specific transcription factors
Includes steroid hormones, thyroid hormones, retinoic acid, and vitamin D
Receptors are transcription factors that bind to DNA promoter elements in genes and alter
expression. Steroid hormones often have zinc finger DNA binding domains.
The hormone needs to be transported in the blood bound to carrier proteins such as serum
albumin, steroid hormone binding protein or thyroid hormone binding globulin.
Some receptors primarily reside in the nucleus. Others, such as glucocorticoid receptor are found
in the cytosol until the steroid interacts with it.
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o Membrane initiated steroid signaling
Faster, includes activation of G proteins and stimulation of protein kinases.
Must know steroid nuclear hormone receptor signaling (including Vitamin D, Retinoic acid & Thyroid
hormone) and how it modifies gene expression
o Steroid hormones are synthesized from cholesterol.
Testosterone, estrogen, and progesterone are the sex steroids produced by the gonads.
Corticosteroids from the adrenal gland
Glucocorticoids - stimulate production of glucose
Mineralcorticords- act on the kidney to regulate salt and water balance
Thyroid hormone is synthesized from tyrosine in the thyroid gland; it is important in
development and metabolism. It is transported across the membrane by a carrier protein.
Vitamin D3 regulates calcium metabolism and bone growth. Retinoic acid and related
compounds (retinoids) are synthesized from vitamin A and play important roles in development.
o Know about the zinc-finger DNA binding domain of intracellular steroid receptors
o Know the cellular locations of Thyroid hormone receptors and Glucocorticoid receptors (found in the
cytosol until steroid interacts with it).
Must know the mode of action for aromatase inhibitors and tamoxifen
o Aromastase inhibitors are used in treatment of estrogen responsive breast cancer in postmonopausal
women. Inhibitors of aromastase can reduce estrogen levels significantly and remove the main source of
growth stimulation from estrogen responsive tumors.
Arrest of tumor growth and/or initiation of apoptosis of estrogen-responsive breast tumors occur
as a result of treatment with aromatase inhibitors.
o Tamoxifen works by blocking estrogen receptors in breast, thereby inhibit estrogen-dependent growth
of tumors. Also used in premenopausal women with estrogen-receptor positive breast cancer.
Must know Gs signaling pathway in detail !!!!!
Know how cholera toxin poisoning disrupts Gs signaling

o Cholera toxin alters secretion of salts and fluid in the intestine. A portion of the cholera toxin is an
enzyme that chemically modifies Gs so that it can no longer hydrolyze GTP. Thus cAMP levels stay
high and cells stimulate to secrete salts and water.
o Cholera A toxin is absorbed into mucosal cells where it is processed and complexed with ARF (ADP
ribosylating factor), a small G-protein that is normally involved with vesicular transport.
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o Cholera A toxin is an NAD-glycohydrolase, which cleaves NAD and transfers ADP ribose portion to
other proteins
o If ADP ribose portion of the toxin ribosylates the G s subunit inhibiting their GTPase activity.
Increased cAMP levels are a result of active binding to adenylyl cyclase. The CFTR channel is activated,
resulting in secretion of chloride ion and sodium ion flow into the intestinal lumen.
o The ion secretion is followed by loss of water, resulting in vomiting and watery diarrhea.
Know Gi signaling
o Gi signaling can inhibit adenylyl cyclase activity by the G subunit.
o Gi subunits act mainly by regulating ion channels.
o Whooping Cough and Pertussis toxin
The inhibitory Gi is inactivated by pertussis toxin, Gi can no longer inhibit adenylyl cyclases
and results in an increase in cAMP
Symptoms: similar to cold, usually develop about a week after exposure. Severe episodes of
coughing start about 10-12 days later. In children, coughing often ends with a whoop noise.
The sound is produced when the patient tries to take a breath. Often see uncontrollable fits, each
with 5-10 forceful coughs (machine gun coughing).
Coughing spells may lead to vomiting or a short loss of consciousness. Other pertussis
symptoms include runny nose, slight fever (102 F or lower), and diarrhea.
Know Transducin (Gt) signaling in photoreception and its role in light induced hyperpolarization of rod
cells
o Cyclic GMP (cGMP) is also an important second messenger in animals. cGMP is formed from GTP by
guanylyl cyclases and degraded to GMP by a GMP phosphodiesterase.
o In the vertebrate eye, cGMP is the second messenger that converts visual signals to nerve impulses. This
process is controlled by the rhodopsin, transducin (Gt) and a cGMP phosphodiesterase (will
decrease levels of cGMP).
o Role of transducing (Gt) in photoreception
The photoreceptor in rod cells of the retina is a G protein coupled receptor called rhodopsin
Rhodopsin is activated when light is absorbed by the associated small molecule retinal.
Rhodopsin then activates the G protein Transducin (Gt).
The G subunit stimulates cGMP phosphodiesterase, leading to decreased levels of
cGMP.
cGMP levels are translated to nerve impulses by a direct effect of cGMP on ion
channels.
Must know Gq signaling pathways in detail !!!!!
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o Information: many G proteins use inositol triphosphate and diglycerol as second messengers.
Phospholipase C cleaves PIP2 into inositol triphosphate and DAG
o Know Gq signaling in platelet activation
ADP released from damaged cells activates Gq receptors
This activates PLC
PLC cleave PIP2 IP3 and DAG
IP3 binds and opens calcium channels associated with endoplasmic reticulum
Platelets also released growth factors to tell local cells to proliferate to promote wound healing
and bleeding.
Know that prostaglandins typically activate G protein receptors
o Prostaglandins such as thromboxanes A (TXA2) can stimulate Gq signaling pathways.
o Prostaglandins are synthesized from arachidonic acid.
Cyclooxygenases are key enzyme in the pathway. Aspirin blocks cyclooxygenases, thereby
inhibiting synthesis of thromboxane A2 and other prostaglandins from arachidonic acid. Aspirin
significantly reduces incidence of heart attacks by reducing blood clots.
Know how Methylxanathines (caffeine and theophylline) mediate their effects
o Caffeine from coffee; tea (theophylline); theobromine from chocolate.
o Two modes of action:
1) The principal mode of action of caffeine is as an antagonist of adenosine receptors.
The caffeine molecule is structurally similar to adenosine.
2) Methylxanthines are also cAMP phosphodiesterase inhibitors
which will increase levels of cAMP
Main intracellular targets of cAMP
o cAMP-dependent kinase or protein kinase A (PKA)
Have two regulatory subunits and two catalytic subunits.
When cAMP binds to PKA, the regulatory subunits disassociate from catalytic subunits. The
cAMP pathways are important for regulating glycogen metabolism, heart contraction, blood
clotting, and secretion of salts and water in the gut.
o EPAC (exchange protein activated by cyclic AMP)
Activated by binding of cAMP and acts as guanine nucleotide exchange factors for RAP
GTpases.
o Ion channels
Know the entire pathway from epinephrine binding to glycogen breakdown in detail!!!!
o Epinephrine binds beta-adrenergic receptor
o Gs protein binds GTP, dissociates from other subunits
o Activated Gs binds and activates adenylate cyclase
o Adenylate cyclase produces cAMP from ATP
o PKA binds cAMP and becomes activated
o PKA phosphorylates and activates phosphorylase kinase
o Phosphorylase kinase phosphorylase B active phosphorylase A
o Phosphorylase A catalyzes the phosphorolytic cleavage of glycogen glucose-1-phosphate
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Know glucagon signaling pathway

o Glucagon is a polypeptide hormone secreted by the alpha cells of the pancreatic islets of Langerhans.
Glucagon binds to high affinity G protein-coupled receptors on hepatocytes and stimulates Gs signaling.
(glucagon receptors are NOT found on skeletal muscle)
o This is simultaneously shutting off glycogen synthesis. PKA also phosphorylates the enzyme glycogen
synthase and inactivates it.
Know that PKA can phosphorylate transcription factors
o cAMP inducible gene expression the free catalytic subunit of protein kinase A translocates to the
nucleus and phosphorylates the transcription factor CREB leading to expression of cAMP inducible
genes.
Know that odorant receptors are G-linked couple receptors that stimulate adenylyl cyclase and then
cAMP binds sodium channels
o The largest family of G protein coupled receptors is responsible for odor detection and recognition.
Odorant receptors are encoded by a large multigene family of hundreds of genes. Odorant receptors
stimulate adenylyl cyclase, leading to an increase in cAMP.
o cAMP binds to and opens sodium channels in the plasma membrane, leading to initiation of a nerve
impulse.
Know the general role of protein phosphatases in signaling
o Protein phosphorylation is rapidly reversed by the action of protein phosphatases, which terminates
responses initiated by receptor activation of protein kinases.
Calcium and signaling
o Calcium levels are low in the cytosol. It is active pumped out of the cytosol to the cell exterior. Various
molecules in the cytosol bind free calcium tightly. The frequency of calcium oscillations influences a
cells response. It also controls a wide range of cellular processes.
o In electrically excitable cells of nerve and muscle, voltage gated calcium channels are opened by
membrane depolarization. The resulting increase of intracellular calcium signals the further release of
calcium from the ER by opening of ryanodine receptors (calcium channels). These channels are
important for the amplifying of calcium response.
The ligand is calcium binding of calcium induces the calcium channel to open.
o Know Malignant Hyperthermia
Is an autosomal dominant condition in which there may be a dramatic adverse response to the
administration of many commonly used inhalational anesthetics (e.g. halothane) and
depolarizing muscle relaxants such as succinylcholine.
40
Malignant hyperthermia is most frequently associated with mutations in the gene for the
ryanodine receptor 1
Soon after induction of anesthesia, patient develops threatening fever, sustained muscle
contraction, and attendant hypercatabolism.
Increased levels of ionized calcium in the sarcoplasm of muscle after drug exposure. This
increase leads to muscle rigidity, elevation of body temperature, rapid breakdown of muscle
(rhabdomyolysis), and other abnormalities.
The condition is an important if not common cause of death during anesthesia.
Know Ryanodine receptors
o Interaction of the alpha subunit of calcium channel, DHPR, and the ryanodine receptors (RyR1)
connections both membranes, T-tubule, and SR membranes. This connection is responsible for
electromechanical coupling. Calcium release from the SR via the RyR1 triggers muscle contraction and
multiple cellular effects by binding of calcium to a variety of other target proteins.
Know how calmodulin functions during calcium signaling
o In the cytosol, calcium binds to proteins like calmodulin. Calmodulin is one of the most important Ca
binding proteins in the cell and can constitute as much as 1% of the total protein mass. When calmodulin
is activated after binding calcium, it targets and regulates kinases and phosphatases.
o Calcium-calmodulin dependent protein kianses (CaM-kinases) mediate many of the responses to Ca
signal in animal cells
CaM-kinases are a family of serine-threonine kinases. Some CaM-kinases phosphorylate gene
regulatory proteins such as CREB proteins
CaM-kinase II is enriched in the nervous system and is concentrated in synapses.
Know the entire pathway of acetylcholine stimulation of endothelial cell to smooth muscle cell
relaxation!!!
o Acetylcholine binds to heptahelical receptor and triggers Gq signaling in endothelial cells.
o Calcium is released into the cytoplasm from the sER and binds to calmodulin
o Calmodulin activates NO synthase and creates NO and citrulline from arginine
o NO diffuses out of the endothelial cells and into the smooth muscle cell
o NO binds and activates guanylyl cyclase synthesizes cGMP from GTP
cGMP phosphodiesterase keeps cGMP in check
o cGMP activates protein kinase G resulting in smooth muscle relaxation and vasodilation.
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Information on NO: paracrine signaling molecule in the nervous, immune, and circulatory system which alters
the activity of enzymes. NO is synthesized from arginine by nitric oxide synthase. Its action is restricted to local
effects because it is unstable and has a short half-life. NO stimulates guanylyl cyclase to make cGMP
Must know how nitroglycerin and Sildenafil effect NO and cGMP signaling
o Nitroglycerin for angina to release constricted coronary arteries. Glycerol trinitrate decomposes to NO,
which activates a guanylyl cyclase. This will relax arterial smooth muscle cells (vasodilation).
o Sildenafil (Viagra) is an inhibitor of cGMP specific phosphodiesterase (breadown of cGMP).
Nitric oxide released by the neurons in the penis results in the blood vessel dilation responsible
for penile erection.
Viagra helps maintain elevated cGMP in erectile tissue, this pathway is stimulated for a longer
time period following NO release.
Know how carbon monoxide acts a signaling molecule
o CO also functions as a signaling molecule in the nervous system. It acts similarly to NO as a
neurotransmitter and mediator of blood vessel dilation. CO can stimulate guanylate cyclase, which also
represents the major physiological target of CO signaling.
Know of receptor protein-tyrosine phosphatases and receptor guanylyl cyclases
o
Know that Insulin receptor, EGF receptor, FGF receptor and VEGF receptors are all examples of tyrosine
kinase receptors
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Three major signaling branches of receptor tyrosine kinase

o Ras/MAPK signaling pathway
o Phospholipase C-gamma signaling pathway
o PI 3-kinase/AKT signaling pathway
Regulatory proteins with SH2 domains recognize and bind to phosphorylated tyrosines on
receptor. This binding stimulates regulatory proteins.
Three types of intracellular signaling complexes associated with receptors
o Assemblies help simplify and localize signaling through receptors. Assembly of signaling complexes
depends on various conserved interaction domains, which are found in many intracellular signaling
proteins.
the activation of RTKs
o ligand binds, receptors aggregate, autophosphorylation occurs between receptors. (activated receptor
tyrosine kinases phosphorylate themselves)
o phosphorylated tyrosines on receptor typrosine kinases serve as docking sites for intracellular signaling
proteins. Proteins with either SH2 domains or PTB can bind phosphorylated tyrosines.
SH2 domain is a compact module. Each SH2 domain has distinct sites for recognizing
phosphotyrosine and for a particular amino acid side chain. This variation allows receptor
tyrosine kinases to bind with specificity.
MUST know RAS-MAPK signaling pathway in detail!!!
o GRB2 (contains SH2 domain) interacts with tyrosine phosphorylated receptor, localizing it to the
membrane.
o SOS is associated with GRB2 and becomes active (SOS is a guanine-nucleotide exchange factor)
o SOS stimulates RAS (a monomeric G-protein) to release GDP for GTP
o RAS is now ACTIVE activates RAF (a MAPKKK) activates MEK (a MAPKK) activates
ERK (a MAPK)
o MAPKs phosphorylates transcription factors (such as AP-1)
o Transcription factors induce cells to grow and divide
o RAS is inactivated by GTPase activating protein (GAP)
o Information: this is a common pathway mutated in cancer.
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MUST know Noonan Syndrome

o Is an autosomal dominant condition which is characterized by short stature, distinctive craniofacial
features, congenital cardiovascular disease, and other more variable clinical findings such as
developmental delay and intellectual disability, bleeding tendencies, lymphatic abnormalities and
genitourinary abnormalities. Occurs in ~1-1000 individuals. Often genes enoding Ras/MAPK signaling
proteins are mutated (PTPN11, KRAS, SOS, RAF1).
o Also known as pseudo-turners. There is a characteristic webbing of the neck in both Turners and
Noonans syndrome. Turners syndrome will only be seen in females, but Noonans can be seen in both
male or female. A boy with webbing of the neck will likely have Noonans.
Activation of phospholipase C-gamma by protein tyrosine kinases
o PLC-gamma has SH2 domains that associate with receptor protein tyrosine kinases.
o Tyrosine phosphorylation increases PLC-gamma activity, stimulating hydrolysis of PIP2 to produce IP3
and DAG. (IP3 stimulates Ca relase and DAG stimulates PKC pathway). PKC requires the binding of
calcium and phosphotidylserine.
Must know Calcineurin NFAT signaling pathway
o Stimulated T cells trigger PLC-gamma resulting in calcium being released. Calcium binds and activates
in calmodulin binds and activates phosphatase called calcineurin. Calcineurin dephosphorylates
NFAT (transcription factor) allowing it to go into the nucleus and stimulate IL-2 gene expression.
Cyclosporin A
o
Important immunosuprresant used to prevent rejection of transported organs. Works by
inhibiting calcineurin. (cyclosporine blocks phosphatase ability, thus preventing the transport
into the nucleus and remains in the cytosol).
o IL2 is a very important molecule for triggering T cells to proliferate and stay alive. An increase in the
response will defend the body against infection etc. why would you want to suppress your immune
system? Organ transplantation.
Know PI 3-Kinase/AKT signaling pathway and the role of phosphatase PTEN
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o PI 3-kinase binds phosphorylated tyrosines on receptor and becomes stimulated. PI 3-kinase
phosphorylates PIP2 in membrane. This regulates cell growth, survival and movement.
o PI 3-kinase produces lipid docking sites in the plasma membrane. Also phosphorylates PIP2 into PIP3.
Intracellular signaling proteins can interact with PIP3 via specific interaction domain, such as pleckstrin
homology domain (PH).
o AKT activation
AKT and PDK1 bind to PIP3
Another kinase (usually mTOR) phosphorylates AKT, this causes a conformational change in
AKT that allows PDK1 to phosphorylate AKT
The activated AKT dissociates from the plasma membrane and phosphorylates various targets,
including the BAD protein (involved with apoptosis).
o AKT and forkhead (FOXO)

AKT phosphorylates several transcription factors including members of the forkhead or FOXO
family.
If growth factors are NOT present, AKT is NOT active, and FOXO travels to the nucleus where
it stimulates transcription of genes that inhibit cell proliferation, or induce cell death.
AKT phosphorylation of FOXO sequestered by molecule 14-3-3 and be inactive form
o Role of phosphatase PTEN

The role of PTEN is to dephosphorylate PIP3 (back to PIP2), acting as a negative control on
PKB/AKT activation.
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If a mutation in PTEN renders it unable to carry out its phosphatase function, PIP3 can no longer
be deactivated so it continues to propagate its signal downstream. This may result in the
continued activation of PKB/AKT, which in combination with other factors, could lead to
increased cell growth and possible tumor development.
MUST Know Type I and Type II Diabetes
o Insulin stimulates the uptake of glucose into muscle and adipose cells and by stimulating glycogen
metabolism. It thereby lowers blood glucose levels.
o Type I the inability to produce insulin
IDDM- usually caused by an autoimmune destruction of islet beta cells in the pancreas
(triggered by unknown mechanism). Early sign of abnormality: development of islet
autoantibodies with normal insulin/glucose responses. This period is followed by glucose
tolerance after a meal. With continued loss of beta cells, hyperglycemia eventually
developments, and insulin production falls below a critical period. Patients become dependent
on exogenous insulin supplements and have a propensity for ketoacidosis.
o Type II results from an improper response to insulin. Glucose remains in the blood.
NIDDM- is a multifactorial disorder with genetic and other factors involve. One in six patients
with NIDDM will develop end-stage renal disease or will require lower extremely amputation. 1
in 5 will become legally blind from retinopathy. Patients with NIDDM do NOT develop
ketoacidosis.
o The insulin signaling pathway influences glucose homeostasis by regulating multiple pathways. The
insulin receptor is a multi-subunit receptor tyrosine kinase. When it binds insulin, recruitment and
activation of the IRS1 protein initiates signal transduction, leading to glucose import, stimulation of
glycogen synthesis, and regulation of gene expression.
Must Know the insulin synthesis process
o Know the cellular location for each modification during insulin production
Genes coding for insulin are transcribed to mRNA in the nucleus. In the cytosol, ribosomes bind
to mRNA and translates, but it stops at the leader sequence if SRP is bound. SRP will bring the
strand to a channel structure called a translocon on the rough ER and dock on the cytosolic side
of the rER. The polymer will grow through the translocon into the lumen (preproinsulin). The
signal sequence is cleaved to form proinsulin. Proinsulin is transported from the rER to the golgi
complex where it is cleaved to form the C peptide and insulin. Secretory granules are secreted by
exocytosis, releasing insulin and C peptide.
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Know what is C-peptide and how it is used to monitor for source of insulin
o
Elevated insulin levels with no detectable C-peptide indicate exogenous insulin
If there is a detectable C-peptide, then the increase in insulin is inside the body.
Know insulin signaling pathway
o Example of tyrosine kinase receptor signaling pathway
Insulin binding activates RTK activity in the intracellular domain of the beta subunit of the
insulin receptor.
Tyrosine residues of the beta subunit are autophosphorylated
RTK phosphorylates other programs, for example, IRS
Phosphorylation of IRS promotes activation of other protein kinases and phosphatses leading to
biological actions of insulin
Increase in: glucose uptake, glycogen synthesis, fatsynthesis
Decrease in: gluconeogenesis; glyconeogenesis; lipolysis
o The role of IRS
The activated receptor binds IRS molecules and phosphorylates IRS at multiple sites, thereby
forming binding sites for proteins with SH domains.
o The four branches off IRS (Ras-MAPK, PLC, PI3-kinase, recruiting Glut permeases to cell
membrane)
Insulin signaling recruits GLUT transporters to the cell membrane
Insulin binds to its receptor in the cell membrane
Activated receptor promotes recruitment of glucose transporters from intracellular pool
to cell membrane
Glucose transporters increase insulin mediated uptake of glucose into cell
When insulin levels decrease, glucose transporters move from cell membrane to
intracellular storage pool, where they can be recycled
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Vesicle fuse to form an organelle called the endosome
The four sub-routes off AKT
Action of AKT (PKB) has four important consequences
o First it leads to movement of GLT4 from vesicles in the cytosol to the plasma
membrane, allowing glucose uptake
o Second, AKT can phosphorylate glycogen synthase kinase-3 (GSK-3) reducing
its activity. This leads to an increase in glycogen synthesis.
o Third, it alters TSC/mTOR signaling pathway and increases protein translation
o Fourth, it alters FOXO pathway leading to decreased glucose synthesis
MUST Know TSC/mTOR signaling
o mTOR stands for mammalian target of rapamycin
rapamycin is an immunosuppressive drug which was recently discovered to also prolong life in
mice (anti-aging effect).
The mTOR containing complexes mTORC1 and mTORC2 are central components of nutrient
and hormone sensitive pathways that regulate cell growth and proliferation.
Abnormal regulation of these pathways has been linked to diabetes and tuberous
sclerosis
Drugs, such as mTOR inhibitor temsirolimus, (Torisel), target this pathway to help treat
cancer
o Know Tuberous Sclerosis
Inherited disorder whose key features include multipl facial angiofibromas, hypopigmented
macules, periungual fibromas, seizures, shagreen patch, cardiac rhabdomyoma, and renal
lesions. Many patients have ocular and neurologic manifestations, including mental retardation.
The disorder results from a mutation in either TSC1 or TSC2
TSC2 mutations account for the majority of sporadic cases. These individuals usually show a
more severe phenotype that includes renal lesions and neurologic deficits.
o Know that the immunosuppressant drug rapamycin complexes with FKBP12 and inhibits mTORC1
mTORC1 is a component of nutrient and hormone sensitive pathways that regulate cell growth
and proliferation.
o Know AMPK signaling
AMPK is a pivotal regulatory molecule in the metabolism of carbohydrates and fats. Examples
of hepatic targets of AMPK:
mTORC1and TSC2- decreased protein synthesis
TORC2- protein is sequestered in the cytoplasm when phosphorylated by AMPK
leading to DECREASED expression at the transcriptional level of gluconeogenic
enzymes
AMPK can be activated in several ways, all of which depend on increased AMP levels within
the cell.
Activated by phosphorylation by LKB1 or calmodulin kinase kinase
The drug metformin activates AMPK via LKB1
Must know Metformin
A drug used for treatment of type 2 diabetes
Reduces blood glucose levels by inhibiting hepatic gluconeogenesis, which is active in
these patients because of the livers resistance to the effects of insulin.
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Metformin also reduces lipid synthesis in the liver. Also, it stimulates glucose uptake by
the muscle which is significant for reducing circulating blood glucose levels.
Activation of CREB
o Under normal conditions, increased levels of hepatic cAMP levels (in response to glucagon) activates
CREB, which in combination with TORC2, leads to increased transcription of genes required for
gluconeogenesis.
o Under conditions of insulin resistance, this pathway remains stimulated, even in the presence of insulin
Action of metformin on gluconeogenesis
o Metformin stimulates the activation of AMPK via LKB1.
o AMPK phosphorylates TORC2 and sequesters it in the cytoplasm, thereby decreasing synthesis of
gluconeogenic enzymes and reducing hepatic output of glucose.
Know the term Dominant Negative Mutation

o Have an altered gene product that acts antagonistically to the wild-type allele. These mutations usually
result in an altered molecular function (often inactive) and are characterized by a dominant or semi-
dominant phenotype.
Know Achondroplasia
o Also known as short limbed dwarfism; characterized by small stature with short limb, large head, low
nasal bridge, prominent forehead, lumbar lordosis. (Incidence is ~1-1000).
o Pathology: failure of cartel age cell proliferation at the epiphyseal plates of the long bones, resulting in
failure of longitudinal bone growth, causing short limb.
o Mutation in fibroblast growth factor receptor 3 (FGFR3) gene;
~80% of patients are due to new mutations; increased risk with late paternal age
Know TGF(beta) receptor signaling pathway
o Example of Serine/threonine kinase receptor- transforming growth factor beta receptor
TGF ligand binds a complex of type I and II receptors
Type II receptor phosphorylates type I receptors
Type I receptors phosphorylates an R-Smad (Smad2 or Smad3)
R-smad complexes with smad4
In the nucleus, the smad complex associates with transcription factors to modulate gene
expression
Other types of enzyme linked receptors
o Protein-tyrosine phosphatases remove phosphate groups from phosphotyrosine, counterbalancing the
effects of protein-tyrosine kinases.
Example: Cd45
Following antigen stimulation, cd45 dephosphorylates a phosphotyrosine that inhibits
the enzymatic activity of SRC family members, which stimulates nonreceptor protein
tyrosine kinase
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o Receptor guanylyl cyclase ligand binding to receptors stimulates the cyclase activity of the receptor to
synthesize cGMP from GTP.
Know JAK-STAT signaling pathway
o Information: Cytokine receptors activate the JAK-STAT signaling pathway, providing a fast track to the
nucleus. Tyrosine kinase associated receptors called JAK-STAT are often used by cytokines to regulate
the proliferation of certain cells involved in the immune response. The receptor itself has no intrinsic
kinase activity, but it binds the tyrosine kinase JAK (janus kinase). STAT proteins are located in the
cytosol and are referred to as latent gene regulatory proteins because they only migrate into the
nucleus and regulate transcription after they are activated. There are many different STAT proteins.
Receptors for different cytokines bind different STATs.
o Signaling pathway
Binding of cytokine cross-links adjacent receptors and JAKs cross-phosphorylate each other on
tyrosines.
Activated JAKs phosphorylate receptors on tyrosines
After STATs dock on specific phosphotyrosines on the receptor, then JAKs phosphorylate them.
STATs dissociate from receptor and dimerize via their SH2 domain
STATs translocate to nucleus, bind to DNA and other gene regulatory proteins, and activate gene
transcription
Know Notch signaling pathway - latent gene regulatory protein

NOTCH signaling is very important during development. For example, when individual
epithelial cells begin to develop as neural cells, they signal to their neighbors not to do the same
through Delta binding receptor NOTCH
Upon binding Delta, NOTCH is cleaved on the other side of the plasma membrane
The freed cytoplasmic tail of NOTCH then migrates to the nucleus, associates with other
regulatory factors bound to promoter elements and modulates gene transcription.
Know Wnt/beta-catenin signaling pathway
o Wnt proteins are secreted signal molecules that act as local mediators and morphogens to control many
aspects of development
o Wnt proteins bind to frizzled receptors and inhibit degradation of beta-catenin
o APC (destruction complex) is a key protein in the regulation of beta-catenin (APC is often mutated in
some forms of colon cancer)
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o When Wnt is present, APC is turned off, and beta-catenin accumulates and translocates to the nucleus,
displaces groucho and associates with coactivator.
o When Wnt signaling is NOT present, APC is active and phosphorylates beta-catenin, degrades it, and
keeps its level low.
Hedgehog signaling
o The receptor for hedgehog is a transmembrane protein (patched) that inhibits second transmembrane
protein (smoothened) by an unknown mechanism. Binding of hedgehog inhibits patched, leading to
activation of smoothened, which initiates a signaling pathway leading to activation of a transcription
factor.
Know NF-kB-dependent signaling pathway and its feedback regulation
o The NF-kB proteins are latent gene regulatory proteins that are present in most animal cells and are
central to many stressful, inflammatory, and innate immune responses.
o Upon cell signaling by TNF, ikB kinase becomes active and phosphorylates ikB
o The inhibitory protein (ikB) dissociates from NF-kB in the cytosol.
o NF-kB migrates to the nucleus, where it interacts with coactivators to alter gene transcription.
Feedback inhibition of NK-kB

o This regulation is critical because extent and duration of NJ-kB activity can determine the transcriptional
response of the cell. Some target genes are induced by transient NF-kB, but induction of other genes
requires several hours of sustained NF-kB signaling.
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o NF-kB is activated as a result of phosphorylation and degradation of IkB, but one of the genes activated
by NF-kB encodes IkB, generating a feedback loop that inhibits NF-kB activity.
Know integrin signaling via FAK
o Integrin signaling
Binding of integrins to the extracellular matrix leads to activation of FAK (focal adhesion
kinase), a non-receptor protein-tyrosine kinase.
Phosphorylation of FAK by SRC provides binding sites for several signaling molecules
including grb2-sos complex, leading to activation of Ras, PL3-kinase, and PLC-gamma.
Know the role of Rac, Rho & Cdc42 in actin remodeling
o Members of the rho subfamily of small GTP-binding proteins (including RAC, RHO, and CDC42) play
central roles in regulation of the organization of actin cytoskeleton.
o Stimulation of actin polymerization by Rho family proteins
Rho family members are activated by integrin signaling and growth factor receptors. Multiple
target proteins mediate cytoskeletal changes. All of the rho family proteins promote actin
polymerization.
Rho also targets protein-serine/threonine kinase called ROCK, which promotes the formation of
stress fibers
Know signaling networks and crosstalk
o Signaling networks do not operate in isolation, and intracellular signal transduction is really an
integrated network of connected pathways. The activity of signaling pathways is controlled by feedback
loops such as NF-kB.
o Crosstalk is the interaction between signaling pathways, such as junctions between Ca and cAMP
signaling between the cAMP and ERK pathways, and between integrin signaling and receptor protein-
tyrosine kinases.
the downstream signaling pathways activated by receptor tyrosine kinases or by g-protein linked
receptors overlap and are interconnected
disruption of growth factor signaling can lead to cancer
o Ras mutations are often associated with cancer.
o Epidermal growth factor receptor mutations can lead to breast cancer, glioblastoma, and fibrosarcoma
(cancer of long bones)
o TGF beta type 1 receptor mutations occur in 1/3 of ovarian cancers
o TGF beta type 2 receptor mutations occur in many colorectal cancers
o SMAD4 mutation occur in ~50% of pancreatic cancers
Presentation # 2: Extracellular Structures and Cell Junctions

o Know that bacterial cell walls are composed of peptidoglycan
The principal component of all bacterial cell walls is a peptidoglycan: linear polysaccharide
chains cross-linked by short peptides.
o Know the difference between gram positive and gram negative bacteria
Bacteria are divided into two large classes based on cell wall structure.
Gram negative bacteria have a dual membrane system, with a thin cell wall in between
Gram positive bacteria have a single membrane, surrounded by a much thicker cell wall
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o Must know mode of action for penicillin

Inhibits the enzyme DD-transpeptidase that forms the cross-links, preventing cell wall synthesis
and bacterial growth.
Two main ways in which animal cells are bound together
o In connective tissues, the main stress-bearing component is the extracellular matrix. In epithelial tissue, it
is the cytoskeletons of the cells themselves, linked from cell to cell by anchoring junctions. Cell-matrix
attachments bond epithelial tissue to connective tissue beneath it.
Some of the ECM matrix functions include: adhesion, filtration, structure, solubility, migration, and sensor
mechanisms.
o ECM consists of 3 classes of molecules: structural proteins (collagen, elastin); protein-polysaccharide
complexes (proteoglycans); adhesive glycoproteins (fibronectins)
Collagen
o Responsible for the strength of the ECM. Two defining characteristics include a rigid triple helix and
unusual amino acid composition (high % glycine). The stability of collagen fibril is reinforced by
hydrogen bonds that involve hydroxyl groups of hydroxyproline and hydroxylysine residues in the
alpha chain. The combination of alpha chains determines the type of collagen.
o Composed from triple helices that are wound around one another like a ropelike structure. The triple helix
consists of repeats of the amino acid sequence gly-X-Y (a glycine every third position).
Glycine is the smallest amino acid, and allows polypeptides to pack closely together.
o Proline is frequently found in X position and hydroxyproline in the Y position; they stabilize the helices
o Types of collagen
Collagen I: skin, tendon, vascular ligature, organs, bone (main component of bone)
Collagen II: cartilage (main component of cartilage)
Collagen III: reticulate (commonly found alongside type I)
Collagen IV: forms bases of cell basement membrane
Formation of hydroxyproline
o Hydroxyproline is formed in the endoplasmic reticulum by modification of proline in collagen
polypeptide chains. Enzyme reaction requires vitamin C. hydroxyl groups are thought to stabilize the
triple helix by forming hydrogen bonds.
MUST Know the basic steps of collagen synthesis and assembly, locations where they occur, and the key
enzymes involved
Nucleus
Formation of mRNA for each type of alpha chain
Lumen of the endomembrane system
Synthesis of alpha chains of pre-procollagen with registration peptides (terminal non-collagenous
domains).
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Hydroxylation of specific prolyl and lysyl residues in the endoplasmic reticulum vitamin C
dependent
Attachment of soluble galactosyl and glucosyl to specific hydroxylysyl residues
Assembly of pre-procollagen strands into procollagen molecules (triple helix)
Transport of soluble procollagen to golgi complex Packaging of soluble procollgen in
secretory vesicles Secretory vesicles assisted by microtubules and microfilaments transport
soluble procollagen molecules to cell surface
Extracellular space
Discharge of procollagen molecules to extraceullular space. Procollagen peptidases cleave most
of the nonhelical registration peptides, transforming procollagen into insoluble tropopcollagen,
which aggregates to form collagen fibrils.
Fibrillar structure is reinforced by the formation of covalent cross links between the
hydroxylysines of tropopcollagen molecules catalyzed by the enzyme lysyl oxidase (copper
dependent enzyme).
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o Know collagen-related disorders
Osteogenesis imperfecta: charac features of OI vary greatly from person to person, even among
ppl w/same type of OI, & even w/in same fam
Type 1: MC & mildest type. AD. Bones predisposed to fracture. Most fractures occur
before puberty. Normal or near normal stature. Loose joints and low muscle tone. Sclera
(whites of eyes) have blue, purple, or gray tint; triangular face; tendency toward spinal
curvature; bone deformity absent or minimal; brittle teeth possible; hearing loss possible;
often beginning in early 20s-30s; collagen structure usually normal, but amt <than
normal.
Type 2: Most severe form; frequently lethal at or shortly after birth, often due to
respiratory problems; numerous fractures and severe bone deformity; small stature with
underdeveloped lungs; collagen is improperly formed; frequent missense mutation in
glycine (closer to carboxy end) causing disruption to form collagen.
Ehlers-Danlos syndromes: array of disorders that involve collagen and connective tissue.
Commonly associated with loose or hypermobile joints and hyper elasticity.
Scurvy (Know the role of vitamin C in collagen synthesis)
At risk: infants, elderly men, alcoholics, smokers
Vitamin C is easily lost via cooking, sensitive to heat
Deficient for 20-40 days
Swollen, bleeding gums, joints, loose teeth, pinpoint hemorrhages around hair
follicles (petechiae), gums, nails, soreness and stiffness of joints and lower extremities,
slow wound healing, anemia, fatigue; alveolar bone resoprtion results in loss of teeth;
perifollicular hemorrhages; patients have difficulty walling off infections to form
abscesses, so infections spread more easily; in children, vitamin C deficiency leads to
growth failure and collagen-rich structures such as teeth, bones, and blood vessels
develop abnormally.
Rebound scurvy- immediate halt to excess vitamin C supplements
Menkes dis: inborn error of metabolism that markedly decreases the cells ability to absorb
copper; Dxd by looking at victims hair, which appears both whitish and kinked when viewed
under a microscope. hair shorter & thinner on sides & back of head. hair can be reminiscent of
steel wool cleaning pads. x-linked recessive trait; disorder of copper transport caused by
mutations in the copper-transporting ATPase gene (ATP7A)
lysyl oxidase affected (dependent on copper), which is an EC enzyme that acts to
establish crosslinking
Sulfhydril oxidase that crosslinks keratin also requires copper.
Specific clinical features include:
o Distinctive facial features
o Pectus excavatum (midline depression in the bony thorax)
o Skin laxity particularly on the nap of the neck and trunk
o Umbilical or inguinal hernia
o Hypotonia, neurodeval delays, & failure to thrive, manifest at 3-6 mos age.
Know the structure and function of elastin
o Connective tissue with elastic fibers is common in organs that stretch and return to shape, such as the
lungs. Elastic fibers are mainly elastin (cross-linked into a network). Crosslinking is completed by
covalent bonds between lysine residues. Elastin is glycine and proline rich.
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o Microfibrils are composed of elastin and other proteins such as fibrilin.
One of the consequences of fibrilin mutation is increased activity of TGF(beta)-1
Know Marfans syndrome
Autosomal dominant disorder (1/10,000 individuals)
Symptoms: (S)keletal, (H)eart, (E)yes
o Skeletal: characterized by skeletal abnormalities. Heart: cardiovasculuar
diseases. Dilation of ascending of aorta leading to aortic rupture or congestive
heart failure. Eyes: ocular abnormalities (myopia and detached lenses)
Caused by a mutation in the gene for fibrillin-1 (FBN1), a key component of connective
tissue.
Typical signs of marfan syndrome
Wrist (walk-murdoch) sign: overlapping the complete distal phalanx of the thum and 5 th
finger when wrapped around the opposite wrist
Thumb (Steinberg) sign: extension of the entire distal phalanx of the thumb beyond the
ulnar border of hand when opposed across the palm
Elongated feet and hands as symptoms of marfans syndrome
Know the structure and function of proteoglycans
o Glycoproteins in which a large number of glycosoaminoglycans are attached to a single protein molecule.
Core protein with varying number of carbohydrate chains attached. Proteoglycans can be linked
directly to collagen fibers.
Key function: trap water (act like sponges and can hold up to 50x their weight); Proteoglycan
networks are resistant to compression and regain their shape if distorted.
o Know what are glycosaminoglycans (GAG)
Long polysaccharide chains, usually consisting of two monosaccharaides or monosaccharide
derivatives in strictly altering order. Hydrophilic molecules with many negatively charged sulfate
and carboxyl groups attract water and cations, thereby forming hydrated, gelatinous matrix.
Know how hyaluronate is different from other GAGs
Most GAGs exist as a component of proteoglycans and not as free glycosaminoglycans.
Hyaluronate is an exception and CAN occur as a FREE molecule
Hyaluronate is synthesized directly from cell surface by an enzyme complex embedded in
the plasma membrane. It can exist freely because it is not sulfated, while all other
GAGs are synthesized in the golgi.
Lubricating properties help reduce friction in joints. Abundant in dividing tissues to help
cells migrate.
Know fibronectin and laminins, and their primary functions
o Adhesive glycoproteins (fibronectin and laminins) bind proteoglycans and collagen in the ECM and also
with membrane receptors (integrins).
o Fibronectins
Bind cells to the matrix and guide cellular movement. Structure is two very large polypeptide
units joined by disulfide bonds at their carboxyl ends.
RGD sequence is recognized by integrins.
Effects of fibronectin on cell shape and cell movement: attaches cell to ECM, helps guide
migration.
Effects of fibronectin on wound healing: soluble plasma fibronectin promotes blood clotting by
binding fibrin and platelets
o Laminins
Usually restricted to the basal lamina. Laminins bind cells to the basal lamina. Consists of
alpha, beta, and gamma polypeptides with multiple binding sites, held together by disulfide
bonds.
o The ECM is modified by the matrix metalloprotease family, which digests a variety of matrix proteins,
including collagens, laminin, perlecan, and cell surface receptors and adhesion molecules.
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Know the structure and function of the basal lamina
o Also referred to as the basement membrane. The basal lamina is a thin sheet of specialized extracellular
material that underlies epithelial cells thereby separating them from connective tissue.
o Properties: structural support of tissue; permeability barrier; organize proteins on cell membranes and
helps regulates differentiation; all forms of basal lamina contain type IV collagen, proteoglycans,
laminins, and sometimes entactin or nidogen.
o Organized in one of three ways: surround certain cells, underlie epithelia, or interposed between two
cell sheets
Know what are integrins and their role in interactions between cells & ECM
o Structure of integrins: Two large transmembrane polypeptides, alpha and beta that associate non-
covalently. Specificity is determined by the alpha subunit. Cytoplasmic side forms complex that interacts
with cytoskeleton. RGD sequence in fibronectin helps interaction with integrin (can switch between
active and inactive conformation).
o Func: Mediate interactions between the ECM and cytoskeleton; regulating cell movement and
attachment.
o Signaling
Cell signaling can lead to integrin clustering. Integrins can also activate intracellular signaling.
Kinases recruited to clusters by adaptor proteins include focal adhesion kinase (FAK) and
integrin-linked kinase (ILK)
Cancer cells often have constitutively active FAK activity.
o Know cell-ECM junctions: Focal adhesion & Hemidesmosomes
Focal adhesions- migratory and non-epithelial cells.
Clustered integrins that interact with bundles of actin microfilaments via linker proteins
talin, vincilin, and alpha actinin
Can be very stable or turn over rapidly as the cell moves. Integrins can reversibly bind
matrix components due to the ability to change conformation between active and inactive
states.
Hemidesosomes- epithelial cells
Integrins are attached to IFs (keratin) via plakin family of linker proteins
Diseases of hemidesmosomes
o Bullous pemphigold- skin blistering autoimmune disease
o Junctional epidermolysis bullosa- severe blistering disease of the skin caused
by mutation in the beta4 subunit.
Know cell adhesion molecules (CAM) and cadherins
o CAM: Member of IgSF and interacts in a calcium dependent manner. N-CAM (neuronal) and L1-CAM
involved in the outgrowth and bundling of neurons.
o Know E-cadherin and anchor protein beta-catenin
Interaction in a calcium DEPENDENT manner and is characterized by a series of structural
similar extracellular subunits. Tissue specificity (e-cadherinepithelial; n-cadherinnervous
tissue; p-cadherinplacenta)
Play important roles in development; metastasizing cells often lose expression of cell surface
cadherins
Cadherins are linked to actin filaments indirectly by linking proteins such as beta-catenin
Know the step of leukocyte migration from blood vessels into infected tissue
o Selectins binding to carbohydrates involved with loose attachment
o Interactions of leukocytes with endothelial cells lining blood vessels are mediated by selectins near sites
of inflammation. Selections (and lectins) recognize and bind to carbohydrates on the cell surface.
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o ICAM interactions with integrins for firm adhesion (more stable adhesion)
Know the basic structure & function of adhesive junctions- link adjoining cells to each other:
o Adherens Junction (Zonula adherens)
Cadherin mediated adhesive junctions connected to the cytoskeleton by actin filaments. In
epithelial cells, adherens junctions typically form a continuous adhesion belt.
At junctions, cadherens interact with beta-catenin alpha-cateinin actin
Beta-catenin & p120 are members of armadillo protein family. bind to cadherin & help
maintain stability.
o Desmosomes (Macula adherens) button like points of adhesion
Gives structural integrity.
Desmosome core: extracellular space between two membranes
Desmogleins and desmocollins
Desmosome plaque: cytoplasmic
Desmoplakins and plakoglobin
Plakoglobin binds to desmocollin and also to desmoplakin. Desmoplakin attaches to
tonofilaments.
Know structure and function of Tight (Zonula Occludens)

o Occludin, Claudin and Junctional Adhesion Molecule (JAM)
Interact with zona occludens proteins that link to the actin filaments
o Tight junctions prevent the movement of molecules across the cell layers. Structure of tight junctions:
connected by sharply defined ridges.
o Claudins and occludins in the plasma membrane of adjoining cells make contact across the intracellular
space and bind tightly to each other, thereby connecting the cells together.
o Role of tight junctions in blocking lateral movement of membrane proteins. Helps to polarize the cell.
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Know structure and function of Gap Junctions

o Allows direct electrical and chemical communication between cells.
o A gap junction is a region at which the plasma membrane of two cells are aligned and brought in close
contact. the gap provides a point of cytoplasmic contact between the two cells.
o Connexins- hollow cylinders composed of 6 connexin units
o Regulated by calcium
Open at low calcium concentrations; closed at high concentrations; found in most cell types,
especially the heart and cerebellum
Know Charcot-Marie-Tooth disease

Collection of neuropathies caused by mutations in numerous genes. CMT is an inherited disease leading
to progressive degeneration of peripheral nerves, with slow loss of muscle control and eventual muscle
degeneration.
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CMTX1 is caused by an inherited connexin protein mutation (Cx32)
Recognize components (junctions) of the Junctional complex in an electron micrograph image
o Junctional complex
It is the whole assembly which includes tight junctions, adherens junctions and desmosomes
ZO- zona occludens; ZA- zona adherins; D- desmosome
Macule adherentes (MA - desmosomes)
Presentation # 4: The Nucleus, Chromosomes, and the Human Genome

Learn about the structure and function of the nucleus, nuclear pores, nuclear lamina and the nucleolus
o A double membrane nuclear envelope surrounds the nucleus
Inner and outer membranes separated by a perinuclear pore
The inner membrane rests on the nuclear lamina (a network of supporting fibers)
The outer nuclear membrane is continuous with the lumen of the ER
The nuclear envelope (phospholipid bilayer permeable only to small nonpolar molecules) has
specialized openings called nuclear pores
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these are the sole channels for small polar molecules, ions, proteins, and RNA to pass
through the nuclear membrane.
The nucleoplasm is the material in the interior space of the nucleus (except nucleolus)
o Must know how to recognize the above structures in an electron micrograph
o Arrows are pointing to nuclear pores
Learn about transport of materials between the nucleoplasm and cytoplasm through the nuclear pores
o Know the role of nuclear localization signal, karyopherins (importins, exportins), and monomeric
GTPase Ran
Passive diffusion of small molecules thru nuclear pores, but most proteins >60,000 daltons cannot
readily diffuse into nucleus. Active transport of large proteins and RNA thru nuclear pores. These
proteins possess 1 or + nuclear localization signals (NLS) to enable transport into the nucleus
(recognized by importin). Some proteins also have nuclear export signals called exportin.
Importin and exportin are members of nuclear transport receptors called karyopherins.
Monomeric GTPase Ran interacts with importin and brings it back to cytoplasm to be reused.
GTPase Ran will be dephosphorylated in the cytoplasm by Ran GAP protein & release importin.
Reg of nuclear import of transcription factors 1 mechanism is to associate with cytoplasmic
proteins that mask their NLS, so they remain in cytoplasm until dephosphorylated, bound to
importin, & brought into the cell.
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Traffic of RNA between the nucleus and the cytoplasm

o RNAs are transported as ribonucleoprotein complexes (RNPs).
o rRNAs are associated with ribosomal proteins and specific RNA processing proteins in the nucleolus.
mRNAs are associated with at least 20 proteins during processing and eventual transport to the
cytoplasm.
o Small RNAs (snRNAs and snoRNAs) are transport to the cytoplasm by an exportin (Crm1) where they
associate with other programs to form snRNPs and return to the nucleus
The nuclear matrix and nuclear lamina are supporting structures of the nucleus
o Nuclear matrix: the insoluble fibrous network in the nucleus after chromatin has been removed. The
nuclear matrix helps maintain shape of nucleus and provides an organizing skeleton for the chromatin
fibers.
o Nuclear lamina: is a thin, dense meshwork of fibers that lines the inner surface of the inner nuclear
membrane. It is constructed from intermediate filaments made of the protein lamin.
mutations in lamin genes cause several different inherited tissue specific diseases including
Hutchinson-gilford progeria
Mammalian cells have three lamin genes. Two lamins interact to form a dimer in which the alpha
helical regions of two polypeptide chains wind around each other to form coiled coil. Lamin
dimers associate to form the nuclear lamina.
Lamins and associated proteins also bind to chromatin, extending to form a loose meshwork
throughout the interior of the nucleus
Know Hutchinson-Gilford Progeria
o This disease causes premature aging and death. This disease results from a mutation in the LMNA gene
coding for Lamin A protein.
o Symptoms include abnormal growth, scleroderma-like skin, loss of hair, and cardiovascular problems.
The nuclei of HGP cells show dramatic changes in structure and changes to chromatin are similar to that
seen in the normal aging process.
Know the role of the acrocentric chromosomes 13, 14, 15, 21 & 22 in the formation of the nucleolus
o Nucleoli are memb free organelles in nuc. DNA sequences encoding rRNA reside in nucleolus. Nucleoli
contain nucleolus organizer region (NOR) stretch of DNA carrying multiple copies of rRNA genes.
Learn about ribosomal RNA transcription, processing and ribosomal subunit assembly in the nucleolus
o 45S-rRNA precursor synthesized by RNA polymerase I and cleaved into 5.8S, 18S and 28S rRNA
molecules
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o The nucleolus is the site of rRNA transcription and processing, and some aspects of ribosome assembly.
The nucleolus is NOT surrounded by a membrane.
o Robertsonian translocation: long arm fusion of one and another long arm. The individual can have 45
chromosomes and be okay if they had this translocation. During a Robertsonian translocation, the
participating chromosomes break at their centromeres and the long arms fuse to form a single
chromosome with a single centromere. The short arms also join to form a reciprocal product, which
typically contains nonessential genes and is usually lost within a few cell divisions.
o Processing of pre-rRNA
Processing of pre-rRNA includes addition of methyl groups to bases & ribose residues, &
conversion of uridine to psuedouridine. Nuclueoli contain >300 proteins and many snoRNAs that
function in RNA processing.
o Ribosome assembly
Formation of ribosomes requires assembly of pre-rRNA with ribosomal proteins and 5S rRNA.
Ribosomal proteins are imported to the nucleolus from the cytoplasm, where they assemble with
the pre-rRNA prior to cleavage. 5S rRNAs are similarly assembled into preribosomal particles
elsewhere in the nucleolus. Additional ribosomal proteins and the 5S rRNA are incorporated as
cleavage and processing proceeds.
Know the central dogma of molecular biology
o DNA RNA protein
Learn how DNA was first discovered to be the source of genetic information
o Know and recognize the phenomenon of transformation
Griffith identifies genetic transformation in pneumococcus
Living S (smooth) strain kills
Living R (rough) strain OK
Heat killed S OK
Heat killed S + living R kills
o Avery et. al experiments
Discovered that DNA from S strain caused transformation
They were first to rigorously document that DNA can carry genetic information. S strain nucleic
acid fraction is capable of transformation. DNase activity eliminates transformation ability.
Know the structure of DNA
o Antiparallel chains in the DNA double-helix held together by hydrogen bonds
Know how to determine the sequence of the complementary DNA strand if given the sequence of a single
strand of DNA
Learn Chargaffs rule and how to apply it
o A=T and G=C, hence the # of purines = # of pyrimidines (A+G = T+C)
o You can predict % of other bases if given a % of one base.
o Example: if a DNA sample from an organism has 21% guanine
it has 21% cytosine (G=C)
A+T = 100 42 = 58%
A = 29% and T = 29%
o Know if a DNA sample does NOT agree with Chargaffs rule, it is not double-stranded and most
likely single stranded
Learn how base composition affects melting temperature
The higher guanine + cytosine content of DNA, the higher the melting temperature
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DNA can be interconverted between relaxed and supercoiled forms
o Topoisomerases catalyze the stepwise relaxation of supercoiled DNA.
o DNA gyrase is a type II topoisomerase that can induce as well as relax supercoiling (require ATP to
generate supercoiling, but not to relax an already supercoiled molecule).
Learn about the human genome and about senquencing of the human genome
o The human genome is contained in the mitochondria and nucleus. The mitochondrial DNA contains 37
genes essential for normal mitochondrial functioning, and originates from the mother.
o Short arm of chr are designated w/ p for petite. Long arms of the chromosome are designated with q.
o Classification of chromosomes based on centromere location
Metacentric- centromere near central region of chromosome.
Submetacentric- centromere is off center and arms of clearly different lengths
Acrocentric- centromere near one end (13,14,15,21,22); contain small, distinctive masses of
chromatin called satellites attached to their short arms by narrow stalks which contain hundreds
of copies of ribosomal RNA genes.
o Genetic linkage analysis and maping of cDNA showed that the human genome is only 20,000-25,000
genes. Alternative splicing in human genes allows a single gene to specify more than one protein. Less
than 2% of the human genome are unique coding sequences.
Learn about fluorescent in situ hybridization
o Several thousand human genes had already been identified and mapped by methods such as in situ
hybridization of probes labeled with fluorescent dyes (FISH).
Learn the basic structure of a gene (introns & exons)
o Coding sequences (exons) are separated by noncoding sequences (introns). The entire gene is transcribed
to RNA and the introns are then removed by splicing. Only exons are included in the mRNA.
o Introns almost all histone genes lack introns. Introns are not found in most genes of simple eukaryotes
such as yeasts, but are present in rare genes of prokaryotes. Introns also allow exons of a gene to be
joined in different combinations, resulting in different proteins form the same gene alternative splicing
Know the acrocentric chromosomes
13,14,15,21,22
Learn about the major classes of DNA sequences
o Simple sequence repeats- tandem arrays of short sequences, from 1-500 nucleotides. Such repeat
sequences band as satellites separate from the main band of DNA, so they are called satellite DNAs.
They are not transcribed but some play important roles in chromosome structure.
Learn about tandem and interspersed repetitive sequences and their medical importance
o Tandem repeats multiple copies arranged next to each other in a row. 10-15% of mammalian genome.
abundant in centromeres and telomeres
alpha satellite DNA- moderately complex structures (5-171bp); clustered around centromeres of
chromosomes; no known function.
Minisatellite- moderately sized (0.1-20 kb); hypervariable minisatelliate DNA (VNTRs); often
found near telomeres
Microsatellite- small arrays of tandem repeats, simple in sequence (1-4 bp); highly polymorphic;
CG dinucleotide (CpG) repeats are prone to mutation due to methylation of C residues followed
by deamination; expansion of trinucleotide repeats associated with certain inherited diseases
(CAG repeats in Huntington disease)
o Short tandem repeats are microsatellites that are important for forensic testing because they can be
easily PCR amplified from small amounts of DNA.
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o Interspersed repetitive sequences- this type of DNA is scattered around the genome. A large portion of
interspersed repeated DNA is from the Alu family; transposable elements (self-replicating molecular
parasites, thus considered selfish DNA). Many are capable of transposition (hence considered
transposable elements). Transposable elements are found in prokaryotes and eukaryotes.
LINE vs SINE
SINE
o Know that Alu elements are ~300 bp in length and that they are examples of
SINE
o Members of the family are related, but not identical. 500,000 Alu family
members in genome. Make up several percent of DNA.
LINE
o Long repeats (up to 6kb); 100,000 copies per genome
o Plentiful in some regions, sparse in others. Transpositionally active and can
encode for reverse transcriptase.
o Medical important of interspersed repeats
Alu and L1 sequences have been implicated as the cause of mutations in hereditary diseases by
retrotranposition. Repeats may insert into a gene and disrupt function.
Aberrant recombination events between different copies of dispersed repeats can also cause some
genetic diseases. Abnormal recombination between different copies of the repeats can lead to
deletion or rearrangement of the gene.
o Retrotransposons vs DNA-only transposable elements
Reverse transcriptase- enzyme using RNA transcript as a template for a DNA daughter strand.
Transposase- enzyme which catalyzes movement of genetic elements.
DNA-only transposable elements
Can be recognized in chromosomes by the inverted repeat DNA sequences present at
their ends. These sequences are all that is necessary for the DNA between them to be
transposed by the particular transposase associated with the element.
Retrotransposons
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oKnow Fragile X Syndrome

X-linked dominant pattern. The fragile site on X chromosome (Xq27.3) is a non-staining gap
observed under cell culture condition of folate or thymidine depletion.
CGG trinucleotide repeat expansion triggering hyper-methylation and decreased FMR1 (fragile X
mental retardation) expression
CGG repeats in the 5 UT region, <52 = normal
>52 - < 200 permutation
When CGG repeats exceed 200, it triggers hyper-methylation of 5 UT and the promoter, thus
shutting down hyper-transcription.
Clinical features
Mental retardation and macroorchidism (large balls); deep plantar creases
FMRP is a RNA-binding protein that associates with polyribosomes to suppress the translation of
proteins from its RNA targets. These targets appear to be involved in a number of processes likely
to underlie the clinical features of fragile x patients.
Learn about gene families
o Many eukaryotic genes are present in multiple copies, which are needed to produce RNAs or large
quantities of proteins such as rRNAs or histones.
o Members of a group of related genes (gene family) may be transcribed in different tissues or at different
stages of development.
Ex: alpha and beta subunits of hemoglobin are encoded by gene families and different members
are expressed at diff stages of life. Alpha is expressed in the fetus and beta is expressed in adults.
o results are proteins optimized for different functions, but some mutations will result in the loss of
function.
Learn about the two origins of pseudogenes
o Psuedogenes are nonfunctional gene copies. There are more than 20,000 psuedogenes in the human
genome.
o Can arise in two ways:
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Duplication of segment of DNA results in transfer of block of DNA to a new location in genome.
Duplication by reverse transcription of mRNA, followed by integration of cDNA copy into new
chral site.
Duplication of gene by reverse transcription usuaully yields an inactive gene copy called
processed psudeogene they lack introns & normal sequences that direct transcription.
Learn about centromeres and telomeres
o The centromere is a specialized region of chromosome that helps ensure the correct distribution of
duplicated chromosomes of duplicated chromosomes to daughter cells during mitosis. Centromeres are
DNA sequences to which proteins bind, forming a kinetochore.
o Telomeres are sequences at the ends of chromosomes, and are required for the replication of linear DNA
molecules. The sequences are similar among eukaryotes, with repeats containing clusters of G residues on
one strand. They are repeated hundreds or thousands of times and end with a 3 overhang of single
stranded DNA. The telomere sequences of some organisms form loops at the ends. They bind a protein
complex (shelterin) that protects the chromosome termini from degradation.
The ends of linear chromosomes cant be replicated by DNA polymerase. Telomerase, which uses
reverse transcriptase activity, replicates telomeric DNA sequences.
Learn about the structure and packaging of DNA
o The nucleosome, 30 nm fiber, loops, condensed chromosomes, chromosomal territories, fractal
globules
Nucleosomes form beadlike structures on DNA. Partial digestion of chromatin generates 200 bp
ladder evidence that proteins are clustered at 200 bp intervals. We will see this again, DNA
laddering will be a hallmark of apoptosis.
MUST know which (and# s) histones are found in the core nucleosome
The nucleosome core particles contain 147 base pairs of DNA wrapped 1.67 times
around a histone core consisting of pairs of H2A, H2B, H3, and H4.
A chromatosome consists of 166 base pairs of DNA wrapped around the histone core
and held in place by H1 (a linker histone).
Know that core nucleosomal histones protect bound DNA from digestion from
nucleases such as micrococcal nuclease
Know about the role of histone H1 in the formation of 30 nm chromatin fibers
DNA wrapped in nucleosomes can form slightly thicker fibers called the 30-nm
chromatin fiber or solenoid requires histone H1
The solenoid can be folded into loop domains averaging 50-100 kb in length. Looped
domains are attached to the chromosomal scaffold.
Know the how chromatin is packaged into fractal globules
Packaging of heterochromatin into fractal globules has recently been reported. Allows for
easy and reversible access to tightly packaged DNA without getting tangled.
Learn about heterochromatin and euchromatin
o Heterochromatin is highly folded chromatin that is usually transcriptionally inactive. Heterochromatin is
highly organized and unusually resistant to gene expression. DNA is packaged so tightly that transcription
is inhibited.
Methylation of cytosine and heterochromatin
o Euchromatin is genomic DNA that is more loosely packed and diffuse. Most of the transcriptionally
active genes in euchromatin. Transcriptionally active genes often have acetylated histones associated with
their DNA.
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o During interphase, heterochromatin remains highly condensed and is transcriptionally inactive. The
euchromatin is decondensed and distributed throughout the nucleus.
Learn about DNA replication factories, nuclear speckles and Cajal bodies
o Replication factories DNA replication appears to take place in distinct functional bodies with multiple
replication complexes.
o Nuclear speckles the mRNA splicing machinery is concentrated in discrete nuclear bodies. They can be
seen with immunofluorescent staining with antibodies against snRNPs and splicing factors.
o Cajal bodies contain the characteristic protein coilin and are enriched in small RNPs. They may
function as sites of RNP assembly and processing.
Learn about epigenetic regulation
o The term that refers to any factor that can affect gene function without change in the genotype. Some
typical epigenetic factors involve alterations that change genome structure and affect gene expression
without changing the primary DNA sequence. (DNA methylation, chromatin structure, histone
modifications, transcription factor binding).
Learn about the Histone Code and the importance of acetylation and euchromatin
o Methylated DNA is often associated with heterochromatin. Methylation on cytosine of 5-CG-3 usually
in 5 noncoding sequences. Methylation patterns are inherited after DNA replication.
o X chromosome inactivation one of the X chromosomes in cells from females is highly condensed,
transcriptionally inactive heterochromatin.
o The core histones are covalently modified at many different sites. Acetylation is assocated with increased
transcription.
o Loss of H1 leads to unfolding of the 30 nm fiber. Use of different histone variants.
Learn about the chromatin organization of the centromere
Learn about mitochondrial DNA
o DNA molecules that resides in mitochondria and chloroplasts are devoid of histones and are circular
o The human mitochondrial genome is a 16.6 kb circular DNA encoding about 37 genes. most polypeptides
encoded by mitochondrial or chloroplast genomes are components of multimeric proteins that also
contain subunits encoded by the nuclear genome
Learn about polymorphisms
Learn how to analyze a karyotype
Learn about polypoidy
o Know the most common cause of triploidy and for tetraploidy
The most common cause of triploidy is poly-spermy. Two sperm fertilizing one egg.
The most common cause of tetraploidy (4n) results from failure of completion of an early
cleavage division of the zygote.
Learn about aneuploidy
o A cell with the number of chromosomes which is not a multiple of the haploid number.
Monosomy an autosomal cell with only 1 copy of a chromosome (lethal abnormality)
Trisomy a cell with 3 copies of a chromosome
o Know that most cases caused by nondisjunction during meiosis
Aneuploidy is a result of nondisjunction the failure of homologous chromosomes or sister
chromatids to separate normally during mitosis or meiosis. More common in meiosis I in females.
o Know Turner Syndrome (Karyotype: 45, XO)
Short stature, gonadal dysgenesis, webbed neck, low posterior hairline, broad chest with widely
spaced nipples; renal and cardiovasicular anomalies; at birth, infants have edema of the dorsum of
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the foot; coarctation of the aorta; lymphedema in fetal life, causing cystic hygroma, which is the
cause of the neck webbing; average to above intelligence; worse social cognition skills if X
chromosome comes from the mother.
Turner syndrome should be suspected in any newborn female with edema of the hands and feet or
with hypoplastic left sided heart or coarctation of the aorta.
The diagnosis should be considered in teenage years for girls with primary or secondary
amenorrhea, especially if they are of short stature.
Learn about Bioinformatics and Systems Biology
o RNAi screens
With the availability of complete genome sequences, libraries of double stranded RNAs can be
designed and used in genome wide screens to identify all of the genes involved in any biological
process.
RNAi process involves a double stranded RNA entity. It is going to be processed with RISC
enzyme, and one of the two strands will be lost. RISC complex targets complementary strand and
blocks production of protein.
Learn about polymorphisms (SNPs & CNPs)
o Polymorphism genetic difference found in more than 1% of the population.
Learn about genome wide association SNP screens
o The genome of two unrelated people differ in about 1 in every 1000 bases mostly in the form of single
base changes or single nucleotide polymorphisms (SNPs).
o Genome wide association scans have used SNPs to identify genes associated with inherited differences in
susceptibility to several common diseases
o The DNAs of thousands of patients and normal controls are hybridized to microarrays containing both
alleles of up to 500,000 common SNPs.
Presentations # 6 a&b: Intracellular Compartments
Must read all of Chapter 10, and Chapter 11 of The Cell, A Molecular Approach
Should be able to recognize the various intracellular structures and compartments in an electron microscope
picture
Understand how proteins are trafficked in the cell to the various organelles
o Free ribosomes in the cytosol proteins found in cytosol, nucleus, peroxisomes, and mitochondria.
o RER bound ribosomes proteins found in endomembrane system, plasma membrane, and secreted such
as to the lysosome.
Understand that most organelles cannot be constructed de novo:
o They require information in the organelle itself; proteins and organelles need to be passed on from parent
cell to progeny.
Know the road-map of the biosynthetic-secretory and endocytic pathways
o Endoplasmic reticulumthe golgi complexendosomeslysosomesnuclear envelopeperinuclear
spacetransport vesicles
Understand what are COP I, COP II and clathrin-coated vesicles and where they are formed
o COPI coated vesicles
Composed of COPI and ADP ribosylation factor (ARF). fuzzy coats, NOT lattices; COPI
facilitates retrograde transport of proteins from the golgi BACK to the ER. They DO NOT BUD
FROM THE ER.
Assembly is mediated by ARFI
o COPII coated vesicles transport of materials from the ER to the golgi
o Formation of clathrin coated vesicles requires: clathrin, GTP binding protein ARF1, and adapter
proteins (GGA and AP1).
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Assembly of coat introduces curvature and produces uniformly sized buds adapter proteins
bind clathrin triskelions and membrane bound cargo receptors mediating selective recruitment
clathrin coat rapidly lost about vesicle formation (by uncoated ATPase). Dynamin is a
cytosolic GTPase required for coated pit constriction and closing of the budding vesicle.
Understand that phosphoinositides mark organelles and membrane domains
o Membrane lipids can also be tagged to help vesicles reach their proper destination. Tag is usually one or
more phosphate groups added to phosphatidylinositol (PI). Inhibition of inositol kinases perturbs vesicle
trafficking to the endomembrane targets.
o Phosphoinositide head groups are recognized by proteins domains that discriminate between different
forms. Phosphorylation pattern of PI determines which proteins binds to it.
Know the role of Rab Monomeric GTPase in vesicle transport and targeting
o Rab proteins play a central part in the specificity of vesicular transport. They are monomeric GTPases.
Each Rab protein is associated with one or more membrane enclosed organelle. Helps ensure that the
vesicles interact with the correct target membranes.
o Rab effector proteins interact with Rab-GTP on the target membrane and establish the first connection
between two cells.
*Understand how Monomeric GTPase are regulated
o Off when bound to GDP
o On when bound to GTP
o Know the roles of GEFs and GAPs
Recognize both the smooth and rough ER in an electron microscopic image
Know what translational elements are in the ER
Understand the process of co-translation in the rough ER and how multipass integral membrane proteins
are produced by start and stop signals
o Co-translation
As the sequence is being inserted into the translocon, translation is continuing inside of the lumen
of the ER.
o Multipass membrane proteins are proteins that span the membrane multiple times and are thought to be
inserted by an alternating series of internal signal sequences and transmembrane stop-transfer sequences.
A combination of start-stop transfer signals determines topology of multipass transmembrane proteins.
o Role of ribosomes, SRP and signal peptide
Most proteins entering the endomembrane system are inserted into the rough ER lumen co-
translationally. Signal recognition particle (SRP) directs ER signal sequences to a specific
receptor on the rough ER. The SRP inhibits further translation and targets the entire complex to
the rough ER by binding to the SRP receptor on the ER membrane.
o The role of signal peptidase cleaving the signal peptide in the process of co-translation
o Know what type of proteins are produced in the rough ER
Proteins bound for the endomembrane system, plasma membrane or to be secreted are produced
in the rough ER
Understand the process of N-linked glycosylation in the lumen of the rough ER
o Dolichol carrier and core carbohydrate structure
o A precursor oligosaccharide complex is transferred en bloc to proteins in the ER. The precursor core
complex is constructed on a special lipid molecule (dolichol)
The core complex 14 sugars: 2 GlcNACs, 9 mannoses, and 3 glucose
Understand the role of N-linked glycosylation and protein folding and the possible fates of misfolded
proteins in the ER
o The glucose tag and calnexin and calreticulin- ER chaperones that recognize core oligosaccharide
structures missing two of the 3 glucose molecules. After the 3 rd glucose is removed, calnexin and
calreticulin can no longer bind and the properly folded protein can leave the ER.
Proteins that do NOT fold correctly are translocated back into the cytosol, where they are
deglycosylated, ubiquitylated, and degraded in proteasomes.
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o The pattern of N-linked glycosylation is used to indicate amount of protein folding so that proteins only
leave the ER when properly folded.
o Glucosyl transferase is the crucial enzyme that determines whether or not the protein is folded properly or
not. If the protein is still incompletely folded, the enzyme transfers a new glucose to the core structure.
Allows calnexin and calreticulin to bind and help refold misfolded protein.
Know that the Rough ER is the site of hydroxylation of proline and lysine during collagen synthesis
Understand how GPI-linked proteins are produced in the ER
o Immediately after the completion of protein synthesis, the precursor protein remains anchored in the ER
membrane by a hydrophobic C terminal sequence.
o An enzyme in the ER cuts the protein from its membrane bound C terminus and simultaneously attaches
amino group of preassembled GPI intermediate to the new C-terminus.
Know the Unfolded Protein Response and the role of BiP in the process
o Hsp70 chaperone BiP is thought to bind to an unfolded polypeptide chain as it crosses the membrane,
then mediate folding and assembly of multi-subunit proteins.
o If an excess of unfolded proteins accumulates, signaling via BiP initiates the unfolded protein response.
o UPR includes 1) an increase in degradation of mRNAs, 2) general inhibition of protein synthesis (PERK),
and 3) increased expression of chaperones.
PERK inhibitions general translation of proteins apoptosis. This pathway is constitutively
active in misfolded protein disease.
o Lipid synthesis
Smooth ER plays a central role in membrane biosynthesis because it is the primary source of
membrane lipids, including phospholipids, cholesterol, and steroid hormones.
Biosynthesis of phospholipids is restricted to the cytosolic monolayer of the ER and occurs in a
step-wise fashion from water-soluble precursors (glycerol).
Synthesis of phospholipids on the cytosolic side allows hydrophobic tails to remain buried in the
membrane.
New phospholipids are only added to the cytosolic half of the ER membrane. Some must be
transferred (flipped to the other side).
Role of scramblases and flippases
Scramblases transfer phospholipids nonspecifically from the cytosolic monolayer to the
outer monolayer (luminal) of the ER bilayer.
Flippases flip phosphotidylserine and phosphotidylethanolamine from the extracellular to
cytosolic leaflet asymmetry.
Production of ceramide
Ceramides are made by condensing a serine with a fatty acid to form amino alcohol
sphingosine. A 2nd fatty acid is added to form CERAMIDE
Ceramide is exported to the golgi and serves as precursor for glycosphingolipids and
sphingomyelin both of these are exclusively found in the noncytosolic monolayer of
the PM
Understand the functions of the smooth ER
o Detoxification and Phase 1 drug metabolism
Drug detoxification makes hydrophobic compounds more hydrophilic to promote excretion.
Hydroxylation is common for drug detox and steroid biosynthesis.
Electron transport system in smooth ER transfers electrons from either NADPH or NADH to
cytochrome p450 reduced p450 can donate electron to oxygen and activate it for hydroxylation
reactions.
Assisted by p-448 aryl hydrocarbon hydroxlase (AHH)
In chronic alcoholics or newborns, large amounts of anesthesia are needed (which may be
dangerous) because cytochrome p450 has been activated by detoxifying either alcohol or
breakdown of fetal hemoglobin.
Cytochrome P450s and mixed function oxidases
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Role in ethanol metabolism
o Carbohydrate metabolism
Hormone stimulated adenylyl cyclase pathway breakdown of glycogen into glucose 1P
Glucose 1P Glucose-6-phosphate
Catalyzed by glucose-6 phosphatase Role in dephosphorylating glucose before it is
exported from the cell
o Calcium storage
Important for signal transduction & the muscle contraction process
Specialized subdomains of smooth ER often serve as sites for calcium storage. Calcium ions are
pumped into ER by ATP dependent calcium pumps (such as the sarcoplasmic reticulum by ATP
dependent calcium pumps).
Understand how proteins are targeted and recycled either to the endoplasmic reticulum or the Golgi
o KDEL tags
When a protein containing a retrieval tag binds to its receptor, the receptor undergoes a
conformational change and the receptor ligand complex is packaged into a transport vesicle for
return to the ER
In the ER the pH is higher (7.1) than in the CGN (6.4), which promotes dissocation of protein
from receptor.
Understand N- and O-linked glycosylation process in the ER and Golgi
Know ERGIC
o COPII coated vesicles carry secretory proteins from ER to the ERGIC and on to the golgi apparatus.
Know how and where lysosomal proteins are marked with mannose-6-phosphate tags
Know the structure and function of the Golgi
Know the various layers of the Golgi
o Cis-Golgi Network (CGN)
o Cis-cisternae
o Medial-cisternae
o Trans-cisternae
o Trans-Golgi Network (TGN)
Know how and where sphingomyelin and glycolipids are synthesized
o Glycolipids are synthesized from ceramide in the golgi by addition of CHOs. Sphingomyelin is
synthesized by transfer of a phosphorylcholine group from phosphatidylcholine to ceramide.
Know the structure and function of lysosomes, recycling endosomes, early and late endosomes.
o Know how lysosomes and peroxisomes can be cytochemically located
Lysosomes: stain for acid phosphatase activity
Peroxisomes: stain for catalase activity
o Know that pH of these organelles are controlled by proton pumps
o Understand transport of materials to and from endosomes and lysosomes (M6P) (I-cell disease)
o Know the difference between heterophagy and autophagy
o Know lysosomal disorders Tay Sachs, Gaucher, Pompe, Hurlers and Hunters
o Know how multivesicular bodies are formed
o Know how recycling endosomes can serve as intracellular storage sites for membrane proteins such as
GLUT permeases
Know the different types of endocytosis
o Phagocytosis, receptor-mediated endocytosis, clathrin-dependent and independent pinocytosis
o Know Familial Hypercholesterolemia
Role of LDL and LDL receptor
Must know slides showing the types of LDL receptor mutations
Know the different classes of LDL receptor mutations
Problem with clathrin-dependent, receptor-mediated endocytosis
o Know the three fates of receptors after endocytosis, including transcytosis
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o Know Clathrin-independent endocytosis via caveola
Caveolin
Understand the process of exocytosis
o Constitutive vs regulated secretion
o Know how synaptic vesicles are formed
Know the structure and function of peroxisomes
o Know the role of catalase
DAB staining for catalase activity
Know the current model for peroxisome biogenesis
o Peroxisome vesicles are thought to bud from the ER. Vesicles may then fuse with one another or with
existing peroxisomes. The peroxisome membrane contains important receptor proteins.
o Cytosolic ribosomes synthesize peroxisomal proteins and then important them into the organelle.
Presumably, the lipids required for growth are also imported, although some may derive directly from the
ER into the membrane of peroxisome precursor vesicles.
o Know that peroxisomal proteins are usually translated in the cytosol and have PTS sequences such as
SKL to target them to the peroxisome
Know the structure and function for each compartment of mitochondria
o Outer membrane
Phospholipid synthesis, fatty acid desaturation, fatty acid elongation.
o Inner membrane
Contains proteins with 3 types of functions: 1) carry out oxidative reactions of the electron
transport chain, 2) the ATP synthase makes ATP in the matrix, 3) transport proteins that allow
passage of metabolites into and out of the matrix
o Intermembrane space
Compartment acidified by ET chain for use by ATP synthase
This space contains several enzymes that use the ATP passing out of the matrix to phosphorylate
other nucleotides.
Know that the anti-apoptotic protein BCL-2 is found in the outer membrane of the mitochondria
Know that mitochondrial DNA is maternally inherited
o Know Lebers Hereditary Optic Neuropathy
Age of onset occurs 16-50 years of age; typically encounter acute loss of vision in one eye
followed by loss of vision in the other eye within weeks to months. Sometimes both eyes
encounter simultaneous loss of vision
Individuals at risk should avoid alcohol and smoking. Results from an inherited mutation in
mitochondrial DNA from mother.
o Recognize the typical pattern of a family with a mitochondrial disorder
If the mother has the disease, so do all of her children. A man never transmits the disease to the
next generation
Understand protein import mechanisms use by mitochondria
o The N terminal signal of the mitochondrial precursor protein is recognized by receptors of the TOM
complex protein is translocated through TIM23 signal is cleaved to form mature protein
o Integration of porins into the outer mitochondrial membrane
TOM complex chaperone SAM complex porin
o Protein import from the cytosol into the inner mitochondrial membrane and intermembrane space
TOM interacts with TIM23 a series of cleavages and signals occur OXA complex inserts
protein into the inner membrane
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Must Know Diseases and Disorders For Block 2

1. Achondroplasia
2. Charcot-Marie-Tooth disease
a. CMT is a collection of neuropathies caused by mutations in numerous genes. CMT is an inherited disases
leading to progressive degeneration of peripheral nerves with slow loss of muscle control and eventually
muscle degeneration.
3. Cholera
4. Diabetes, Type I
5. Diabetes, Type II
6. Ehlers-Danlos syndromes
7. Fragile X syndrome
8. Hutchinson-Gilford Progeria
9. Malignant Hyperthermia
10. Marfan syndrome
1. Autosomal dominant disorder (1/10,000 individuals)
2. Symptoms: (S)keletal, (H)eart, (E)yes
a. Skeletal: characterized by skeletal abnormalities. Heart: cardiovasculuar
diseases. Dilation of ascending of aorta leading to aortic rupture or congestive
heart failure. Eyes: ocular abnormalities (myopia and detached lenses)
ii. Caused by a mutation in the gene for fibrillin-1 (FBN1), a key component of connective
tissue.
iii. Typical signs of marfan syndrome
1. Wrist (walk-murdoch) sign: overlapping the complete distal phalanx of the thum and 5 th
finger when wrapped around the opposite wrist
2. Thumb (Steinberg) sign: extension of the entire distal phalanx of the thumb beyond the
ulnar border of hand when opposed across the palm
3. Elongated feet and hands as symptoms of marfans syndrome
11. Know the structure and function of proteoglycans
12. Menkes disease
1. Menkes disease is an inborn error of metabolism that markedly decreases the cells ability
to absorb copper.
2. The disease can often be diagnosed by looking at a victims hair, which appears to be
both whitish and kinked when viewed under a microscope.
a. The hair is shorter and thinner on the sides and back of the head. The hair can be
reminiscent of steel wool cleaning pads.
3. Transmitted as an x-linked recessive trait; is a disorder of copper transport caused by
mutations in the copper-transporting ATPase gene (ATP7A)
4. The lysyl oxidase enzyme is affected (dependent on copper), which is an extracellular
enzyme that acts to establish crosslinking
5. Sulfhydril oxidase that crosslinks keratin also requires copper.
6. Specific clinical features include:
a. Distinctive facial features
b. Pectus excavatum (mindline depression in the bony thorax)
c. Skin laxity particularly on the nap of the neck and trunk
d. Umbilical or inguinal hernia
e. Hypotonia, neurodevelopmental delays, and failure to thrive, typically manifest
at three to six months of age.
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13. Noonan Syndrome
a. Is an autosomal dominant condition which is characterized by short stature, distinctive craniofacial
features, congenital cardiovascular disease, and other more variable clinical findings such as
developmental delay and intellectual disability, bleeding tendencies, lymphatic abnormalities and
genitourinary abnormalities. Occurs in ~1-1000 individuals. Often genes enoding Ras/MAPK signaling
proteins are mutated (PTPN11, KRAS, SOS, RAF1).
b. Also known as pseudo-turners. There is a characteristic webbing of the neck in both Turners and
Noonans syndrome. Turners syndrome will only be seen in females, but Noonans can be seen in both
male or female. A boy with webbing of the neck will likely have Noonans.
14. Osteogenesis imperfect
1. The characteristic features of OI vary greatly from person to person, even among people
with the same type of OI, and even within the same family.
2. Type 1
a. Most common and mildest type of OI. Autosomal dominant disorder. Bones
predisposed to fracture. Most fractures occur before puberty. Normal or near
normal stature. Loose joints and low muscle tone. Sclera (whites of the eyes)
usually have a blue, purple, or gray tint; triangular face; tendency toward spinal
curvature; bone deformity absent or minimal; brittle teeth possible; hearing loss
possible; often beginning in early 20s or 30s; collagen structure is usually
normal, but the amount is less than normal.
3. Type 2
a. Most severe form; frequently lethal at or shortly after birth, often due to
respiratory problems; numerous fractures and severe bone deformity; small
stature with underdeveloped lungs; collagen is improperly formed; frequent
missense mutation in glycine (closer to carboxy end) causing disruption to
form collagen.
15. Scurvy
1. At risk: infants, elderly men, alcoholics, smokers
2. Vitamin C is easily lost via cooking, sensitive to heat
3. Deficient for 20-40 days
4. Swollen, bleeding gums, joints, loose teeth, pinpoint hemorrhages around hair
follicles (petechiae), gums, nails, soreness and stiffness of joints and lower extremities,
slow wound healing, anemia, fatigue; alveolar bone resoprtion results in loss of teeth;
perifollicular hemorrhages; patients have difficulty walling off infections to form
abscesses, so infections spread more easily; in children, vitamin C deficiency leads to
growth failure and collagen-rich structures such as teeth, bones, and blood vessels
develop abnormally.
5. Rebound scurvy- immediate halt to excess vitamin C supplements
16. Tuberous Sclerosis
a. Is an inherited disorder whose key features include multiple facial angifibromas, seizures, shagreen patch,
cardiac rhabdomyoma, and renal lesions. Many patients also have ocular and neurologic manifestations,
including mental retardation.
b. The disorder results from a mutation in either TSC1 or TSC2
c. 2/3 of cases may be due to spontaneous genetic mutations. TSC2 mutations account for the majority of
sporadic cases. These individuals usually show a more severe phenotype that includes renal lesions and
neurologic deficits.
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17. Turner syndrome
i. Short stature, gonadal dysgenesis, webbed neck, low posterior hairline, broad chest with widely
spaced nipples; renal and cardiovasicular anomalies; at birth, infants have edema of the dorsum of
the foot; coarctation of the aorta; lymphedema in fetal life, causing cystic hygroma, which is the
cause of the neck webbing; average to above intelligence; worse social cognition skills if X
chromosome comes from the mother.
ii. Turner syndrome should be suspected in any newborn female with edema of the hands and feet or
with hypoplastic left sided heart or coarctation of the aorta.
iii. The diagnosis should be considered in teenage years for girls with primary or secondary
amenorrhea, especially if they are of short stature.
18. Whooping Cough
19. Familial Hypercholesterolemia- results in the formation of atherosclerotic plaques and atherosclerosis; genetic
predisposition to high blood cholesterol levels and heart disease caused by an inherited defect in the gene
encoding for the LDL receptor. LDL is NOT taken up by the cells and this induces the cell to produce cholesterol.
LDL receptors recognize APO-B100.
a. Characterized by elevated levels of plasma cholesterol. in heterozygotes, total plasma cholesterol is 300-
600 mg/dL and LDL cholesterol is >160; in homozygous patients, total cholesterol is 600-1200 mg/dL.
b. Cholesterol accumulation in soft tissues and skin, producing xanthomas is often seen in FH.
20. Gauchers disease- lysosomal enzyme missing glucocerebrosidase; substance accumulating in the inclusion
bodies glucocerebroside; symptoms: erosion of bonesl fractures; crumpled paper inclusions (characteristic
macrophages).adult form (liver and spleen enlargement) and osteoporosis; infantile form: mental retardation;
frequently fatal
21. Hunter- x-linked recessive; deficient enzyme: iduronate sulfatase; clinical features similar to Hurlers, but NO
CORNEAL CLOUDING; mental retardation is mild to severe with physical deformity.
22. Hurler- alpha-L-iduronidase deficiency
a. Clinical presentation: corneal clouding; coarse facies; linear growth ceases by year 3; progressive and
profound mental retardation; death by cardiorespiratory failure.
23. I-Cell disease- loss of glycosylation. A severe autosomal recessive lysosomal storage disease. Caused by defect in
enzyme that transfers a phosphate group to mannose residues. a defect in protein trafficking; golgi-specific
phosphotransferase.
a. Clinical presentation: unusual facial features; skeletal changes; mental retardation; many of the acid
hydrolases are found in the bodily fluids; similar in appearance to hurlers syndrome
24. Lebers Hereditary Optic Neuropathy age of onset occurs 16-50 years of age; typically acute loss of vision in
one eye followed by the other weeks or months later; results from an inherited mutation in mitochondrial DNA
from mother. Individuals at risk should avoid alcohol and smoking.
25. Pompe accumulate excessive amounts of glycogen in the liver, heart, and skeletal muscle. Defective form of
alpha-1,4 glucosidase.
26. Tay-Sachs disease cherry red spots in the retina with progressive neurological deterioration until death (2-4
years). Pseudodeficiency alleles do not cause disease at all. This disease is missing hexosaminidase A, responsible
for cleaving G(m2) leads to accumulation of ganglioside G(m2) and has a high incidence in Ashkenazi jews.
27. X-linked adrenoleukodystrophy- (affects boys); leads to adrenal failure, neurological impairment, and death.
Defect in the transport of very long chain fatty acids into the peroxisome. The fatty acids accumulate in the CNS
and destroy the myelin sheath. Defective integral membrane protein responsible for transporting very long chain
fatty acids into the peroxisome for beta-oxidation. Generally resembles ADD or hyperactivity.
28. Zellwegers syndrome (neonatal adrenoleukodystrophy) missing key protein essential for targeting
peroxisomal enzymes for uptake by the organelle peroxisomal enzymes remain in the cytosol.
a. Symptoms are birth: lack of muscle tone and inability to move. It is a rare hereditary disorder affecting
infants, and usually results in death. Unusual problems in prenatal development such as enlarged liver and
high levels of iron and cooper in the blood.
Must Know Drugs For Block 2

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1. Aromatase inhibitors
a. Blocks the production of estrogen and reduces levels of estrogen.
2. Caffeine
a. This is a type of methylxanthine in coffee (tea theophylline; chocolate theobromine). Methylxanthines
have two modes of action: adenosine receptor inhibitor and a cAMP phosphodiesterase inhibitor.
3. Cyclosporin A
a. Important immunosuprresant used to prevent rejection of transported organs. Works by inhibiting
calcineurin. (cyclosporine blocks phosphatase ability, thus preventing the transport into the nucleus and
remains in the cytosol).
4. Cytochalasin B and Phalloidin
5. Insulin
6. Metformin
a. A drug used for treatment of type 2 diabetes. Metformin reduces blood glucose levels by inhibiting
hepatic gluconeogenesis, which is active in these patients because of the livers resistance to the effects of
insulin. Also reduces lipid synthesis in the liver. Also stimulates glucose uptake by the muscle, which is
significant for reducing circulating blood glucose levels.
7. Nitroglycerin
a. For angina to release constricted coronary arteries. Glycerol trinitrate decomposes to NO, which activates
a guanylyl cyclase. This will relax arterial smooth muscle cells (vasodilation).
8. Penicillin
9. Rapamycin (sirolimus)
a. A potent immunosuppressant used to prevent transplant reject. Mode of action: binds to FK-binding
protein 12 (FKBP12). The rapamycin-FKB12 complex binds and inhibits complex1 (mTORC1)
10. Tamoxifen
a. Blocks estrogen receptors
11. Viagra (sildenafil)
a. An inhibitor of cGMP-specific phosphodiesterase (cGMP breakdown). Nitric oxide released by the
neurons in the penis results in the blood vessel dilation responsible for penile erection.
b. Viagra helps maintain elevated cGMP in erectile tissue; this pathway is stimulated for a longer time
period following NO release.
Presentation # 5: DNA Structure, Replication and Repair

MUST Know the cell cycle
o highly ordered, resulting in duplication and division of the cell
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o M (mitotic) phase chromosome segregation, cytoplasmic division
o Interphase
G1 (gap 1) transition from M to S phase; synthesis of proteins that would be required during S
phase.
G0 cells that become arrested in G1 (quiescence)
o S (synthetic) phase DNA synthesis
o G2 (gap 2) transition from S to M; synthesis of proteins needed during M phase
Know the structure of the nucleotides and different bases
o Know how ddNTPs are different than dNTPs

The ribose in RNA has hydroxyl groups on 2 and 3 positions on ring. Extra hydroxyl group
makes RNA more chemically reactive than DNA, thus RNA can serve as catalytic agent
(ribozyme) in ribosomes and mRNA splicing.
The deoxyribose in DNA is missing a hydroxyl group on position 2 on the ring. To denote the
difference between these molecules we use NTP to refer to ribonucleic acids and dNTP to refer to
deoxyribonucleic acids. The dideoxynucleotides do not have a hydroxyl group on either the 2 or
3 carbon.
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o Know how antiviral drug ZDV works
ddNTPs do not have a hydroxyl group on either the 2 or 3 carbon. They can be converted to
dideoxynucleoside triphosphates in cells, and like ZDV, terminate chain growth when
incorporated into DNA. Chain termination results from the absence of a hydroxyl group on the 3
group. This can be used to interfere with bioreplication. Cancer cell wont be able to replicate.
Patients with AIDS are often treated with ZDV (formerly called AZT); this drug is an analog of
thymine nucleotide found in DNA.
This drug is a thymine nucleotide found in DNA. ZDV lacks a 3-OH group no additional
nucleotides can be attached through a 5 3 bond. Thus, chain elongation of the DNA is
terminated.
Reverse transcriptase has a higher affinity for ZDV that does normal human cellular DNA
polymerases, enabling the drug to target viral replication more specifically than cellular
replication.
ddNTP is used as an anticancer nucleotide analog
o Know which bases are found in DNA and in RNA

Thymine in DNA, Uracil in RNA
Know that DNA synthesis is semi-conservative

o DNA undergoes semiconservative replication (half of the parent molecule is retained by each daughter
molecule). In semiconservative replication of DNA, the two strands of parental DNA separate, and each
serves as a template for synthesis of a new daughter strand by complimentary base pairing.
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Know the different major DNA polymerases in prokaryotes and eukaryotes, and how they synthesize DNA
53
o In the DNA polymerase reaction, incoming nucleotides are covalently bonded to the 3 hydroxyl end of
the growing DNA chain. Each successive nucleotide is linked to the growing chain by a phosphoester
bond between the phosphate group on its 5 carbon and the hydroxyl group on the 3 carbon of the
nucleotide added in the previous step
o The DNA strand is read in a 35 direction, but synthesized in a 53 direction.
o The main types of bacterial DNA polymerases (I, II, III, IV, and V)
DNA polymerase III is the primary polymerase (workhorse)
Polymerase I is responsible for removing RNA primers, proofreading, DNA repair and filling in
gaps left by DNA Pol III (finishing the DNA replication process)
o the main types of eukaryotic DNA polymerases (alpha, beta, gamma, delta, and epsilon)
polymerase gamma found ONLY in mitochondria
polymerase alpha involved with initial synthesis of DNA strands off RNA primers (part of the
primase complex)
polymerase beta DNA repair
polymerase delta and epsilon synthesize leading and lagging strands
o Know proofreading activity of prokaryotic DNA polymerases
(3 5 exonuclease activity)
Exonuclease chews the ends of primers. 35 exonuclease is the proofreading activity.
Incorrect nucleotide incorporated about 1 in 100,000 nucleotides. Cell needs process to lower
mutation rate.
Endonuclease vs. exonuclease

o Exonuclease cuts from the ends of the chain, releasing single nucleotides. Endonucleases cleave within
the chain to produce single stranded nicks.
Know about prokaryotic origins of replications and eukaryotic replicons
o The ORI is the starting site for DNA synthesis in prokaryotes. Prokaryotic genomes usually have a single
origin of replication. Most circular DNA molecules are replicated in a bidirectional process from a single
origin, and have a theta replication orientation (see in bacteria, mitochondria, chloroplasts, and some
viruses). 1 ORI and 2 replication forks are form.
o Eukaryotes: replicons units of replication on linear DNA. Typical chromosomes may contain several
thousand replicons. Large number of replicons allows eukaryotic to replicate their DNA faster.
Eukaryotic pre-replication complex
Initiation process requires formation of pre-replication complex. Origin recognition
complex (ORC) binds to replication origin. MCM complex (with helicase) binds next
with helicase loaders.
When these have attached, the DNA has been licensed for replication. The process of
licensing makes sure the cells only replicate their DNA only once.
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MUST KNOW all the steps and enzymes involved with DNA synthesis
o binding of initiator protein to replication origin binding of DNA helicase to initiator protein loading
of helicase onto DNA strand opening of DNA enables entry of RNA primase RNA primer synthesis
enables DNA polymerase to start first new chain of DNA initiation of three additional DNA chains and
formation of replication works (two replication forks moving in opposite directions towards and away
from the leading strand (lagging)). Single stranded binding proteins help keep strands apart.
Topoisomerase (gyrase) prevent/controls supercoiling.
o Leading vs Lagging strands
The leading strand is synthesized continuously in the direction of replication fork. The lagging
strand is synthesized in small pieces (Okazaki fragments) away from the replication fork. The
okazaki fragments are joined by the action of DNA ligase. Ligase activity will join nicked strands
together.
o Initiators
o helicases (unwinds DNA)
o topoisomerases (decreases supercoiling caused by unwinding)
type I cuts one strand then seals the strand
type II cuts both strands to allow uncoiling
o DNA gyrase (a type II topoisomerase used by bacteria)
o single-stranded binding protein (bind to exposed single strands and stabilizes the DNA for DNA
replication)
o primase- an enzyme that synthesizes RNA fragments about 10 base pairs long using DNA as a template.
In bacteria, primase is a part of primosome complex. In eukaryotes, primase is tightly bound to
polymerase alpha (to initiate DNA replication). In prokaryotes, DNA polymerase III will take over. In
eukaryotes, DNA polymerase alpha and delta.
o DNA polymerases- cannot begin DNA replication from scratch. (However, RNA polymerases are able
to).
o RPA
o Sliding clamp- keeps the polymerase firmly on the DNA when it is moving, but releases it as soon as the
polymerase runs into a double stranded region of DNA; forms a ring like structure around DNA
o clamp loaders- the assembly of the clam requires clamp loaders that hydrolyze ATP as it loads the clamp
onto a primer template junction
prokaryotes use a beta subunit clamp
eukaryotes PCNA
o RNase H and FEN1 work similarly to polymerase I in prokaryotes.
Know that primers are needed to start synthesizing Okazaki fragments and the leading strand
Know how RNA primers are removed
Prokaryotes by DNA Polymerase 1
Eukaryotes by FEN1 and RNaseH
Know how to determine the RNA primer sequences if given a DNA template sequence and vice-
versa
Know that Quinolones inhibit DNA gyrase
o Constitute a large class of synthetic antimicrobial agents that are highly effective in the treatment of many
types of infectious diseases, particularly those caused by bacteria.
Examples nalidixic acid; ciproflaxin; norfloxacin
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o Quinolones and fluoroquinolones are drugs that inhibit DNA gyrase preventing DNA replication and
transcription.
Know that Doxorubicin, etoposide and teniposide inhibit human topoisomerase II
o Used in treatment of several neoplastic diseases. These drugs act by enhancing the rate at which target
topoisomerase II cleaves DNA and by reducing the rates at which these breaks are resealed.

o Helicase, primase, and two molecules of DNA pol III carry out coordinated synthesis of both leading &
lagging strands of DNA. both molecules of DNA polymerase are associated w/clamp loading protein,
which is bound to the helicase at the fork. Topoisomerase acts as a swivel ahead of fork. Behind the fork,
RNA primers are removed by DNA polymerase I and okazaki fragments are joined by DNA ligase.
Eukaryotic replication fork
o DNA polymerase in eukaryotes does not associate into a dimeric complex. Replication protein A (RPA)
binds the ssDNA preventing reannealing. Replication factor C (RPC) induces binding of proliferating
cell nuclear antigen (PCNA). Chromatin remodeling proteins are also involved w/eukaryotic DNA
replication (they help to move nucleosomes).
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Know what are telomeres and how they are maintained by telomerase (reverse transcriptase)
o Found at the ends of eukaryotic chromosomes; 10-15 kb of tandem hexanucleotide repeat units found at
telomeres. Added by telomerase enzyme. Function to protect ends of telomeres and provide a means of
replication.
o Lagging strand cannot properly replicate the very 5 ends of chromosomes. Telomerase is a protein/RNA
complex that can synthesize telomeres found at the ends of chromosomes.
o Know that telomerase has reverse transcriptase activity
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Telomerase is a reverse transcriptase. It carries its own template RNA, which is complementary to
the telomere repeat sequence. Multiple copies may be generated in order to maintain teleomeres.
RNA template allows telomerase to extend the 3 end by one repeat unit beyond its original
length. The complementary strand can then be synthesized by the polymerase alpha-primase
using an RNA primer provided by telomerase. Removal of the RNA primer leaves an
overhanging 3 end, which can form loops at the ends of eukaryotic chromosomes.
o Telomerase found in germ cells and tumor cells but NOT in differentiated cells. Cells lacking
telomerase activity undergo shortening every time they divide. This results in cell aging (senescence)
and/or disruption of the cell cycle. Cancer cells are able to senescence through activation of telomerase.
Tumor cells, for example.
Know the key differences between eukaryotic and prokaryotic DNA synthesis
o EUKARYOTES have multiple origins (replicons) versus single ORI in PROKARYOTES
o EUKARYOTES undergo DNA synthesis during S phase in cell cycle
o EUKARYOTES remove RNA primers using FEN1 and RNaseH
o EUKARYOTES have telomeres at the ends of their LINEAR DNA molecules, PROKARYOTIC genomes
are usually circular and do not have telomeres.
o 53 exonuclease: prokaryotes polymerase I; eukaryotes NONE
o Stability of ssDNA: prokaryotes SSB; eukaryotes - RPA
Presentation # 1: DNA Repair
General facts: most mutations occur in somatic cells and are never passed to offspring. Many mutations are
detrimental. An average gene retains a mutation every 200,000 years.
Know the major types of DNA mutations and how they are repaired
o 1. DNA replication mismatches mismatches
o 2. Chemicals (nitrous acid) base alteration
o 3. Radiation (UV and high energy radiation) thymine dimer and double strand breaks
o 4. Spontaneous loss of nucleosides base alternation
o Depurination
Creates an AP site
Repaid mechanism AP endonuclease will replace with nucleotide
o Deamination
cytosine can be deaminated [uracil glycosylase] uracil
5-methyl cytosine thymine
o UV-induced pyrimidine dimers distorts the structure of the DNA and blocks transcription or
replication past the site of damage.
Repair mechanism: (direct repair of thymine dimers) photoreactivation; energy from visible
light is used to break ring structure, reversing dimerization reaction.
Placental mammals (including humans) lack this mechanism of DNA repair. Mammalian
cells use NER to correct dimers.
o Base excision repair- repair single damaged bases.
o Uracil glycosylase- when DNA is damaged through deamination reactions, cytosine is converted to
uracil. Error is repaired by uracil DNA glycosylase.
Damage repair explains why DNA contains thymine instead of uracil. DNA repair mechanisms
would be unable to distinguish normal uracil from mutated forms.
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o AP site DNA repair AP site is a sugar with no base attached. Purine bases can be lost spontaneously as
in depurination. Can also be formed by excision of a base uracil glycosylase. These sites are repaired
by AP endonuclease (with the help of phosphodiesterase) the deoxyribose is removed and the gap is
filled by DNA polymerase and ligase
o AP endonuclease two key roles: 1) repair nucleotides missing bases caused by spontaneous events,
such as depurination. 2) repair nucleotides missing bases caused by target repair process, such as uracil
glycosylase
o Nucleotide excision repair- removal of damaged bases followed by replacement with newly synthesized
DNA. Repair major distortions in DNA double helix. Pathway that corrects thymine dimers. Mutations in
pathway causes xeroderma pigmentosa.
Steps in NER
1) identification of the mismatched or mutated DNA strand
2) nick the mismatched DNA or mutated strand by endonuclease
3) 53 DNA polymerase fill the gap
4) DNA ligase forms phosphodiester linkage
Helicase is required to unwind the DNA for excision. An excinulcease is an enzyme
complex that can directly excise an oligonucleotide.
Mammalian cells
Disrupted base pairing is recognized by XPC, followed by cooperative binding of XPA,
TFIIH, and RPA. Subunits of TFIIH, XPB and XPD, act as helicases to unwind about 25
base pairs. XPG and XPF/ERCC1 are recruited and act as endonucleases to cleave
DNA on the 5 and 3 end of the damaged region.
o DNA Mismatch Repair
In bacteria, proteins recognize the parental strand because it is methylated and replace the
segment with newly synthesized (and unmethylated) DNA containing the mismatched base.
The mechanism in humans is not well understood
In eukaryotic cells, presence of single stranded breaks in newly replicated DNA appears to
specify the strand to be repaired.
Eukaryotic homologs of mutS and mutL bind to mismatched bases and direct excision. Mutations
in the human homologs can cause HNPCC.
Sometimes inaccurate in fixing C to T transition
When there is a mismatch, either one of the strands can be excised. When polymerase
and ligase fill and seal the gap, a new segment would have been included.
o Transcription-coupled repair
Nucleotide excision repair during transcription. When RNA polymerase is stalled by DNA
damage in mammalian cells, it is recognized by CSA and CSB. Then recruits other proteins to
repair damage.
Know Cockaynes Syndrome
Defective CSA and CSB proteins and unable to recruit other proteins for transcription
coupled repair.
Rare inherited disorder in which people are sensitive to sunlight, have short stature, and
have the appearance of pre-mature aging (as in progeria). In type I (classical), symptoms
are progressive and typically become apparent after 1 year of age. Early onset (type II) is
apparent at birth. NOT linked to cancer
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After exposure to UV radiation, people with CS can no longer perform transcription
coupled repair. This type of repair is occurring as the protein is being replicated.
6
o O -methylguanine methyltransferase
DNA damage can result from alkylating agents reactive compounds that transfer methyl or
ethyl groups to a DNA base. Methylation of the O6 position of a guanine forms O6-
methylguanine, which can bond with thymine instead of cytosine.
Repaired by O6 methyl guanine methyltransferase transfers the methyl group from O6-
methylguanine to a cysteine residue in its active site.
Know Xeroderma Pigmentosum and Nucleotide excision repair of pyrimidine dimers
o Causes extreme sensitivity to UV radiation and multiple skin cancers; autosomal recessive inheritance
o Half of affected individuals demonstrate from early infancy acute sun sensitivity, sever sunburn with
blistering or persistent erythema on minimal sun exposure; marked freckling of the face; most patients
develop xerosis (dry skin), and other skin conditions.
o Atrophy of the skin of the eye lids results in ectropion, entropion, or complete loss of the lids.
Know Hereditary nonpolyposis colon cancer (HNPCC) caused by mutation in DNA mismatch repair pathway
o Most common mutations: MLH1 and MSH2
o Often shows microsatellite instability and may cause other genes to be compromised. MSH2 (40%),
MSH1 (50%), and MSH6 (%10) are the most common mutations.
Know why CpG islands are hotspots for mutations in our genome
o Know the steps for C to T transitions
Transitions occur more frequently at CpG islands because the cytosine is prone to methylation at
position 5, and spontaneous deamination of 5-methylcytosine to thymine.
o 30% of point mutatiosn in known inherited diseases, involve CT transitions, at sites containing CG
dinucleotides (CpG islands)
Know how double-stranded DNA breaks are repaired
o Nonhomologous end-joining- uses a set of proteins that bind to the ends of the two broken DNA
fragments and joins them together. This is error prone.
Ku heterodimers grasps the broken chromosome ends and help recruit other proteins to fix the
break. Results in a loss of bases at ends.
o Homologous recombination- intact chromosome acts as a template to guide repair of the damaged
chromosome. Breast cancer genes BRCA1 and 2 are involved
BRCA1 and 2 are tumor suppressor proteins. Abnormal BRCA1 confers high risk for type 1
ovarian cancer.
BRCA2 gene confers high risk of breast cancer (not ovarian) type 2
Know the basic steps of homologous recombination
o Recombination is also the key to generating genetic diversity. Homologous recombination is the exchange
of information between DNA molecules that share sequence homology over hundreds of bases. This
occurs during both meiosis and DNA repair.
o Recombination results from the breakage and rejoining of two parental DNA molecules. Overlapping
single strands are exchanged via base pairing between homologous DNA, molecules, leading to the
formation of a heteroduplex region.
o Imported bacterial DNA is on a short linear piece of DNA called exogenote. Homologous recombination
produces an exchange of DNA between linear exogenote and region on bacterial chromosome.
o Homologous recombination requires:
Several genes worth of homology or near homology between the strands
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A series of recombination enzymes/factors: recA-D; recA is absolute requirement
Function of recA: promotes the exchange of strands between homologous DNAs. recA
binds to single stranded DNA to form protein-DNA filament, then binds to double
stranded DNA. then, specific base pairing between single stranded and its
complement, and strand exchange
Holliday model and junction
o Model for recombination. Initiated by nicks at the same position on the parental DNA molecules. The
nicked DNA s trands partially unwind, and each invades the other molecule by pairing with the
complementary broken strand. Ligation of the broken strands then produces a cross-strand intermediate
known as a holliday junction. Resolved by cutting and rejoining strands to yield recombinant molecules
or nonrecombinant heteroduplexes.
Branch migration and resolution of holliday junctions
In E. coli, holliday junctions are resolved by a complex of ruvA, ruvB, and ruvC. ruvA
recognizes the junction, recruits ruvB, ruvC cleaves crosslinked DNA strands, and
rejoined by ligase.
MUST KNOW:
o Site specific recombination integration of a DNA molecule into another DNA molecule with which it
has no homology except for a small site on each DNA (called an attachment, integration, or insertion
site).
Requires restriction endonucleases and appropriate sites on DNA. Integration process rather
than an exchange.
Three major roles: 1) integration of a fertility factor to make an Hfr cell. 2) Integration of
temperate phage DNA to create a prophage. 3) movement and insertion of transposons
o Know the basic steps of bacterial transformation (uptake of DNA, recombination, recA, B, C)
Gene transfer resulting from the uptake of DNA from a donor.
Factors affecting transformation: DNA size and state; competence of the recipient
Sensitive to nucleases
Free DNA from dead bacterial cells taken up by living competent cells in the same environment.
To produce stable genetic change, fragment must be integrated into bacterial cell genome via
recA
Competence encoded by chromosomal genes that become active under certain environmental
conditions. For example, pneumococci secrete a competence factor that induces cells to
synthesize special proteins that expose the cell membrane to DNA binding protein.
o Know the basic steps of generalized and specialized transduction
Gene transfer from a donor to a recipient by way of a bacteriophage
Requires bacteriophages (virus that infects bacteria)
Resistant to environmental nucleases
Generalized transduction in which potentially any donor bacterial gene can be transferred.
Infection of donor phage replication and degradation of host DNA assembly of
phage particles release of phage infection of recipient homologous
recombination OR the phage may integrate at special sites of attachment into the bacterial
genome phage becomes lysogenic and remains dormant later time prophage
detaches from the bacterial chromosome carrying a number of bacterial genes then
goes lytic high frequency of recombinants
87
Mapping: if a single virion incorporates two genes from the host, then you know those
genes are in close proximity to each other.
Specialized transduction in which only certain donor genes can be transferred.
o Know the basic step of conjugation between and F+ and F- bacteria
Involves cell to cell contact; occurs between two living bacterial cells; especially important in
gram negative; mediated by F pili and allows transfers of plasmids
Mating types in bacteria
Donor
o F factor (fertility factor) = F (sex) pilus encoded by TRA genes of the
conjugative plasmid.
Pilus retracts to bring donor and recipient into contact to form conjugal
bridge for gene transfer. Interacts with F- to transfer a copy and convert
F- into F+ cell.
Recipient
o Lacks F factor
+
o Know how a F cell can become a Hfr cell
o Know the basic steps of conjugation between and Hfr and F- bacteria
o Know that the conjugation mating bridge is sensitive to physical stress

Must Know DNA rearrangement for the immunoglobulin and T cell receptor loci
o Immunoglobulin consists of pairs of identical pairs of heavy and light polypeptide chains, with C-
terminal constant regions and N-terminal variable regions. Each antibody is encoded by unique genes
formed by site specific recombination during B lymphocyte development.
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o Rearrangement of immunoglobulin light chain genes
Genes that encode light chains have 3 regions: V (associated with the N terminal end of the
variable region), J (associated with the C terminal end of the variable region), C (associated with
the constant region).
During B lymphocyte site specific recombination, one V region is joined to one J region.
o Rearrangement of immunoglobulin heavy chain genes
Heavy chain genes include forth region, D (diversity).
2 recombination occur: 1) D and J recombine; 2) V recombines with the DJ segment
o Know the roles of RAG1, RAG 2 and terminal deoxynucleotidyl transferase (TDT) in the process
V(D)J recombination is mediated by RAG1 and RAG2 proteins. They recognize recognition
sequence adjacent to coding sequence and introduce a double stranded nick between RS and CS.
When the double strands are rejoined, bases are lost at the ends of the strand, and bases are
additionally added by TDT (terminal deoxynucleotidyl transferse).
Know the basic steps of immunoglobulin class switch recombination
o Results in rearranged V(D)J regions with different heavy (constant) chains.
o Humans produce five classes of immunoglobulins, IgM, IgG, IgD, IgA, and IgE.
o Heavy chains are encoded by a variable region igM antibody is produced when initial V(D)J is joined
to C(mu). Rearranged variable region is transferred to a new downstream constant region, with
deletion of the intervening DNA.
o Recombination occurs between highly repetitive sequences in switch regions (S) located upstream of C
regions.
o Switch regions are WITHIN introns, so class switch does not affect the immunoglobulin coding
sequence.
Know the role AID in class switch and somatic hypermutation of immunoglobulin loci
o Somatic hypermutation (only in B lymphocytes); produces multiple mutations within rearranged
variable regions of both heavy and light chains.
o Activation induced deaminase (AID) plays a key role in both class switch and somatic hypermutation.
AID catalyzes deamination of cytosine in DNA to form uracil.
C U mutations appear to stimulate processes by removal of U by base excision repair.
Presentation # 2: Transcription: Synthesis of RNA

Know the basics of RNA transcription
o DNA template read 3 to 5 direction and RNA synthesized in 5 to 3 direction
Know what are the template and coding strands
o Know how to identify the RNA sequence if given either the coding or the template strand
o Know how to identify either the coding or the template strand if given an RNA sequence
Know the major types of RNA (mRNA, tRNA, microRNA & rRNA)
Know the differences between Eukaryotic and Prokaryotic transcription
Know that prokaryotes use more than one type of sigma factor to selectively regulate gene expression
Know that sigma factors recognize two key prokaryotic promoter consensus sequences: Pribnow box (TATAAT
box) at -10 and the -35 TTGACA sequence
Know the stages of prokaryotic transcription
Know Rho-dependent and Rho-independent termination of prokaryotic transcription
Know how Rifampin and Actinomycin interfere with transcription
Know that transcription and translation can occur simultaneously in prokaryotes
Know what are trans-acting factors and cis-acting elements
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Know what is an operon
Know what is positive and negative gene expression regulation
Inducible vs repressible negative regulation mechanisms
MUST KNOW the mechanisms of positive and negative regulation of the inducible Lac operon
o Know the three genes in the Lac operon polycistronic message
Lac Z = Beta- galactosidase
Lac Y = Galactoside permease
Lac A = Transacetylase
o Know the cis-acting elements: LacO (operator), LacP (promoter), and CAP/CRP binding site
o Know that a form of lactose binds to the repressor
o Know the consequence of lactose & glucose levels on Lac operon expression
Know how CAP/CRP is regulated by cAMP and how Lac I is regulated by lactose
Must know the repressible gene expression regulation of the trp operon
o Know that tryptophan is the corepressor responsible for binding the apo-repressor
Must know what is attenuation
o Know the trp operon example
Know what are riboswitches and how they influence transcription or translation
Know the three major eukaryotic RNA polymerases
o RNA Pol I, RNA Pol II & RNA Pol III
Know what are their primary products, cellular location and which is sensitive to amanitin
(derived from mushrooms)
Recognize amanitin poisoning
Know the basic structure of the human protein-coding gene
o Know promoters, core promoter, enhancers, silencers, activators and repressor, and how they are involved
with transcription
o Know the terms upstream and downstream when referring to gene structure
Know the function of general transcription factors TFIID and TFIIH
o Importance of the core promoter and the TATA box
o Importance of TFIIH phosphorylating CTD of RNA Pol II in order to release RNA Pol II to initiate
transcription
Know how eukaryotes terminate transcription
Know the common DNA-binding domains found in transcription factors
o (helix loop helix, helix turn helix, leucine zipper, zinc fingers
o Know that steroid hormone receptors have zinc finger DNA binding domain, an activation domain and a
ligand binding domain
Know what is an activation domain
Know the importance of Hox gene system in human body pattern formation
o Know that Hox proteins are master regulatory transcription factors for development and are mainly
responsible for controlling craniocaudal segmentation along the main body axis of the embryo
o HOXD13 Mutations and Synpolydactyl
Know the ways activators can increase rates of transcription
Know steroid signaling
Know that transcription factors can be regulated by phosphorylation
o Know Gs/cAMP/PKACREB
Know what are homeotic genes and their importance in development
Know what are enhancers and the different type of coactivators
Know how activators bound to enhancers recruit coactivators
o Know that activators bound to enhancers cause looping of the DNA
o Know about the three categories of coactivator: mediators, histone acetyl transferases, ATP-dependent
chromatin remodeling complexes
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Know what are genetic insulators & barrier sequences
Know the following techniques to identify DNA binding sites for transcription factors: DNA footprinting,
Electrophoretic-mobility shift assay, Chromatin immunoprecipitation assays, reporter-gene transfer assays
Know how transcription factors can be isolated by DNA-affinity chromatography
Know what are heterochromatin and euchromatin
o Know the impact of methylation status on transcriptional activity
Know the two mechanisms of chromatin remodeling
o ATP-driven chromatin remodeling complex
o Acetylation
Know the roles of histone acetyltransferase and histone deacetlyase
Know the impact of acetylation status on transcriptional activity and chromatin structure
Know DNA methylation and its role in heterochromatin formation
Know the process of X-inactivation (Barr body = inactive X chromosome)
o The role of long noncoding RNA XIST, macroH2A and methylation to form heterochromatin
Know that XIST is expressed from the X inactivation center from the X chromosome undergoing
inactivation
Know the mechanism long noncoding RNA induced transcriptional silencing
Know the 4 different mechanisms for transmitting epigenetic patterns to daughter cells
MUST KNOW Genomic Imprinting
o Know Prader-Willi Syndrome and Angelman Syndrome
Know how siRNAs can mediate heterochromatin formation and regulate transcription
Know what is a polytene chromosome
Know how mRNA precursors are processed
o Splicing (including alternative splicing), 5 methyl cap, 3 poly A tail and RNA editing
Know the key enzymes & factors involved with each process
Guanylyl transferase methyl cap
Role of SnRNPs and U2AF in splicing
Splicing occurs in the nucleus
Know any key regulatory sequences involved with each process
AAUAAA and poly A tail
Splice donor and acceptor sequences
Know that a significant # of beta thalassemia-causing alleles have splicing mutations (but NOT
all cases)
Know and recognize alternative splicing
Know that alternative splicing increase the diversity and # of different types of proteins a
cell can produce
Must know what is RNA editing and be able to recognize examples of such
Must know RNA editing of apolipoprotein B mRNA(Apolipoprotein B48 vs B100)
Know Systemic Lupus Erythematosus
Know the negative and positive control of alternative splicing
Must Know Beta Thalassemia
o Know what are HbA, HbF, and HbA2
Know the role of methyl cap and poly A tail in protecting the mRNA from degradation
Know the importance of mRNA localization in the cytoplasm
Know prokaryotic and eukaryotic ribosomes and their rRNAs
Know how rRNA precursors are processed
o Degradation of transcribe spacers, methylation, pseudouridine
Know how tRNA precursors are processed
o Removal of 5 leader
Role of RNase P
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o 3 ends with CCA,
o removal of introns,
o chemical modifications generating bases such as inosine and ribothymidine
Presentation # 3: Translation: Synthesis of Proteins
Know the key features of the genetic code

o Specificity, universality, nonoverlapping, and degenerate
Know how to read a codon table
o Know the start codon and the three stop codons
o Know how to predict amino acid sequence from nucleotide sequence
Know how to find an open reading frame
Know the basic information flow through protein synthesis
Know of codon anticodon interactions and their role in determining amino acid order during protein synthesis
Must know how to calculate the # of amino acids in a protein from a length of DNA nucleotide sequence, or visa
versa
Must know and recognize missense, nonsense, silent, nonstop and frameshift mutations
Must know sickle cell anemia disease and molecular mutation
o caused by a missense mutation
o Know what is HbS
Know nonsense-mediated mRNA decay
Know the composition and sizes of prokaryotic and eukaryotic ribosomes and their subunits
o Know the role of rRNAs during translation,
especially prokaryotic 23S and 16S rRNA
eukaryotic 28S
Know the basic structure of tRNA
Know how aminoacyl-tRNA synthetases add an amino acid onto tRNAs
o Know the consequences of attaching the wrong amino acid to a given tRNA
Know the wobble hypothesis
Know the key differences between prokaryotic and eukaryotic translation
o Know what is the Shine-Dalgarno sequence
o Prokaryotes have coupled transcription and translation, whereas eukaryotes do not.
o Prokaryotic initiator amino-acyl tRNA has N-formyl-Met, whereas eukaryotes use MET
MUST KNOW the processes of translation initiation, elongation and termination in prokaryotes and eukaryotes
o Know the key eukaryotic initiator factors eIF-4G, eIF-4E, eIF-2 & eIF-2B and their role during initiation
of translation
o Know the elongation factors;
prokaryotic EF-Tu, EF-Ts & EF-G
eukaryotic EF-1alpha, EF-1beta/gamma, EF-2
o Know how release factors work
o Know what is peptidyltransferase activity and that the prokaryotic 23S rRNA and the eukaryotic 28S
rRNA have innate peptidyltransferase activity to form peptide bonds
Know the role of methyl cap and poly A tail in proper initiation of translation of mRNA
Know the major antibiotics that disrupt translation
Know how diphtheria toxin and pseudomonas Exotoxin A interfere with translation
Know about polyribosomes
Know the four high-energy bonds used to create a peptide bond
o Two from 1 ATP to attach an amino acid onto the tRNA
o One from GTP for EF-Tu (EF-1)
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o One from GTP for EF-G (EF-2)
Know the role of IRE and iron in regulating initiation of ferritin translation
Know the role of IRE and iron in regulating the stability of the transferrin receptor mRNA
MUST KNOW what are microRNAs, how they are processed and how they work
o Know the differences in regulation between microRNAs that are completely complementary to their
mRNA targets, compared to microRNAs that are only partially complementary to their mRNA targets
o Know what are P-bodies
MUST know what is RNA interference and what are siRNAs
o Know how siRNAs can mediate heterochromatin formation
MUST know what is RNA interference and what are siRNAs
Know how siRNAs can mediate heterochromatin formation
Know how eIF2 phosphorylation status influences translation
o Know how heme levels regulate eIF2 activity
Know the role of 4EBPs, eIF4E and S6 in the regulation of translation
o Cell signaling via mTORC1
Know the role of Poly A tail length and translation regulation
Know the mechanisms of mRNA decay
Know the following chaperones and their role in protein folding
o Hsp60,
o Hsp70,
o Chaperonin,
o Protein disulfide isomerase,
o Peptidyl prolyl isomerase
Know the major posttranslational modifications
o Proteolysis
Know insulin processing
o Carboxylation
Know about the carboxylation of blood clotting factors on glutamate residues to produce -
carboxyglutamate.
Know that the enzyme responsible for carboxylation of blood clotting factors requires vitamin K
and is inhibited by the drug warfarin
Must know about vitamin K deficiency
Especially in newborns
o Addition of lipids
o Phosphorylation
o Binding of small molecules (GTP)
o Protein-protein interactions
o Nitrosylation
o N- and O-linked glycosylation,
o Phosphorylation, Hydroxylation, Biotinylation,
Know how proteins are marked for destruction by proteosomes after being marked with ubiquitin
o Know the example of Cyclin B regulation
o Know the proteasome inhibitor Bortezomib
Know what is SUMO and the othe cellular roles of ubiquitin
Know Autophagy
Presentation # 4: Cell Cycle, Mitosis & Meiosis

Learn about Somatic and Germ-line cells
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Germ line cells propgate genetic information into then ext generation
Somatic cells form the body of the organism and all of the cells in the body except germline. They are
necessary for the survival of the germline, but do not themselves leave any progeny.
Learn the key terms of homologs, chromatids, gene locus, alleles, centromeres, kinetochores, synaptonemal
complex, homozygote and heterozygote
Homologs members of a pair of chromosomes. Carry matching genetic information and are the sames
genes in sequence.
Gene locus is the place on a chromosome that contains the DNA sequence for a particular gene.
Alleles are slightly different forms of genes at same locus
Heterozygous two different alleles; homozygous two identical alleles
Genotype genetic make-up; phenotype physical expression or make up
Two different genotypes can result in a similar phenotype!
Learn all the phases of the cell cycle
G1 transition from M to S phase; synthesis of proteins that would be required for the S phase
G0 cells in G1 phase that become arrested enter the G0 phase. Liver cells enter the G0 phase
but on damage, enters G1 phase and cell cycle continues.
S
Synthesis of DNA and histones
End of each chromatid marked by telomeres specialized DNA sequences that ensure integrity of
chromosomes during division. DNA replication happens at thousands of origins of DNA
replication.
G2 transition from S to M phase; synthesis of proteins required for the M phase. (brief phase)
M chromosome segregation and cytoplasmic division.
Know the number of chromosomes and chromatids at the various phases
Know how cellular DNA content can be analyzed by flow cytometer or fluorescence activated cell sorter
(FACS).
Know how to determine stage of cell cycle from DNA content graph
Cellular DNA content can be determined by incubation of cells with a fluorescent dye that binds to DNA,
then analysis of fluorescence intensity of individual cells in a flow cytometer or fluorescence activated
cell sorter.
Animal cells in G1 are diploid, so their DNA content is 2n; during S phase, replication increases the DNA
content of the cell to 4n.
Learn about centriole replication
At some point in G1, two centrioles of the pair separate. During S phase, daughter centriole grows
perpendicular to the mother centriole, and elongation is completed by g2 phase.
Know how normal somatic cell cycle and embryonic cell cycle differ from each other
Learn all the phases of Mitosis and the importance/consequence of each phase
Prophase- condensed chromosome becomes visible; start moving apart; nucleolus starts disappearing;
formation of the mitotic spindle;
Prometaphase- nuclear membrane breaks down; condensation of chromosomes continues and
congression occurs ; chromosomes move to point midway between spindle poles.
Metaphase- chromosomes maximally condensed and align at the metaphase plate; arranged at equatorial
plate line; balanced forces of microtubules from opposite poles at kinetochores.
Remember how to interpret a karyotype image
Anaphase- sister chromatids separate and move towards opposite poles
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Telophase & cytokinesis nucleus reassembly occurs; cytokinesis occurs; chromosomes will
decondense and nuclear membrane forms.
Learn how the actin & myosin contractile ring forms a cleavage furrow
Cleavage begins as a slight indentation in cell surface deepens into a cleavage furrow.
Actin contractile ring and myosin motors drive the process. Cytoplasm cleaves separating
daughter cells.
The formation of the contractile ring is activated by aurora and polo-like kinases at a
location determined by the position of the mitotic spindle. Cleavage proceeds as
contraction of the actin-myosin filaments pull the plasma membrane inward, eventually
pinching the cell in half.
Learn about Cdks, cyclins, CDKI, Wee1 kinase, Cdc25, APC/C and SCF
The progression of the cell cycle through various phases is regulated by transition through specific check
points mediated by cyclin dependent kinases (CDKs).
When damage is sensed, cells arrest at specific phases and allow time to repair the damage.
Learn the roles of Aurora and Polo-like kinases during mitosis
Learn how Aurora A is critical for the proper formation and functioning of centrosomes, and its
importance for recruiting gamma tubulin.
Growth factors
TGF-beta causes an increase in CDK inhibitor p15 and p21 p21 plays a role in preventing cells
containing damaged DNA from passing through the G1 check point.
CDK1 is capable of substituting for all the other CDKs
CDK and cyclins
Activation of CDKs requires association with cyclin. This contributes to specificity and levels of different
cyclins vary during the cell cycle.
The activity of the cyclin-CDK complex is further subjected to positive or negative regulation (i.e.
phosphorylation, inhibitors of CDKs, proteolysis of cyclins and inhibitors)
4 mechanisms of CDK regulation
Cyclin synthesis and degradation
Activation of cyclins by CAK (composed of CDK7 and cyclin H)
Inhibitory phosphorylation catalyzed by Wee1 protein kinase (activated by dephosphorylation by
cdc25 protein phosphatase).
Binding of inhibitory proteins CDK inhibitors (CKI) also impair CDK activating kinase.
Learn in detail the three key regulatory pathways of the cell cycle
Start check point regulated by Rb
G2/M check point controlled by MPF and is blocked until DNA is replicated
Spindle check point anaphase promoting complex makes sure all the chromosomes are properly
attached to microtubules.
G1/S
A very important regulatory step to ensure repair of genome damage before initiation of DNA
replication.
Know that growth factor signaling through the Ras/Raf/Mek/Erk pathway stimulates cyclin D
synthesis. In this stage, D cyclins associate with CDK4/6. D-type cyclins are produced as long as
the growth factor is present. Can be rapidly degraded when the levels of growth factor fall.
Know the roles of Rb, [Cdk4/Cyclin D, Cdk6/Cyclin D] and [Cdk2/cyclin E], p21, p27, CAK,
E2F, DP1
Know that Rb is regulated by phosphorylation by Cdk4/6 and Cyclin D.
Hypophosphorylated Rb can bind transcription factor E2F and block G1 to S
phase transition
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Hyperphosphorylated Rb releases E2F to promote transcription
G2/M
Know the roles of MPF (Cdk1/cyclin B)
MPF controls the G2/M checkpoint. It is activated by a multistep process and its
consequences include nuclear membrane break up and chromosome condensation.
Know how MPF is regulated
MPF is regulated by the phosphorylation and dephosphorylation of cdk1. Cyclin b is
synthesized and complexed with cdk1 during G2 phase.
Cdk1 is phosphorylated and inhibited through G2 phase. Dephosphorylation activates
cdk1, leading to initiation of M phase
Cyclin B is ubiquinated and degraded
Know Cohesins and Condensins
Chromatin condensation is driven by condensins. Condensins and cohesins contribute to
chromosome segregation. Cohesins bind to DNA in S phase and maintain the linkage
between sister chromatids. Condensins are activated by CDK1/cyclin B and Aurora B,
and replace most of the cohesins, so sister chromatids only remain linked at the
centromere
Spindle assembly check points
MPF controls the spindle assembly check point by activating the anaphase promoting complex.
Know the roles of APC/C
At the SAC, progressing to anaphase is mediated by activatin of the APC/C; also
mediated by Mad/Bub proteins that inhibit CDC20, a required component of APC/C.
Securin, Separase, Cohesin, Mads, Bubs, Cdc20, MPF (Cdk1/cyclin B)
Know enzymatic activity (if any) and targets of these proteins as mentioned in class
APC/C triggers the breakdown of securin securin binds and inhibits separin separin can
target and cleave cohesins freeing sister chromatids so they can separate from each other.
Separase degrades cohesions which breaks the link between sister chromatids, allowing
them to segregate and move to opposite spindle poles.
when the chromosomes are lined up on the spindle (as in metaphase), CDC20 will activate
APC/C and two things will happen: 1) cyclin B will be degraded and 2) separase securing
separin degradation of cohesins
APC triggers breakdown of MPF cyclin (IMPORTANT signal for end of mitsosis)
Kinetochores also play a role in the completion of mitosis
Mad and Bub inhibit the APC by controlling CDC20, preventing initiation of anaphase
Once the cell has progressed through anaphase, APC/C triggers degradation of aurora and polo-
like kinases.
Learn about cell cycle arrest in response to DNA damage
Know the roles of p53, ATM/ATR, Mdm2, p21, Chk1, Chk2 Myc, Arf
MDM2 controls the levels of p53 in the nucleus by binding p53 and shuttling it out of the nucleus
where it is destroyed by the ubiquitin dependent pathway.
If p53 is phosphorylated by ATM or ATR and is acetylated, it will no longer interact with MDM2
and intracellular levels of p53 increase
P53 levels increase if there is excessive stimulation of mitogenic pathways
Mitogenic activated pathways upregulation in transcription factor Myc production
of ARF ARF inactivates MDM2 no MDM2 results in increased p53 levels
Know that p53 is a transcription factor that responds to DNA damage by inducing cell cycle
arrest
P53 has important role in cell cycle arrest in response to DNA damage. When DNA is
damaged, ATM and ATR phosphorylate p53 and PREVENT its degradation.
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P53 p21 (CDK inhibitor) cell cycle arrest!
Know how p53 levels are maintained low in the nucleus under normal conditions
ATM is activated by double strand breaks, ATR is activated by single stranded or unreplicated DNA
they phosphorylate and activate checkpoint kinases CHK2 and CHK1
CHK2 and CHK1 phosphorylate and inhibit cdc25 which is required to activate cdk1 and cdk2
Inhibition of cdk1 stuck in G2
Inhibition of cdk2 stuck in G1/S
Know Li-Fraumeni Syndrome- a mutation in the TP53 gene that encodes for p53. Mutations in both alleles
required for inactivation of the TP53 gene. This disease occurs at an usually early age.
Know Ataxia-Telangiectasia
Learn all the phases of Meiosis and the importance/consequence of each phase
Must know that DNA replication occurs prior to meiosis 1
Prophase I- homologous chromosomes become paired and exchange DNA; divided into 5 phases;
formation of synaptonemal complex and crossing-over occurs
Must know the 5 stages of Prophase 1
Leptotene- chromosome begins to condense, but they cannot be distinguished.
Zygotene- synapsis occurs with attachment by the synaptonemal complex
Pachytene- recombination occurs with bivalent-tetrad formation
Diplotene- synaptonemal complex disappears; bivalents begin to separate, but remain
attached at the chiasmata.
Diakinesis- stage of max condensation
Metaphase I- bivalents align at the spindle equator; kinetochores facing the same side and chromosomes
held together by chiasmata; nuclear membrane disappears and spindle forms;
Anaphase I- homologous recombination move to opposite spindle poles; disjunction occurs and members
of each bivalent move apart. Homologs separate! Nondisjunction may occur at this point leading to
aneuploidy.
Telophase and cytokinesis I- two haploid cells are produced
Meiosis II resembles mitotic division; Prophase II- brief; Metaphase II- kinetochores face in the
OPPOSITE directions to pull sister chromatids a part; Anaphase II- sister chromatids separate
Learn about the genetic consequences of normal and abnormal meiosis
If given a karyotype, genotype or RFLP Southernblot of an aneuploidy situation, determine when and in
which parent nondisjunction occurred
Know the consequences on chromosome numbers in gametes if nondisjunction occurs in either meiosis 1
or meiosis 2
Know that crossing over and recombination, and independent assortment of chromosomes at anaphase
during meiosis are responsible for the genetic diversity of the haploid daughter cells
Know the differences between mitosis and meiosis
Mitosis converts one diploid cell into four haploid cells. There is one round of DNA synthesis followed
by two rounds of chromosomal segregation. Meiotic crossing over or recombination also occurs here.
Meiosis II is like mitosis without a preceding DNA replication; sister chromatids separate in this stage.
Know the key stages of human gametogenesis, especially for oogenesis
Spermatogenesis
Sperm found in seminiferous tubules after sexual maturity. Tubules lined with spermatogonia in
different stages of differentiation.
Oogenesis
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Occurs only during prenatal development. Oogonia in ovarian cortex originate from primordial
germ cells. No primary oocytes develop after 5 months of fetal life.
At sexual maturity, individual follicles mature oocyte completes meiosis I one secondary
oocyte (ovum) meiosis II begins, but is arrested in metaphase until fertilization.
If fertilization takes place, secondary oocyte will complete 2 nd meitotic division. During the
arrest, the oocycte chromosomes decondense and are actively transcribed, and cell growth and
materials for early embryonic growth are stock piled.
Know in which tissues meiosis occurs
Know the two arrest points for oogenesis
Diplotene of Prophase I
Metaphase II
Know when the X and Y chromosomes separate from each other during spermatogenesis and when their sister
chromatids separate as well
Know that the SRY/TDF gene on the Y chromosome near the pseudoautosomal region is important for
development of phenotypic males
Abnormal crossing over during spermatogenesis moving the SRY gene from the Y chromosome
to the X chromosomes can result in XX males and XY females
Know the roles of Cdk1/Cyclin B, and Mos during oogenesis
Mos is an essential component of cytostatic factor (CSF); it is synthesized in oocytes at the completion of
meiosis I and is required for maintenance of cdk1/cyclin B activity.
Action of Mos results in activation of ERK MAP kinase, but ERK activates RSK this maintains
activity of MPF by inhibiting cyclin B degradation.
Learn the key events during fertilization
At fertilization, the sperm binds to a receptor on the surface of the egg and fuses with the egg plasma
membrane. Fertilization leads to mixing of paternal and maternal chromosomes, and it induces changes in
the egg cytoplasm. Binding of the sperm to its receptor signals an increase in calcium levels, probably
from stimulation from hydrolysis of PIP2. (This induces surface changes that prevent additional sperm
from fertilizing the egg). APC/C becomes activated and meiosis can go to completion.
Asymmetric cytokinesis gives rise to a second small polar body. After completion of oocyte meiosis, the
fertilized egg (zygote) contains two haploid nuclei (pronuclei), one derived from each parent.
Know the Law of Segregation- states that the alleles of each gene separate from each other during gamete
formation. Usually results when the alleles separate from each other during meiosis I
Know the Law of Independent Assortment- states that the alleles of each gene separate independently of the
alleles of other genes
Develop an awareness about the process of homologous recombination and crossing-over
Presentation # 5: Cell Death & Cell Renewal
Learn about the key differences between Necrosis and Apoptosis

o Necrosis swelling and rupturing of injured cells, typically involving many cells at the tissue level.
Characterized by severe destruction, cytoplasmic organelle destruction, loss of membrane integrity,
heterophagy, accompanying inflammatory response.
Accidental cell death from acute injury
o Apoptosis- genetically controlled cell death that occurs in response to environmental, developmental, or
other stimuli and is usually at the single cell level; minimum damage to surrounding cells, WITHOUT
accompanying inflammatory response.
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Characterized by DNA fragmentation, chromatin condensation, fragmentation of the nucleus and
cell. Programmed cell death during embryogenesis and hormone dependent involution in
the adult.
Know some of the key features of apoptosis:
o Why is it important? Ensure homeostasis of all tissues; prevent tumor growth; control mechanism in
maintaining immune system.
o DNA Laddering caused by endonucleases is NOT normally seen in necrosis
o Blebbing
o Presentation of eat me signals such as phosphatidylserine on the cell surface loss of membrane
asymmetry: appearance of phosphotidylserine (a phospholipid), thrombospondin, on outer plasma
membrane help recognition of apoptotic cells by macrophages. In normal cells, phosphotidylserine is
restricted to the inner leaflet of the plasma membrane.
o Stages of apoptosis: cell shrinkage, chromatin condensation, surface blebbing, membrane-bound
apoptotic bodies containing cytoplasm and tightly packed organelles.
Learn about the role of caspases during apoptosis.
o Caspases are zymogens that remain inactive until an apoptosis signal initiates the activation of one
initiator caspase which will cause a cascade leading to activation of other caspases (effectors).
o Initiator vs effector caspases
Initiator caspases 8, 9, 10, 12
Effector (executioner) caspases 3, 6 ,7
Know which caspases are initiator caspases for the innate apoptotic pathway and know
which are initiator caspases for the death receptor-mediated pathway of apoptosis
Learn about IAPs and anti-IAPs

o Caspases are regulated by a family of proteins called IAP (inhibitor of apoptosis)
o They either inhibit caspase activity or target caspases for ubiquination and degradation in the
proteasome.
o IAPs prevent accidental apoptosis caused by the spontaneous activation of procaspases, and are located in
the CYTOSOL
o When the intrinsic apoptotic pathway is stimulated, anti-IAPs are released and block inhibitory activity
of IAPs apoptosis can be induced.
Apoptotic threshold is established when the level of expression of survival (proapoptotic vs apoptotic) exceeds the
level of expression of survival factors, leading to activation of caspases. Once the apoptotic threshold is reached,
the physiology of the mitochondria changes which leads to the release of calcium and key mitochondrial proteins
found in the intermembrane space.
o Cytochrome C, diabo/smac, apoptosis-inducing factor (AIF), endo G, htra2/omi
Learn in detail two examples of mitochondrial pathway of apoptosis
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o DNA damage
Induced by intracellular damage, DNA damage or growth factor withdrawal. Release of key
mitochondrial proteins into the cytosol from intermembrane space leads to activation of
apoptosis.
ATM/p53/bax & puma/bcl2/cytochrome c/apaf-1/caspase9/caspase 3 pathway
Role of p53 as an apoptosis regulator
ATM and CHK2 phosphorylate p53 when the cell has extreme DNA damage. P53
activates/increases production of noxa, PUMA, and other factors activates Bax and Bak
these interact with mitochondria chytochrome C and other factors are released into the cytosol
cytochrome C recruits Apaf-1, procaspase-9, and ATP or dATP into a complex (apoptosome)
caspase 9 then stimulates caspase 3 ACTIVATES APOPTOSIS
o Deprivation of survival factor
Loss of signaling via PI-3 kinase/AKT/phosphorylated BAD/14-3-3/etc.
Many growth factors that signal cell survival activate receptor protein tyrosine kinases
activation of PI 3-kinase formation of PIP3 activation of AKT AKT phosphorylates
proteins that contribute to cell survival 14-3-3 will sequester Bad when phosphorylated
maintained in INACTIVE state
IN THE ABCENSE OF AKT: activation of Bad promotes apoptosis and activation of FOXO
stimulates transcription of Bim
Bad and related pro-apoptotic factors are dephosphorylated and translocates to mitochondria
and interacts with bcl-2 or bcl-x loss of normal mitochondrial physiology release of
cytochrome C and other factors activation of caspase 9 pathway
Learn about the 3 classes of BCL-2 family of proteins
o Must know anti-apoptotic BCL-2 and that it resides on the outer mitochondrial membrane
Binds Bid and blocks formation of channels that allow cytochrome C release from the
mitochondria.
o Know that Bax and Bak form oligomers in the outer membrane of mitochondria, leading to release
of cytochrome c
Death signals result in activation of a BH3 only protein such as Bid, which can lead to
mitochondrial pore formation, swelling, and release of cytochrome C.
Bid binds Bax cytochrome C release binds Apaf-1 leads to formation of apoptosome
o These proteins play tissue-specific roles in addition to signaling pathways. The tissue specificity is
overlapping.
o Anti-apoptotic: BCL2
o Proapoptotic channel forming: Bax, Bak, Bok
o Proapoptotic BH3-only: Bad, Bid, Bim
Know the following five molecules released from the mitochondria and their roles in apoptosis:
cytochrome c, SMAC/Diablo, HtrA2/Omi, AIF and Endonuclease G
o Calcium can lead to activation of caspases associated with ER
o SMAC/diablo (ANTI-IAP) directly interacts with IAP and block their activity
o HtrA2/omi blocks IAP (can cleave too); promotes caspase independent apoptosis via its serine
protease activity.
o Apoptosis inducing factor (AIF) GOES TO NUCLEUS; stimulates large DNA fragmentation and
condensation of chromatin
o Endonuclease G directly mediate nuclear DNA fragmentation in NUCLEUS
Learn in detail two examples of death receptor pathway of apoptosis
o TNF
TRADD & FADD/caspase 8/caspase 3 pathway
TNF can initiate apoptosis if associated with TRADD and FADD
TNF can intitiate survivial response if TRADD is associated with TRAF-2 and RIP
RIP interferes with FADD interaction with TRADD and thus no apoptotic signal
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Stimulates NF-kB pathway
o Fas ligand
FADD/caspase 8/caspase 3 pathway
Learn about cytolytic granule mediated apoptosis

o Can circumvent requirements of FAS or TNF receptors. A,B,H,K, and M granzymes and porins released
by cytotoxic T cells or NK cells activate effector caspases inside the cell.
Can bind other proteins such as Mcl-1 and Bid mitochondrial apoptotic pathways
Learn about cell death via autophagy
o Autophagy can be used as an alternative to apoptosis. It does not require caspases. It can be activated by
cellular stress and provide an alternative to apoptosis when apoptosis is blocked. These cells die via
autophagy are characterized by accumulation of lysosomes
Know that abnormal regulation of apoptosis is associated with oncogenesis
o Example: overexpression of BCL2 in some forms of cancer
Must know example: Follicular B-cell Lymphoma chromosomal translocation leads to up-
regulation of Bcl-2
Learn the following examples of differentiated cells that retain ability to proliferate:
o Fibroblasts (role of PDGF) fibroblasts in connective tissue can proliferate quickly in response to PDGF
released at the site of the wound
o Endothelial Cells (role of VEGF)- endothelial cells that line blood vessels can proliferate to form new
blood vessels for repair and regrowth of damaged tissue. These are triggered by VEGF which is produced
by cells that lack oxygen.
o Liver cells- normally arrested in the G(o) phase of cell cycle can be stimulated to proliferate if large
numbers of liver cells are lost. Numerous factors including TNF-alpha, EGF, and hepatocyte growth
factor regulate liver regeneration.
Know what are stem cells
o Less differentiated, self-renewing cells present in most adult tissues; retain capacity to proliferate and
replace differentiated cells throughout the lifetime of an animal; the key property divide to produce
one daughter cell that remains a stem cell and one that divides and differentiates.
Learn the following examples of tissues replaced by proliferation of stem cells:
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o Blood cells, and Epithelial cells of skin, hair and digestive tract have short life spans and must be
continually replaced by proliferation of stem cells.
o Hematopoietic (blood forming) stem cells
o Epithelial cells lining the intestines live only for a few days before they die by apoptosis; new cells are
derived from the slow continuous division at the bottom of intestinal crypts.
o Skin and hair epidermis, hair follicles, and sebaceous glands are all maintained by their own stem cells.
o Learn about skeletal muscle stem cells called satellite cells
Normally has little turnover, but in response to damage, can regenerate rapidly. Regeneration is
mediated by proliferation of satellite cells the stem cells of adult muscle
Learn about embryonic stem cells
o pluripotentcy the capacity to develop into all of the different types of cells in adult tissues.
Learn about somatic cell nuclear transfer, therapeutic cloning
o Dolly arose by the process of somatic cell nuclear transfer. This type of cloning in mammals is a
difficult and inefficient process.
o Therapeutic cloning nucleus from an adult human cell would be transferred to an enucleated egg. The
resulting embryo could produce differentiated cells by transplantation therapy. This would bypass the
problem of tissue rejection.
Learn about induced pluripotent stem cells
o Definition: programming somatic cells to resemble embryonic stem cells. The action of four transcription
factors is sufficient to reprogram adult mouse somatic cells.
o We can reprogram human fibroblasts into pluripotent cells (transcription factors required: Oct4, Sox2, and
Nanog) form autoregulatory feedback loop. Can be introduced via viral vectors.
Learn about transdifferentiation
o Process of reprogramming differentiated cells such as fibroblasts into another type of differentiated cell.
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Must Know Diseases and Disorders For Block 3
Angelman Syndrome- phenotype: unusual facial appearance, short stature, severe mental retardation, spasticity,
and seizures. The abnormal chromosome 15 is inherited from the mother. Both boys and girls can get it!
Prader-Willi Syndrome- phenotype: obesity; excessive and indiscriminate eating habits, small hands and feet,
short stature, hypogonadism, and mental retardation; the abnormal chromosome 15 is inherited from the father.
Both boys and girls can get it!
Li Fraumeni disease- rare cancer family with history of many different forms of cancer; usually afflict patient at
unusually early age; inherited in autosomal dominant pattern; mutations in both alleles are necessary to inactivate
the P53 gene.
Ataxia-Telangiectasia caused by inherited mutation in the gene encoding ATM; ataxia marks the beginning of
progressive degeneration of the cerebellum that gradually leads to a lack of muscle control. Telangiectasia tiny
red spider veins, which appear in the corners of the eyes or on the surface of the ears and cheeks exposed to
sunlight. Immune system problems; pneumonia; predisposition to cancer;
Diphtheria toxin - prevents the translocation by inhibiting eEF-2
Follicular B-cell Lymphoma- chromosomal translocation leads to up-regulation of Bcl-2
Sickle Cell Anemia example of inherited disorder resulting from a missense mutation; the mutation is in the 6 th
codon of the beta globin gene (glutamate valine missense); hemoglobin with two normal alpha chains and two
of the sickle cell mutant beta chains is called HbS; the mutation causes HbS hemoglobin to polymerize under
deoxygenated conditions. (thus sickle shaped)
o Anemia (Hb levels 6-10 g/dL); splenomegaly; painful swelling of hands or feet; repeated infection, which
is major cause of death.
Synpolydactyl- HOXD13 mutation; semidominant condition in which heterozygotes have interphalangeal
webbing and extra digits in their hands and feet; homozygotes are more severe with additional bony
malformations; mutations usually by expansion of polyalanine tract; normal -15; mutation 22-24.
Systemic Lupus Erythematosus (SLE)- chronic systemic autoimmune disease characterized by the production
of autoantibodies and loss of self-tolerance; Anti-SM and anti-RNP specifically recognize splicing snRNPs;
prevents splicing
o Common symptoms: butterfly rash; oral ulcers; photosensitivity; renal problems; death often due to
renal failure and infections.
Cockaynes Syndrome- rare inherited disorders in which people are sensitive to sunlight, have short stature, and
have the appearance of premature aging. Classical form (type I) symptoms are progressive and appear AFTER
the first year; type II early onset or congenital that appears at birth.
o After exposure to UV radiation, people with CS can no longer perform transcription coupled repair.
NOT LINKED TO CANCER; defect in CSA and CSB enzymes.
Xeroderma Pigmentosum- XP is characterized by sun sensitivity, ocular involvement, and greater than 1000 fold
increased risk of cutaneous and ocular neoplasms; freckling of the face; dry skin; eye problems; about 30% of
patients have neurologic manifestations, diminished or absent deep tendon stretch reflexes, progressive
sensorineural hearing loss, and cognitive impairment. AN ERROR IN NUCLEOTIDE EXCISION REPAIR
Hereditary nonpolyposis colon cancer (HNPCC)- (and endometrial cancer for females); pathway: DNA
mismatch repair error or mutation;
o Results from the mutation in genes coding for proteins involved in mismatch repair; most common
mutations MLH1 and MSH2
Hereditary Breast and Ovarian Cancer
o BRCA1 and BRCA2 are tumor suppressor proteins; when abnormal:
BRCA1 confers high risk of breast and ovarian cancer (TYPE 1)
BRCA2 gene confers a high risk of breast cancer; not an elevated risk of ovarian.
o BRCA1 and BRCA2 are involved in homologous recombination.
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Vitamin K deficiency alcoholics, newborns, people with prolonged use of antibiotics are at most risk; inherited
disorders are also likely. Vitamin K is a fat soluble vitamin; involved in generating gamma-carboxyglutamate,
which is necessary for blood clotting factors; a new born with vitamin K deficiency is hemorrhaging;
o A true vitamin K deficiency is unusual because adequate amounts are generally produced by intestinal
bacteria or obtained from the diet; inadequate dietary intake of vitamin K does occasionally occur in
conjunction with anorexia.
Beta thalassemia severe hypochromic, microcytic anemia during 1st year of life; HB levels <7 g/dL; mostly
HBb F and HbA2; severe chronic anemia stimulation of erythropesis marked expansion of marrow space
characteristic skeletal changes; skull, long bones, hypertrophy of maxilla, chipmunk facies;
Must Know Drugs and Toxins

Rifampin- used to battle TB; specifically inhibits initiation of RNA synthesis in BACTERIA; interferes with
the first few phosphodiester bonds in RNA chain;
Actinomycin- binds tightly to dsDNA, prevents it from being effective template for RNA synthesis in both
prokaryotes and eukaryotes.
Aminoglycosides
o Must know example: Streptomycin
o Bind to 30S prokaryotic subunit and inhibit initiation on protein synthesis; they can also induce
misreading if translation does occur.
Chloramphenicol- binds to the 50S ribosomal subunit and inhibits the peptidyl transferase activity of the 23S
rRNA.
Clindamycin and Macrolides
o Must know example: Erythromycin
o Both work by binding to the 50S ribosomal subunit and preventing translocation during protein synthesis.
Diphtheria toxin and pseudomonas exotocin (exotocin A)
o Inhibit elongation factor-2 (eEF2) and prevents translocation.
Tetracyclines- bind to the 30S prokaryotic subunit and block the binding of amino-acyl tRNA to the acceptor site
(A-site) on the ribosome. Examples: doxycycline and minocycline
Puromycin mimics the shape of amino acyl tRNA and becomes incorporated into the growing peptide chain,
thus inhibiting elongation.
Alpha-Amanitin an inhibitor of RNA polymerase II; group of complex cyclic polypeptides which damage
tissues by inhibiting RNA synthesis within each individual cell; fatality rate is 50% without prompt care; 4 stages
of onset.
Bortezomib- was the proteasome inhibitor approved for the treatment of a human cancer multiple myeloma;
Warfarin (synthetic analog of dicoumarol) the formation of Gla is sensitive to inhibition by dicoumarol, an
anticoagulant occurring naturally in spoiled sweet clover. (has to do with vitamin K production)
Quinolones/fluoroquinolones- constitute a large class of synthetic antimicrobial agents that are highly effective
in the treatment of many types of infection diseases, particularly those caused by bacteria.
o Examples: ciprofloxacin; norfloxacin; nalidixic acid
o Drugs that inhibit DNA gyrase, preventing DNA replication and transcription.
Doxorubicin, Teniposide, & Etoposide- (DET) to treat several neoplastic disease. These act by enhancing
rate at which target topoisomerase II cleaves DNA & by reducing rates at which these breaks are resealed.
Zidovudine (ZDV)- patients with AIDS are often treated with a mixture of drugs including ZDV; this drug is an
analog of the thymine nucleotide found in DNA; lacks a 3 OH group, and therefore no additional nucleotides can
be attached through a 5-3 bond. This chain elongation of the DNA is terminated.
o Reverse transcriptase has a higher affinity for ZDV than does normal human cellular DNA polymerases,
enabling the drug to target viral replication, more specifically than cellular replication.
o ddNTP is used as an anticancer nucleotide analog.
Cell Biology Block 4 Presentation #1 - Cancer
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Learn about the role of cell proliferation & differentiation in the genesis of tumors
Tumors are produced by uncontrolled cell proliferation in which the balance between cell division and
differentiation is disrupted. This is usually coupled with loss of regulated cell death (apoptosis)
Normally new cell growth is balanced by apoptosis
Tumors (or Neoplasm): a disease process characterized by uncontrolled cellular proliferation leading to a mass
or tumor (neoplasm)
For a neoplasm to be a cancer, it must be malignant, which means its growth is no longer controlled and the
tumor is capable of invading neighboring tissues or spreading (metastasizing)
Cancer: a general term for a malignant tumor
Learn the three main forms of cancer (carcinomas, sarcomas, leukemia)
Sarcomas: Tumors originating in mesenchymal tissue, such as bone, muscle, or connective tissue
Carcinomas: originate in epithelial tissues, such as the cells lining the intestines, bronchi, or mammary ducts
Account for 90% of all cancers
Leukemias and lymphomas
Leukemia: cancer cells reside and proliferate mainly in the bloodstream rather than growing as a solid mass
Within each of the major groups, tumors are classified by site, tissue type, histological appearance, and degree of
malignancy
Learn the differences between benign and malignant tumors
Benign tumors do not metastasize; instead they tend to grow in a confined local area
Metastatic tumors have the ability to spread to other tissues in the body
Learn that most cancers are clonal, somatic and sporadic in origin
Most cancer cells are clonal (derived from a single abnormal cell), somatic (non-germ line cells), and sporadic
(random) in origin
Learn about cancer stem cells
Cancer stem cells are defined as those cancer cells that can self-renew to produce additional malignant stem cells
& at the same time generate non-tumorigenic cells such as transit amplifying cells
Cancer stem cells can arise from normal stem cells that have sustained mutations to make them cancerous or
from more differentiated cells that have undergone mutations or epigenetic changes that give them stem cell
properties
A small population of cancer stem cells maintain many tumors
Cancer stem cells are often difficult to eradicate by conventional treatments because they replicate more
slowly
To cure cancer we need to find better ways to target and kill cancer stem cells
Learn the importance of PI3-K/PTEN in growth signaling
The PTEN tumor suppressor gene encodes a lipid phosphatase that dephosphorylates PIP 3 PIP(2) and PTEN
inhibits PI 3-kinase and AKT
Both can act as oncogenes by promoting cell survival, and stimulating cell proliferation.
The role of PTEN is to dephosphorylate PIP3, acting as a negative control on PKB/Akt activation.
Under normal growth, stimulatory signals from the insulin receptor activate PI 3-Kinase, which
phosphorylates PIP2 to generate PIP3
If PTEN is mutated and doesn't work, PIP3 can no longer be deactivated so it continues to propagate its signal
downstream.
Can result in continued activation of PKB/Akt; along other factors can lead to excessive cell growth and
the development of tumors
Learn that cancer cells are anchorage-independent and Insensitive to population density
Most normal cells need a surface in order to proliferate and survive. Tumor cells do not.
Normal cells undergo apoptosis if not attached and is a safeguard to prevent normal cells from floating away
and colonizing other tissues
Cancer cells therefore exhibit anchorage-independent growth
Cancer cells also exhibit a reduced sensitivity to density-dependent inhibition of growth
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Normal cells in tissue culture will usual grow until a single layer of cells is formed, then cell division stops
Cancer cells grow unrestrainedly on top of each other
Learn how cancer cells become immortalized by reactivating telomerase
Normal cells divide about 50-60 times when placed in culture
Stop dividing and undergo degenerative changes
Cellular senescence may be due to loss of telomerase activity
Cancer cells have reactivated telomerase activity maintain their telomeres.
By maintaining telomere length above a critical threshold, cancer cells retain the ability to proliferate
indefinitely
Telomerase expression reappears in transformed cells and in many tumors
An alternative mechanism for maintaining telomere length employs enzymes that exchange DNA information
between chromosomes
Learn about the importance of angiogenesis during oncogenesis
Angiogenesis is the formation of new blood vessels
When a tumor reaches about a million cells, new blood vessels are required to supply oxygen and nutrients -
cancer cells secrete growth factor for their formation (VEGF)
The new capillaries are easily penetrated by the tumor cells, providing access to the circulatory system and
metastasis.
The roles of VEGF and matrix metalloproteinases in the process
The hazards posed by cancer cells come from uncontrolled proliferation combined with the ability to spread
throughout the body
Angiogenesis is required for tumors to grow beyond a few millimeters in diameter
Vessel growth is controlled by balance between angiogenesis activators and inhibitors
Activators: VEGF, FGF
These cause endothelial cells to proliferate and produce matrix metalloproteinases which breaksdown the
ECM allowing endothelial cells to migrate
Inhibitors: angiostatin, endostatin & thrombospondin
Tumors increase production of activators and decrease inhibitors
Learn about invasion and metastasis
Spreading of cancer by invasion and metastasis is a complex multistep process
Invasion refers to direct migration & penetration of cancer cells into neighboring tissues
Changes in cell adhesion, motility, and protease production allow cancer cells to invade surrounding tissues
and vessels
Mechanisms that allow for invasion
Alteration in cell surface proteins that cause cells to adhere
E-cadherin levels lower in epithelial cancers
increased motility of cancer cells
Metastasis involves the ability of cancer cells to enter body fluids (blood) and travel to distant sites, where they
form tumors
Either directly or indirectly through the lymph system, cancer cells arrive in the bloodstream - Most do not
survive voyage through bloodstream
Those that do usually have additional changes which make them well suited for metastasizing
Learn about the importance of matrix metalloproteinases in the process
Production of proteases that compromise cell movement barriers & degrade ECM
Breaching the basal lamina in epithelial cancers
Example: plasminogen activator which converts plasminogen into plasmin
Plasmin degrades ECM and basal lamina proteins
Activates matrix metalloproteinases
Facilitates cell migration into blood vessel
Learn why metastasis occur more frequently with certain organs
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Blood-flow patterns and organ-specific factors determine where cancer cells metastasize
Often metastasize the first capillary bed encountered - usually the lungs or liver
Seed & soil hypothesis: some sites provide optimal growth conditions for particular cancer cells, other
sites do not
Prostate cancer commonly metastasizes to bone
Bone cells produce specific growth factors that stimulate prostate cells
Learn about causes of cancer and tumor progression
Cancers are caused mainly by environmental agents and lifestyle factors, most of which act by triggering DNA
mutations
Epidemiological data have shown many causes of cancer to be identified
Cigarette smoking & lung cancer
Carcinogens are cancer-causing agents such as cigarette smoking with lung cancers; Scrotum cancer in
chimney sweeps
Hepatocellular carcinoma & Aflatoxin B1
In many parts of the world hepatocellular carcinoma occurs more frequently due to ingestion of aflatoxin B1,
a potent carcinogen produced by a mold found on peanuts
Aflatoxin has been shown to modify a particular base in the TP53, causing a G to T mutation in codon 249,
converting Arg to Ser in the critically important p53 protein
This mutation is found in nearly half of all hepatocellular carcinomas in patients from parts of the world in
which there is a high frequency of contamination of food by aflatoxin
But not found in similar cancers in patient with low exposure to aflatoxin in food
The Arg-249-Ser mutation in p53 enhances hepatocyte growth and interferes with the growth control and
apoptosis associated with wild-type p53
LOH of TP53 in hepatocellular carcinoma is associated with a more malignant appearance of the cancer
Altho aflatoxin B1 alone is capable of causing HCC, it also acts synergistically w/chronic hep B & C
infections.
Pathogens
Viruses and other infectious agents are responsible for some cancers
Oncogenic viruses
Two ways pathogens trigger cancer
Those that cause tissue destruction and chronic inflammation. Immune response leads to the generation of
free radicals and DNA damage
H. pylori, parasitic flatworm, hepatitis B & C viruses
Virus stimulates proliferation by either introducing a viral gene or modifying host gene expression
EBV, Retroviruses, papillomavirus
Tumor Viruses
Hepatitis B & C Viruses - are the principal causes of liver cancer; the viruses infect liver cells and can lead to
long-term chronic infections, which are associated with a high risk of liver cancer
Polyomaviuses - was recently identified as causative agent for Merkel cell carcinoma - merkel cell
polyomavirus
Polyomavirus has been postulated to cause other cancers such as sporadic colon cancer
Polyomavirus Simian virus 40 (SV40) does not cause human cancer, but have been critically important as
models
They have small genomes and they are good assays for virus replication and transformation
SV40 do not induce tumors in their natural host speciesmonkeys and mice, respectively
In the natural host (permissive cells), infection leads to virus replication, cell lysis, and release of progeny
virus particles. The permissive cell is killed and cant be transformed
In nonpermissive cells the virus replication is blocked.
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The viral genome sometimes integrates into cellular DNA, and expression of specific viral genes results in
transformation of the infected cell.
Papillomavirus - small DNA viruses; about 100 different types infect epithelial cells
Some cause benign tumors (such as warts) and others cause malignant carcinomas, particularly cervical and
other anogenital cancers
Early detection/treatment of cervical cancer is made possible by the Pap smear test
Cell transformation results from expression of two early-region genes, E6 and E7
HPV E6 & E7 - Papillomaviruses that are cancerous from genes E6/E7
Adenovirus
Adenoviruses are a large family of DNA viruses not associated with cancer, but important as models.
Adenoviruses are lytic in cells of their natural hosts but can induce transformation in nonpermissive cells.
Herpes viruses
Herpesviruses are among the most complex animal viruses, with genomes of 100 to 200 kb.
Kaposis sarcoma- associated herpesvirus and Epstein-Barr virus cause human cancers.
Epstein-Barr virus can transform human B lymphocytes in culture, but the mechanisms are not well
understood.
Retroviruses - cause cancer in a variety of animals, including humans.
Human T-cell lymphotropic virus type I (HTLV-I) causes adult T-cell leukemia.
AIDS is caused by the retrovirus HIV
HIV doesn't cause cancer directly but AIDS patients have a high incidence of malignancies
Particularly lymphomas and Kaposis sarcoma via secondary infections
Initiators vs. promoters
Cancer arises through a multi-step process involving initiation, promotion and tumor progression
Successive rounds of randomly inherited changes followed by natural selection
Tumor initiation results from genetic alteration leading to abnormal proliferation of a single cell, which
proliferates into a population of clonal tumor cells.
During initiation, normal cells are converted to a precancerous state
Chemicals that act as initiators often cause DNA damage
Tumor progression continues as additional mutations occur within cells of the tumor population
Promotion then stimulates the altered cells to proliferate and form tumors
Chemicals that act as promoters stimulate cell proliferation
Natural selection will then favor cells with the fastest growth rates - progression towards cells that will
form tumors from excessive growth
Some of the mutations may confer selective advantage, such as rapid growth
Clonal selection: Descendants of these cells become dominant
Clonal selection continues throughout tumor development, so tumors continuously become more rapid-
growing and increasingly malignant
Ames test
Detects mutations that cause bacteria to regain the ability to produce histidine and thus grow
The focus assay
The study of tumor induction by radiation, chemicals, or viruses was advanced by development of in vitro assays
to detect conversion of normal cells to tumor cells in culture, a process called cell transformation.
The focus assay is based on the ability to recognize a group of transformed cells as a morphologically distinct
focus against a background of normal cells on a culture dish
Focus assay takes advantage of three properties of transformed cells: 1) altered morphology, 2) loss of contact
inhibition, and 3) loss of density-dependent inhibition of growth
Learn about the roles of oncogenes and tumor suppressors during tumorgenesis
Oncogenes are genes whose presence can trigger the development of cancer
Often encode for proteins that stimulate excessive cell proliferation &/or promote cell survival
Nonviral oncogenes determined by the oncogene transfection assay
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Oncogene transfection assay is used to analyze ability of DNA to transform a cell
Most common oncogene mutated: Ras
RasH = thyroid carcinoma
RasK = colon, lung, pancreatic, and thyroid carcinomas
RasN = acute myeloid and lymphocytic leukemias and thyroid carcinomas
Tumor suppressor are genes whose loss or inactivation can lead to cancer
Most common tumor suppressor mutated: p53
Learn the five mechanism of converting a proto-oncogene into an oncogene
Proto-oncogenes are normal cellular genes that can become mutated into oncogenes
Five mechanisms of converting a proto-oncogene into an oncogene
1) Point mutation: Ras point mutations keep it in its active form
2) Gene amplification: extra copies of genes leads to excess of protein product
3) chromosomal translocation - example: Burkitts lymphoma
4) local DNA rearrangements
5) Insertional mutations: retrovirus integrates in the wrong location.
Must know translocations cancers
CML - Chronic Myelogenous Leukemia: t(9;22)(q34;q11)
Philadelphia chromosome (Ph1): A translocation between chromosomes 9 and 22
Creates a fusion BCR-ABL gene at 9q and 22q
CML is a cancer of blood cells, characterized by replacement of bone marrow with malignant, leukemic cells.
Many of these leukemic cells can be found circulating in the blood and can cause enlargement of the
spleen, liver, and other organs
Burkitt Lymphoma: t(8;14)(q24;q32)
Burkitt lymphoma is a rare form of cancer predominantly affecting young children in Central Africa, but the
disease has also been reported in other areas. The form seen in Africa seems to be associated with infection by
the Epstein Barr virus, although the pathogenic mechanism is unclear.
Translocation puts the MYC gene under the influence of an enhancer from the immunoglobin genes
Acute promyelocytic leukemia t(15;17)(q22;q12)
In 95% of cases, acute promyelocytic leukemia is characterized by chromosomal reciprocal translocation
involving:
The retinoic acid receptor-alpha gene (which regulates myeloid differentiation) on chromosome 17
(RAR)
The promyelocytic leukemia gene (PML) on chromosome 15 (which encodes a growth suppressing
transcription factor)
Creates a PML/RARa fusion gene.
It produces a chimeric protein that arrests the maturation of myeloid cells at the promyelocytic stage. (It
reduces terminal cell differentiation.) And this leads to the increased proliferation of promyelocytes.
Mutated forms of the retinoic acid receptor (PML/RARa) act as oncogene protein in human acute
promyelocytic leukemia.
The mutated oncogene receptors interfere with the action of their normal homologs, blocking cell
differentiation and maintaining the leukemic cells in an actively proliferating state.
Patients with acute promyelocytic leukemia can be treated by retinoic acid, which induces
differentiation and blocks continued cell proliferation.
Follicular B-cell lymphoma t(14;18)(q32;q21)
BCL2, an antiapoptotic gene, is upregulated by a translocation which brings an immunoglobin promoter and
enhancer in proximity to the BCL2 gene
Differences between gatekeeper and caretaker tumor suppressors
Some tumor suppressors truly suppress tumors by regulating the cell cycle or causing growth inhibition by cell-
cell contact
Suppressor genes of this type are gatekeepers because they regulate cell growth directly
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Other Tumor Suppressor genes, the caretakers, are involved in repairing DNA damage & maintaining
genomic integrity.
Loss of both alleles that are involved in repairing DNA damage or chromosome breakage leads to cancer
indirectly by allowing additional secondary mutations to accumulate either in proto-oncogenes or in other
tumor suppressors genes.
Learn the two-hit origin of cancer for tumor suppressors
The two-hit origin of cancer for tumor-suppressors both alleles of the gene need to be mutated. 1 st hit can be
inherited and 2nd hit can arise spontaneously; epigenetic origin: gene silencing such as X inactivation and
imprinting.
Learn the mechanisms leading to loss of heterozygosity

Individuals who have heterozygous alleles in normal tissue but have tumors that contained alleles from only
one of their two chromosomes
LOH is the common mutational mechanism by which the remaining normal RB1 allele is lost in
heterozygotes (chromosome 13 region)
LOH is a feature of a number of other tumors, both sporadic and heritable, and is often considered evidence
for the existence of a tumor-suppressor gene
Learn the role of epigenetics in oncogenesis
Epigenetic changes are important during the process of oncogenesis
A mechanism by which developing tumor cells can inactivate tumor suppressor genes
Methylation
Chromatin packaging
MUST know Retinoblastoma and Rb
Involved in causing hereditary retinoblastoma - Rb regulates G1 S transition
Found mutated in several common adult nonhereditary cancers
Lung, breast, and bladder cancer
A 110 kilodalton phosphoprotein found in many tissues; RB1 gene is a prototypical gatekeeper tumor suppressor
gene
Loss of Rb allows the cell to proliferate uncontrollably
Rb regulates G1 to S Transition
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It blocks cell cycle progression (G1/S phase) when it is in the hypophosphorylated state. It interacts and
modifies the behavior of nuclear proteins (E2F).
Dephosphorylated Rb interacts with transcription factor E2F. This prevent E2F from stimulating
transcription
When it is hyperphosphorylated it allows the cell to enter S phase.
G1 Cdk-cyclin phosphorylates Rb causing it to be released from E2F and allowing E2F to stimulate
transcription
Must know Li Fraumeni and p53
The most frequently mutated gene associated with cancer
A DNA-binding protein forms a tetramer
Important in the cellular response to DNA damage.
Activates genes that stop cell division and allow DNA repair
P53 also is involved in inducing apoptosis in cells that have irreparable DNA damage
Loss of p53 function, allows cells with damaged DNA to survive and divide, thereby propagating potentially
oncogenic mutations
Li-Fraumeni syndrome results from p53 mutation.
Early onset of cancers - "cancer families"
Learn about BRCA1 & 2 in inherited breast cancer
Strong genetic component.
Increased risk if first degree relative afflicted. More so if onset occurred before age 40
Frequent bilateral disease
Less than 5% of all breast cancer
BRCA1 gene: Chromosome 17q21 (50% of AD familial breast cancer)
BRCA2 gene: Chromosome 13q12.3 (33% of AD familial breast cancer)
Increased chance of male breast cancer (10-20% of all)
MUST KNOW Familial polyposis coli
Colorectal cancer: 150,000 people/year, 15% of all cancer
Small portion due to autosomal dominant (AD) Familial Polyposis Coli (familial adenomatous polyposis (FAP)
Gardner variant is a subvariant - develop osteomas of the jaw and desmoids (tumors in the muscle of the
adominal wall)
FAP heterozygotes can have numerous benign growths (adenomatous polyps) by age 20
Usually one or more polyps become malignant
Colectomy (removal of colon) prevents development of malignancies
The role of APC and b-catenin - Adenomatous Polyposis Coli (APC)
APC gene loci are responsible; located on chromosome 5q and encodes a cytoplasmic protein that regulates b-
catenin.
Two roles of b-catenin
Link the cytoplasmic portion of transmembrane cell adhesion molecules and actin cytoskeleton
Activate transcription
Wnt pathway
Loss of APC leads to accumulation of free B-catenin that is translocated to nucleus and activates transcription
Tumor suppressor APC gene mutations are common in nonhereditary forms of colon cancer
Learn HNPCC and mismatch repair
2-4% colon cancers due to HNPCC - AD onset during early adulthood without the adenomatous polyps of FAP
Heterozygous males have 90% lifetime risk of developing colon cancer
Female heterozygotes have 70% chance, 40% risk of endometrial cancer and 10-20% risk for cancer of the
biliary or urinary tract, and the ovary
DNA isolated from tumors display microsatellite instability
Inherited dfx in genes required for mismatch repair responsible for HNPCC
The genes: MLH1, MSH2, PMSL1, PMSL2, and MSH6
MLH1 and MSH2 together account for 60-70% of HNPCC
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The HNPCC genes are prototypical caretaker tumor-suppressor genes.
Learn about genetic instability and gene amplification
A trait of cancer cells in which abnormally high mutation rates are caused by defects in DNA repair and/or
chromosome sorting mechanisms
Inherited defects in genes required for mismatch repair are responsible for HNPCC
Xeroderma pigmentosum caused by inherited defects in UV damage excision repair pathway
Hereditary breast cancers involves mutated forms of BRCA1 or BRCA2, which are involved in repairing
double-strand breaks within DNA
Genetic instability can cause defects in mitosis and aneuploidy (some cancer cells have an extra chromosome)
Gene amplification: extra copies of genes leads to excess of protein product; common in many cancers
Amplified segments detected by CGH typically as 2 types of cytogenetic changes
Double minutes; very small accessory chromosomes
Homologous staining regions (HSR) that do not band normally and contain multiple, amplified copies of
a particular DNA segment.
Amplified regions are known to contain extra copies of proto-oncogenes (myc, ras, EGFR)
Learn about the hallmarks of cancer
1. Self-sufficiency in growth signals
Do not need to be stimulated to grow -Ras mutation
2. Insensitivity to antigrowth signals
Mutations involving RB or TGF-b/Smad pathway
3. Evasion of apoptosis
Mutations in p53 or Bcl2 which promote cell survival
4. Limitless replicative potential
Mutation allows cell to maintain telomere length
5. Sustained angiogenesis
Blood supply & nutrients are vital for tumor growth
6. Tissue invasion and metastasis
Decrease in cell adhesion (E-cadherin mutation) &/or increase in protease production
7. Cancers induce help from the normal stromal cells in their local environment
8. They are defective in IC control mechanisms that normally stop cell divisions permanently in response to
stress (such as hypoxia) or DNA damage
An enabling trait: Genetic instability
Allows for the generation of genetic mutations to occur
Role of microRNAs and oncogenesis
Approximately 10% of miRNAs have been found to be either greatly overexpressed or down-regulated in
various tumors and are referred to as oncomirs.
One example is the 100-fold overexpression of the miRNA miR-21 in glioblastoma multiforme, a highly
malignant form of brain cancer.
Overexpression of miRNAs can suppress expression of tumor-suppressor gene targets
Whereas loss of function of other miRNAs may allow overexpression of the oncogenes they regulate.
Since each miRNA may regulate as many as 200 different gene targets, overexpression or loss of function of
miRNAs may have widespread oncogenic effects because many genes will be disregulated
Introduction about diagnosis, screening and molecular treatment of cancer
Retinoic acid and APL
The first treatment for a specific oncogene protein was used in acute promyelocytic leukemia.
The gene that encodes retinoic acid receptor (RARa) is fused with PML to form the PML/RARa oncogene.
Treatment with retinoic acid results in remission of the leukemia in most patients.
Herceptin & breast/ovarian cancer
Herceptin is a monoclonal antibody against the ErbB-2 oncogene protein, which is over-expressed in 25
30% of breast cancers - result of amplification of erbB-2 gene.
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It has also been approved for use in treatment of advanced colorectal cancer and head and neck tumors - is
cancer drug that acts through molecular targeting
Gleevec & CML
A specific inhibitor of the Bcr/Abl protein kinase (called imatinib or Gleevec) effectively blocks
proliferation of chronic myeloid leukemia cells
Imatinib also inhibits the PDGF receptor and Kit protein-tyrosine kinases.
Kit is an oncogene in about 90% of gastrointestinal stromal tumors.
Imatinib is also active against other types of tumors in which the PDGF receptor is activated as an oncogene.
CML - Gleevec sits in the ATP-binding pocket of Tyr residue in a substrate protein
This blocks onward transmission of a signal for cell proliferation and survival
Avastin - a monoclonal antibody that binds to and inactivates VEGF
Is an anti-angiogenic therapy that acts by attacking a tumors blood supply
Erbitux and colorectal cancer
Erbitux: is a monoclonal antibody against the EGF receptor of ErbB oncogene
Cancer drug that acts through molecular targeting
Gefitinib and Erlotnib for lung cancer - 2 small molecular inhibitors of EGF receptor which have shown activity
against some lung cancers
lung cancers that responded were those in which mutations resulted in constitutive activation of EGF receptor
tyrosine kinase
Know oncogene addiction
The sensitivity of tumors to inhibition of activated oncogenes is called oncogene addiction.
activated oncogene becomes a major driving force in tumor cell, and proliferation & survival may be dependent
on oncogene, whereas normal cells have alternative signaling pathways that can compensate if any one pathway
is blocked.
113

FA13 Cell Biology Final

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FA13 Cell Biology Final

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Molecular & Cell Biology Fall 2013 Semester

List of Objectives, Key Concepts and Diseases covered in Block 1

Know the following amino acids and their structures:

Cysteine forms disulfide bonds

Proline - Hydroxylated in mature collagen, disrupts secondary structure

Polar uncharged amino acids

Threonine - Phosphorylation, O-linked glycosylation

Positively charged amino acids

Glutamic acid Vitamin K dependent carboxylation, excitatory neurotransmitter in

Understand the levels of organization for protein structure

Understand clinical consequences of certain misfolded proteins and polypeptides

Must know the structure of the bases!!!

Know which bases are complementary with each other

- ceramide is composed of a sphingosine and a fatty acid.

Terpenes (examples: coenzyme Q- electron transport, dolichol- important carrier and

Presentation # 2: Cell and Organelles

o Rough endoplasmic reticulum (rER)*

o Recycling, early and late endosomes

Presentation # 3: Membrane Structure and Dynamics

Presentation # 4: Transport Across the Membrane

Presentation # 5: Biotechnology & Human Diseases

Know Duchenne and Becker Muscular Dystrophies

Tonofilaments Epithelium Keratinizing and

Vimentin Mesenchymal Fibroblasts, chondroblasts,

Desmin Muscle Striated and smooth muscle

Neurofilaments Neurons Most neurons

Glial filaments Glial cells Astrocytes (GFAP)

Know the basic structure of desmosomes and hemidesmosomes

epithelial cells and underlying

Know Epidermolysis bullosa simplex

Microtubule motors and movement

List of diseases you must know for block 1

2) Ibuprofen Ibuprofen and naproxen are NSAIDs that reversibly

Na and K ATPase blockers that elevate intracellular Na

4) Oubain An inhibitor of sodium/potassium pump. Exposure to

5) Colchicine (anti-gout); INHIBIT MICROTUBULE GROWTH

6) Taxol and griseofulvin STABILIZE MICRTUBULES

7) Cytochalasin B Binds to barbed ends and blocks elongation. This can

8) Phalloidin Binds to actin filaments and prevents dissociation. It can be

Presentations # 3: Signal Transduction

Must know Gs signaling pathway in detail !!!!!

Know how cholera toxin poisoning disrupts Gs signaling

Know glucagon signaling pathway

Three major signaling branches of receptor tyrosine kinase

MUST know Noonan Syndrome

o AKT and forkhead (FOXO)

AKT phosphorylation of FOXO sequestered by molecule 14-3-3 and be inactive form

o Role of phosphatase PTEN

Know the term Dominant Negative Mutation

Know Notch signaling pathway - latent gene regulatory protein

Feedback inhibition of NK-kB

Presentation # 2: Extracellular Structures and Cell Junctions

o Must know mode of action for penicillin

Know structure and function of Tight (Zonula Occludens)

Know structure and function of Gap Junctions

Know Charcot-Marie-Tooth disease

ZO- zona occludens; ZA- zona adherins; D- desmosome

Macule adherentes (MA - desmosomes)

Presentation # 4: The Nucleus, Chromosomes, and the Human Genome

Traffic of RNA between the nucleus and the cytoplasm

oKnow Fragile X Syndrome

Must Know Diseases and Disorders For Block 2

Must Know Drugs For Block 2

Presentation # 5: DNA Structure, Replication and Repair