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Bio. 421
Molecular Cell Biology
Spring 2009
Instructor: Dr. Mary Ogilvie
Office: S203F
Phone #: 321-3437
email: mogilvie@cbu.edu
Book: The World of the Cell, 6th ed.: Becker, Kleinsmith, Hardin
Grade will be based on:
4 mid-semester exams ------------------ 100 pts each
-You will be permitted to drop the lowest grade if you miss no more than three lectures and no more than one lab.
1 final (comprehensive) ------------------ 150 Pts.
4 Quizzes ------------------------------------- 20 pts each
1 journal presentation ------------------ 50 Pts.
Total ------------------------------------------- 580 Pts.
Make-up Exams: Should you not be able to attend lab the day of an exam, please call me as soon as possible. You will be asked to present a Drs. Note in order to take a make-up exam.
Grading Scale:
A = 90 -100 D = 60 -69
B = 80 -89 F = Less than 60
C = 70 79
Course Goals:
1. To identify the function of organelles within the eukaryotic cell and to understand how the cell must integrate the functions of all organelles to operate.
2. To appreciate the evolutionary relationship between prokaryotic and eukaryotic cells.
3. To identify the major differences between normal and cancer cells and appreciate the influence that genes have on cellular transformation.
5. To understand the importance of ligand-receptor interactions in the communication of a cell with its environment.
6. To learn some of the roles that genes play in the structure and physiology of a cell.
Cell Presentation Evaluation Sheet
Names of Speakers _________________ and _________________
Topic____________________
(15) Powerpoint slides ________
Comments:
(15) Clarity of talk ________
Comments:
(5) Handled questions ________
Comments:
(10) Degree of complexity ________
Comments:
(5) References ________
Total Pts = ___________
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Cell Biology Course Outline
"The Birth of Complex Cells"
A. The Endosymbiont Hypothesis
1. What is it?
2. What is the strongest evidence for it?
B. Suggested Modification by deDuve
-Host cell had certain eukaryotic characterist. before acquiring endosymbionts
C. Assumptions about Phagocytic Host
1. Much larger than prey
2. Host cell was a heterotroph
a) Digestion by secreted enzymes
b) Stromatolite colonies = oldest fossils
-Support both heterotrophs and autotrophs
3. Host cell lost ability to manufacture cell wall
-Stromatolite colonies provided protection for a cell without a cell wall
4. Surrounded by flexible membrane
a) Evidence: Giant Prokaryotes today with highly convoluted membranes
b) May have led to deep pockets formed with enzymes inside
5. Network of compartments since membranes have self-sealing properties
b) Deep pockets sealed off
i. Specialization of intracellular pockets developed
ii. Development of endomembrane system
6. Primitive internal skeleton
-required to support cell without cell wall
D. Host Cell's Acquisition of Mitochondria
1. May have saved it from extinction
2. May have driven other cells to extinction
E. The Oxygen Holocaust
1. Photosynthesis by Cyanobacteria increased levels of O2
2. Obligate anaerobes died
3. Survivors:
a) Found O2-free environments.or
b) Developed ways of living in the presence of O2
5. Peroxisomes may have been endosymbionts 1st
a) More primitive than mitochondria
b) Removal of toxic O2 biproducts
c) Gave mitochond. time to develop from internalized aerobic prokaryote
G. Gene Transfer from Organelle to Nucleus
1. Not unusual
2. Problem: proteins must be transported from cytosol to specific organelle
3. Solution: Address tags
a) Recognized by specific organelle
i. Peroxisome = SKL
ii. Mitochondria = ~70 AA at N-terminus
b) Bind receptor on organelle
c) Removed in organelle
4. Molecular Chaperones
a) Bind proteins as they are made (keep proteins partially unfolded)
b) Hsp 70
i. Mito. protein chaperone
ii. ATPase activity for:
-Translocation of protein into mitochondria
-Release from chaperone
Chapter 1
Measurements
1. nanometer
2. micrometer
3. Angstom = 0.1nm
4. Have some idea of the size of cells and organelles
5. Dalton = Unit of mass = one H atom
Chapter 3 Macromolecules of Cell
A. Proteins
1. Informational molecules
2. Be familiar with general properties of Amino Acids
3. Primary Structure
a) Platelets require fibrinogen (Fg) for cross-linking
b) Fg binds GP IIb-IIIa on platelet (via RGDS)
c) RGDS peptides cause inhibition of platelet aggregation
-Compete with Fg for binding to GPIIb-IIIa
d) Make peptides w/ amino acid substitutions, test for inhib. of aggregation:
i. RGES = no inhib.
ii. RADS = no inhib.
iii. KGDS = less inhib. than with RGDS
iv. RGDA = complete inhib.
-#4 position is promiscuous
4. Secondary Structure
a) Alpha Helix
-Keratiin
b) Beta Pleated Sheet
- Collagen
5. Tertiary Structure
a) Non-repetitive nature of chain
b) Important bonds?
c) Intrachain disulfides formed by cysteines
6. Quaternary Structure
a) Interchain disulfides
b) What does reducing agent do?
Chapter 7 Membranes
I. Membrane Functions
1. Delineation and Compartmentalization
2. Localization and Organization of Function
3. Regulation of Transport
4. Detection of Signals
5. Transmission of Signals
6. Cell to Cell Communication
a) Gap junctrions
b) Plasmodesmata
II. Fluid-Mosaic Model
A. Lipid Bilayer = Fluid
B. Proteins = Mosaic
1. Integral
a) Amphipathic
b) Bacteriorhodopsin
i.Multipass protein
ii. Function?
2. Peripheral
a) Don't penetrate bilayer
b) Remove from membrane easily
-Change ionic strength of buffer
3. RBC Membrane
a) Intregral
i.Glycophorin
ii.Anion Channel Protein
-Exchange of Cl- and HCO3- in blood
-Multipass
b) Peripheral proteins form shell under membrane
i.Spectrin tetramers
ii. Ankyrin
iii. Actin
b) RBC membrane different from other cells:
i. Not as heterogeneous
ii.More extensive attachment to cytoskeleton
C. Carbohydrates and Membrane
1.Form glycocalyx
a) Glycolipids and glycoproteins
b) Functions?
2. Glycoproteins
a) Zeta Potential due to sialic acid
i.Decreases viscosity of blood
ii. RBCs repelled
b) O-linked carbo. bound to Serine or Threonine
c) N-linked carbo. bound to Asparagine
d) Glycosylation in RER and Golgi
e) Demonstrate asymmetry of glycoproteins in membrane
i. Galactose oxidase oxidizes galactose & galactosamine
ii. 3H-Borohydride reduces sugar
-addition of 3H to galact.
iii. Extract membrane proteins
iv. SDS-PAGE
-Proteins separate by molecular weight
v. Autoradiography = bands on film
vi. If lyse cells before exposure to GO and 3H-BH
-See same pattern of bands on film
D. Lipids
1. Lipid/protein ratio varies in different membranes
2.Triglycerides
a) Glycerol + Fatty acids
- Saturated and unsaturated FA
b) Membrane Phospholipids
i. Asymmetry in membrane
ii. Flip flop very slow in artificial membrane (liposome)
iii. Faster in natural membrane....Why?
iv. Asymmetry of phospholipids established during biogenesis
3. Cholesterol
a) Functions
i. Affects membrane fluidity
ii. Decreases permeability of membrane to small polar mol.
iii. Increases membrane stability
b) Orientation in membrane?
c) Why is this considered a temperature buffer?
E. Membrane Fluidity
1.Sat. F.A.
a) Produce higher transition temp.
b) Tend to be solid at room temp.
2. Unsat. F.A.
i. Lower transition temp.
ii. Double bonds make kinks in membrane
-Less stability
iii. Tend to be oils at rm. Temp.
iv. Double bonds
3. Homeoviscous adaptation in ectotherms
i. Compensate for temp. change
ii. E.coli = expression of desaturase at low temp.
iii. .Arctic fish have increase unsat. FA
4. Homeotherms
-Hibernating animals increase unsat. FA
5. FRAP
a) Use a fluorescent tag on lipids
b) Bleach with laser then watch "hole" fill in
c) Calculate rate of lateral diffusion
c) Why a different diffusion rate in liposomes and cell membranes?
D. Protein Mobility in Membrane
1. Lymphocytes + lectin + Fluor. tag
-Stimulate mitosis
2. Even distribution ---Patching--- Capping---- Endocytosis
3. What property of lectins makes patching and capping possible?
4. Aggregation of lectin receptors in membrane
a) Aggregation of protein kinases on inside of membrane
b) Initiate signal
V. Membrane Asymmetry
A. Carbohydrates
-Always face outwards
B. Proteins = No flip-flop
C. Lipids
1. Different phospholipids in different leaflets
2. Where is each type distributed?
Chapter 4 Properties of Cells
I. Limit to Cell Size
A. Surface/volume ratio
1.Volume increases with cube of cell's dimensions
2. S.A. increases with square of cell's dimensions
3. Be able to demonstrate this concept
4. Larger cells have smaller SA/vol. ratio:
a) Must compensate
b) Need much membrane for exchange of molecules
B. Compensation by Eukaryotes
1. Compartmentalization due to Organelles
2. Infolding of membrane to increase surface area
II. Cytoplasm vs. Cytosol
-What's the difference?
Chapter 12
I. Subcellular Fractionation
A. What are the Factors Determining Rate of Movement?
B. Preparation of Sample in cold, isotonic buffer
1. Homogenize.or
2. Sonication
C. Differential Centrifugation
1. Sedimentation rate
-Svedberg units = Sedimentation Coeff.
2. Density Gradient Centrifug.
a) Use 0.25 M sucrose
b) Centrifuge 1st to remove large cellular debris
c) Remove supernatant and spin again
-at increased speed and time
3. Equilibrium Density Gradient Centrifugation
II. Endomembrane System
A. Components
1. SER, RER
2. Golgi
3. Lysosomes
4. Nuclear envelope
5. Plasma membrane
6. Vesicles
B. Endoplasmic reticulum
1. How does content compare to plasma membrane?
2. Microsomes---what are they?
3. Appearance of RER vs. SER?
C. Smooth E.R. Functions
1.Hydroxylation of Phe -->Tyr
a) Series of reactions
b) NADPH + H+ passes electrons to electron carriers in SER
i. P450 final electron carrier
ii.Passes electrons to oxygen
iii. Oxygen reduced to water
d) Mono-oxygenases pass oxygens to Phe
i. Phe oxidized to Tyr.
ii. Also called mixed function oxidases
e) PKU = Missing mixed func. oxidase
i.Can't eliminate Phe
ii. Mental retardation by 12 mo. if no change in diet
2. Detoxification by Mixed Function Oxidases
a) Toxins made more hydrophilic by hydroxylation
-Easier to eliminate
b) Drug detoxification, i.e. Barbiturates (phenobarb. in rats)
i. See more SER
ii. See more mixed function oxidase activity
iii. Rebound effect
c) Broad specificity
- Increases metabolism of other drugs
3. Glycogen metabolism
a) Role in glycogenolysis
i.Activate via Epinephrine or glucagon?
ii. Receptor shape change------G-protein activation-----activ. Adenylate cyclase -----cAMP made------Cascade ----Glycogen phosphorylase----Phosphoglucomutase------Glucose-6-phosphatase
b) Glucose-6-phosphatase in SER membrane
i. Removal of phosphate
-Why necessary?
ii. Leads to increase or decrease in blood sugar?
4. Phospholipid synthesis and Arrangement
a) Three amphipathic biosynthetic enzymes in ER membrane
-Also in RER
b) Synthesis first in cytoplasmic leaflet
i. Addition of FA to glycerol-3-phos.
- form phosphatidic acid
-by acyl transferase
ii. Removal of phosphate
-by phosphatase
ii. Addition of head group using cpmponents of cytosol
-by phosphotransferase
c) Flippases
i. Catalyze flip-flop
ii. Phospholipid head group specific
iii. How might these account for membrane lipid asymmetry
D. Movement of P-lipids from ER to Other Organelles
1. Endomembrane system
-Vesicles bud and fuse
2. Mitochondria, chloroplast, peroxisome
a) Phospholipid Exchange Proteins
b) Water-soluble carriers of specific P-lipids
-Pick up P-lipid from one membrane and release into another
E. Rough E.R.
1.Types of proteins are made on RER
a) Membrane-bound
b) Secreted
c) Lysosomal
2. Signal mechanism (Chapter 20)
a) Signal peptide on nascent protein
-Characteristics?
b) Signal Recognition particle (SRP)
i. Attaches to:
- Signal peptide
- Ribosome (stops translation)
- SRP receptor on ER
c) SRP and SRP receptor bind GTP
i.Translation restarts
ii. Translocation of signal protein to pore protein
d) What does GTP hydrolysis do?
e) Signal peptidase
i. Cleaves signal peptide
ii. After translation complete
f) Pore protein dissociates
g) Folding of protein in lumen
-also quaternary structure
3. RER Retention Signal
a) KDEL on RER resident proteins
b) Receptor in cis Golgi binds KDEL
c) Recycling back to RER in vesicle
4. Transmembrane protein insertion
a) Stop-transfer peptide
i.hydrophobic
ii. Fed into pore protein
- stops in membrane
iii. C-terminus remains on cytosolic side of ER
iv. Signal peptide cleaved after synthesis of polypep.
b) Departure of pore protein --> diffusion
c) Multi-pass proteins
i. Start and stop signals
-H'phobic
2. Start = sends polypep. domain into lumen
3. Stop = sends polypep. domain into cytosol
(Chapter 12)
5. Core glycosylation (N-linked sugars)
a) High mannose core added to dolichol phosphate
-Sugars found in core?
b) Translocation by flippase
-from cytosolic to luminal leaflet
c) Added as an entire unit to Asn (N-linked)
-by oligoscch. transferase
d) Removal of 3 glucose and one mannose
-by glucosidase and mannosidase
E. Golgi
1. Structure
a) Stacks of 4-8 cisternae
b) Cis Golgi
i."Transition" vesicles from RER to Cis
-Coatomer aids in budding
ii. KDEL receptor found here
c) Medial = "shuttle" vesicles between cisternae
d) Trans Golgi
i."Secretory vesicles" from trans to p.m.
ii. Clathrin aids in budding
e) Each face of Golgi is biochemically distinct
2. Terminal Glycosylation
a) Non-Lysosomal Proteins
i. alpha Mannosidase-1
-removes 3 mannoses from high mannose core
iii. Add Galactose, Sialic acid, etc.
b) O-linked sugars added to Ser and Thr
3. Protein sorting
a) Default pathway= Anterograde (What is Retrograde transport?)
i. Trace labeled amino acids injected into rabbit parotid gland cells
ii. What pathway does label take?
iii. Be familiar with graph
b) Constitutive Secretion
i. Continually occuring
ii. Examples?
c) Regulated secretion
i. Zymogen granules accumulate next to membrane
ii. Proteolysis of some proteins in granules
iii. Exocytosis due to extracellular signal
- Increased Calcium in cytosol
4. Exocytosis
a) Movement of vesicle on microtubules
b) calcium-dependent
c) Calcium-dep. Protein on vesicle binds p.m. protein
d) ATP hydrolysis
e) membrane-fusion
G. Lysosome: Read (De Duve) Section
1. Identify types of acid hydrolases
2. Targeting of Lysosomal enzymes
a) GlcNAc Phosphotransferase
i. in cis Golgi
ii. Phosphorylation of lysosomal proteinsdue to recognition domain on protein
- glycosidase removes GlcNAc
b) alpha-Mannosidase-1
i. Substrate = non-lysosomal proteins
ii.Phophorylation of protein inhibits alpha-Mannosidase
c) Mannose-6-phosphate receptor
i. In Trans Golgi
ii. Accumulation of acid hydrolases into vesicles
-Vesicles bud
iii. Fusion of vesicles into late endosomes
iv. Proton pump in lysosomal membrane
- Decreasing pH ----- dissociation of proteins from receptor
v. Phophatases cleave phosphate from rest of hydrolases
-Activation of acid hydrolases
vi. M-6-P receptor recycled to trans Golgi in vesicles
3. Lysosomal Storage Diseases
a) I-cell Disease (genetic defect)
i. Acid hydrolases secreted from fibroblasts
ii. GlcNAc phospho-transferase missing
- Secretion by default
iii. Grow fibroblasts in culture with phosphorylated mannose
-What happens?
b) Tay Sachs (severe mental retardation)
i. What hydrolase is missing?
ii. Accumulation of gangliosides in lysosomes of brain cells
iii. Autosomal recessive disorder
5. Digestion by Lysosomes
a) Phagocytosis
i. Phagosome ?Secondary lysosome
iii. Residual body
b) Autophagy
i. Autophagic vacuole
ii. Destruction of organelles
c) Autolysis
i. Programmed cell death (apoptosis)
ii.Digit formation during development
d) Extracellular digestion
III. Peroxisome
1. Contains catalase and several oxidases
2. Degradation of Fatty acids
a) By oxidases
b) beta-oxidation of longer FA chains (>16 carbons)
i. CoA attached to FA in cytosol
ii. oxidase cleaves two carbons and attaches them to CoA
iii. Know equation
c) Generation of H2O2
i. Harmful to certain enzymes
ii.Broken down by catalase and coupled to oxidation of potentially harmful organics
-phenol, formaldehyde, alcohols
iii. Know equation
3. Degradation of amino acids
4. Not part of endomembrane system
-Biogenesis = Division of pre-existing perox.
5. Perox. proteins made on what types of ribosomes?
6. SKL is address tag
a) Universal among eukaryotes
b) Experiment to demostrate universal address tag
i.Gene for luciferase from firefly transfected into tobacco cells
ii. luciferase moves into peroxisome
iii. Add luciferin.Result? Conclusions?
IV. Receptor Mediated Endocytosis
A. General Process
1. Receptor + ligand
2. Clathrin-coated pit
3. Clathrin-coated vesicle
-Coat necessary for budding
4. Uncoating of vesicle necessary for fusion
-Uncoated vesicle
5. Fusion with Early Endosome
a) Early endo. Made in trans golgi network
-responsible for sending endocytosed material to right destination
b) ATP-dep. proton pump decreases pH
c) Uncoupling of ligand and receptor
-Usually receptors recycled to plasma memb. (Ex. LDL)
6. Fusion of early with late endosome
a) LDL digested
b) Cholesterol diffuses thru vesicle membrane
B. Alternatives for sorting of uncoated vesicles:
a) Fusion with late endosome
-becomes active Lysosome
b) Receptors degraded
-carried into late endosome (ex. EGFR)
c) Ligand carried to Trans Golgi
-enters endomemb. System
d) Transcytosis to different part of p.m.
-IgA synthesized on basal surface of epithelium
-Endocytosis followed by fusion with early Endo.
-Movement to apical surface of cell and release
C. Epidermal Growth Factor (EGF)
1. Ligand binding necess. for endocytosis
1. Both ligand and receptors degraded in lysosome
2. Down-regulation of receptors
a) Increased EGF leads to decreased receptors
b) One means of regulating the EGF response in the cell
Chapter 14 Mitochondrion
A. General Info.
1. Lgst organelle except for nucleus
-Size?
2. Different #s in different cells
3. Found in parts of cell where intense metabolic activity occurs
-Ex) Muscle myofilaments
B. General Structure
1. Outer membrane
a) Freely permeable to small molecules and ions
-Due to porins
b) Limit = >10,000 Daltons
2. Intermembrane space with small cytosolic molecules
3. Inner Membrane
a) Permeability barrier
b) Carrier proteins in membrane for what molecules?
c) Be familiar with Freeze fracture technique
i. P-face = protoplasmic
ii. E-face = exoplasmic
iii. See hydrophobic interior of membrane
iv. In mito., inner memb. has many more proteins than outer
-Why?
d) Cristae= increase surface area
4. Matrix
a) Many enzymes
-For what processes?
b) Ribosomes
c) Circular chromosome with 12 genes
d) ATP, ADP
e) RNA
C. F0/F1 Complexes on Cristae
1. Be familiar with the Chemiosmotic model
2. Be familiar with experiment to determine function of F0/F1
a) F1 = ATP synthase
i. 3 alpha subunits = holds ADP or ATP
ii. 3beta subunits = catalytic
b) Stalk
i. g, d, e polypeptides
ii. gamma rotates as protons pass thru
-May drive change in a:b configuration
c) F0 = Proton transporter
i. a, b, c polypeptides
ii. Hydrophobic complex
-transmembrane
iii. Channel for proton passage
D. Transport of Proteins into Mito.
1. encoded in nucleus
2. protein synthesized with transit sequence
3. proteins encoded in Mito. are almost always subunits of multimeric proteins
-assoc. in Mito. with proteins encoded in nucleus
4. Molecular chaperones (Hsp70) bind nascent protein in cytosol
a) keep in loosely folded state
b) escort protein to Mito.
5. ATP hydrolysis
a) Release of chaperone
b) translocation to pore.
6. Where is transit removed and by what enzyme?
7. Mitochondrial Hsp 70 binds
-ATP hydrolysis drives translocation into matrix
- " " causes release of Mito. Hsp 70
8. Hsp 60 binds and helps to fold protein in to final state
9. Signal Sequence
a) How is it revealed?
b) Hydrophobic, anchors into inner membrane
c) Rest of polypeptide moves thru membrane into intermembrane space
d) Signal may or may be removed
-final location of this protein?
e) Signal may not be removed
-final location of this protein?
correlated with level of cellular activity
Platelet Structure and Activity
I. Coagulation Cascade
1. Extrinsic pathway
2. Tissue factor from injured cells
a) binds calcium and Factor VII
b) Forms thromboplastin
3. Factor X converted to prothrombinase
-conversion of prothrombin to thrombin
4. Thrombin removes peptides from Fg ? Fibrin
-entraps blood constituents
II. Platelet Origin and Structure
A. Origin
1. Megakaryocyte in bone marrow
2. Release fragments of memb. and cyto.
B. Structure
1. No nucleus
2. Mitochondria, Glycogen granules present
4. Dense tubular system
5. Dense granules
a) ADP
b) Serotonin
6. Alpha granules
a) Thrombospondin
b) Fg
III. Platelet Activation
A. Adhesion
1. Collagen activates platelets
a)when subendothelium exposed
b) Collagen R = GPIb-V-IX
2. Leads to GPIIb-IIIa activation
a) Binds vWF in plasma
b) vWF binds subendothelium
c) von Willebrand's disease
-impaired adhesion
3. Platelet attachment to vessel wall
4. Leads to activation of GPIIb-IIIa
B. Aggregation
1. Primary = IIb-IIIa + Fg
2. Secondary = TSP+Fg+ IIb-IIIa
3. Aggregometer
a) Modified spectrophotometer
b) Use PRP + ADP
c) See two waves of aggregation
i. Primary (reversible)
ii. Secondary (irrevers.)
C. Shape Change
1. Cytoskeletal rearrangements
2. Formation of filipodia
-Increase in surface area
D. Secretion
-alpha granule and dense granule constituents
Chapter 16
Structural Basis of Cellular Info.
I. The Nuclear Envelope
A. Double membrane
1. Inner membrane
2. Outer membrane
a) Connected to RER
b) Often ribosomes associated
3. Perinuclear space
4. Nuclear pore = 3000 - 4000/nucleus
B. Nuclear Pore Complex
1. Structure
a) >100 polypeptides
b) Rings of 8 subunits on each side of envelope
c) Central granule = transporter
~9nm channel
d) Fibers extend outward into cytosol and inward into nucleoplasm
2. Passive Transport
a) Aqueous diffusion channels
b) Eight, 9nm channels around periphery
c) Small ions and mol. pass thru freely
d) Experiments with colloidal gold provided what info.?
e) Experiments with radioactive proteins
-What's the limit on protein size?
3. Active Transport (ATP-dependent)
a) Nuclear localization signals (NLS) on proteins
b) Steps in transporting NLS polypeptides into nucleus
i. NLS protein binding to importin in cytosol
-Fibers may serve as tracks
ii. NLS Protein + receptor bind cytosolic side of pore
iii. Transport thru transporter
-Probably requires GTP hydrolysis
-Requires Ran = GTP-hydrolyzing protein
c) Nuclear Export signal
i. Found in proteins within ribonucleoprotein complexes
ii. Binds exportins in nucleus.
d) How large is transporter thought to be?
II. Nuclear Fibers
A. Nuclear Matrix
1. Analogous to cytoskeleton
2. Questionable existence
-supporting evidence?
3. Possible functions:
a) Maintenance of cell shape
b) Organization of chromosomes
c) Tracks for newly synthesized RNA to nuclear pores
B. Nuclear Lamina
1. Lamins
2. Thin, dense layer around inner membrane
3. Possible functions
a) Support of nuc. envelope
b) Organization of chromosomes
C. Chromatin Fibers
1. Nuclear lamina may bind chromatin to nuclear env.
2. Bound via constitutive heterochromatin
a) Highly condensed
b) Simple sequence repeats
c) Centromere and telomere
-Telomeres appear to be point of attach.
3. What is Facultative heterochromatin?
4. What is Euchromatin?
C. Nucleolus
1. Fibrillar component:
a) DNA = genes for rRNA
b) RNA = rRNA being synthesized and processed
2. Granular component:
-rRNA + proteins ---> ribo. subunits
3. Nucleolar Organizer Region
a) Multiple copies of rRNA genes
b) How many NORs in humans?
c) Located at telomeres of more than one chromosome.
d) During mitosis
i. Disappearance due to packaging
ii. No rRNA transcription
4. Size of nucleolus correlated with level of cellular activity
Chapters 22 Cytoskeleton
I. Actin Microfilaments (MF)
A. Strucure
1. G-Actin ---->F-actin ----> rt. handed double helix
2. 7 nm diameter
3. Inherent polarity necessary for:
a) directionality
b) Indep. regulation of assem/diassembly at either end
4. ATP
a) Slowly hydrolyzed to ADP in F-actin
b) ATP not strict requirement for polymerization
-Evidence?
B. Regulation of Assembly/Disassembly
1. More rapid assem/disassembly at + end
2. Small G-proteins in plasma membrane
a) GTP-binding proteins
b) Activate kinases
i. phosphorylate phophotidyl inositol in membrane
ii. Production of polyphosphoinositides (poly PIs)
c) Poly PIs bind actin-binding proteins
i. Cap Z binds ends of MF..blocked
ii. Poly PIs remove Cap Z from MF
-Assemb./Disassemb. can now occur
3. Profilin = Releases G-actin from thymosin beta 4
a) Transfers G-actin to + end of MF
b) Assembly favored
C. Cell Cortex
1. 3D meshwork of actin and actin-assoc. proteins
-Adjacent to p. m.
2. Function:
i. Cell surface rigidity
ii. Shape change
ii. Locomotion
3. Gel-sol properties depend on degree of x-linking
a) Filamin = dimer of ID polypeptides
b) What happens to cell cortex if more filamin is present?
4. Cytochalasins
i. Alkaloids from fungi
ii. Bind + end of MF and prevents further assembly
iii. Favors disassembly
iv. What does it do to fibroblasts in culture?
D. Functions of Actin MF
1. Cleavage furrow
2. Locomotion of cells
3. Cell cortex
4. Cell shape
5. Contraction in muscle cells
II. Microtubules
A. Structure
1. Diameter = 25 nm
2. Tubulin subunits
i. Dimer of alpha and beta subunits
ii. Both bind GTP or GDP
3. Tubulin --? Oligomers---->13 Protofilaments ----> Microtubule
4. Inherent polarity
-Assembly & disassembly favored at + end
5. Gradual hydrolysis of GTP
-More affinity between tubulins and GTP than GDP
B. Three phases of assembly
1. Lag phase
i. Nucleation of oligomers
ii. Slower process
2. Elongation
i. Addition of tubulins at either end
ii. Faster than lag phase
3. Plateau
i. Concentration of free tubulin limiting
ii. Assembly balanced with disassembly
C. Critical concentration (CC)
1.Conc. of tubulin @ which assembly balances disassembly
2. + end can grow or shrink faster than (-) end
3. CC for + end much lower than that for (-) end
4. Treadmilling demonstrated
-due to differences in CC between + and - ends
D Role of GTP
1. Necessary for polymerization
2. GDP will not support polymerization
3. Hydrolysis not necessary for assembly
- evidence?
E. MTOC
1. Function
a) MT assembly initiated
b) Anchor for one end of MT (generally the minus end)
2. Centrosome
a) Seen only at Interphase
b) 2 centrioles + pericentriolar material
c) Pericentriolar material = nucleation site for MTs
i. Helical rings of gamma-tubulin
ii. Assoc. with pericentrin
d) Minus end of MT attached to MTOC
3. MTOC determines orientation of MTs
(Chapter 23)
4. Nerve cells = Fast axonal transport
a) MTs used as tracks along axon
-Vesicles carried from cell body to axon terminus
b) Driven by MT-Assoc. Motor Proteins (Motor MAPs)
i. Kinesin carries vesicle along tracks via ATP hydrolysis
-Anterograde transport
c) Dynein carries vesicle retrograde
(Chapter 22, Contin.)
b) Cilia
i. Basal bodies at base of cilia = MTOC
ii. (-) end of MTs attached to basal body
F. Cochicine
1. Plant alkaloid
2. Binds tubulin dimers and adds onto + end
3. Destabilizes MT
4. Why used for karyotyping?
Not covered on Tets (in italicst:
G. MT Assembly/Disassembly at Mitosis (Chapter 17)
1. Centrosome replicates prior to Prophase
a) Daughter centrosomes move to opposite poles
b) Serve as poles to mitotic spindle
2. Kinetochore MTs grow out from poles
a) Capture kinetochore
-MTs bind by (+) end
b) Stabilized as kineto. Attached to two (+) ends of opposite MTs
3. Polar MTs (no chromosomes bound)
a) Grow from poles and overlap at equator
b) Proteins x-link polar MTs at equator
-Stabilize
4. Prometaphase = Chrom. movement to equator
a) Kineto. MT pull chrom. toward pole
b) Chrom. pushed away from spindle pole
5. Anaphase A
a) Separation of chromatids at centromere
-Partially do to Topoisomerase II
b) Chrom. pulled centromere 1st , to poles
i. Motor proteins = force for driving kineto. toward pole
-may use kinto. MT as tracks
c) Depolym. of kineto. MT at (+) ends
6. Anaphase B
a) Separation of spindle poles
b) x-linking proteins for polar MTs = motor proteins
c) Polar MTs slide apart
d) Polar MTs lengthen at (+) ends
e) Astral MTs
i. Attach (+) ends of MTs to cell cortex
ii. Motor proteins associated
iii. Exert outward pull on spindle
III. Intermediate Filaments (IF)
A. Gen. Info.
1. Structure
a) Diameter = 8 - 12nm
b) Dimers (intertwined)
c) Two dimers align laterally
-Protofilament
d) Eight protofil. staggered for strength
e) Most stable and least soluble of the cytoskel. components
2. 6 different classes of proteins
-Single family of related genes
3. Cell-type specific
a) "IF Typing" with immunofluorescence
b) Cancer diagnostics
B. Function of IF:
1. Positioning of nucleus
a) Nuclear lamina contains lamins
-IF proteins
b) Phosphorylation of lamins
i. causes disassembly of lamina
ii. Nuclear envelope breaks down
2. Tension-bearing
a) Found in areas subjected to mechanical stress
b) Ex) Tonofilaments in desmosomes
3. May serve as scaffold for rest of cytoskeleton
a) Plectin attaches IF to MF and MT
b) Epidermolysis bullosa simplex
i. Mutant keratins
ii. or missing plectin
Not Covered on TEST (in italics):
Chapter 11 Junctions
I. Adhesive Junctions (3 types)
-Differ in kinds of associated cytoskel. filaments
A. Desmosomes
1. Button-like
2. Found in areas subjected to stress
3. Structure
a) Desmosome core
b) Desmocollins
c) Desmogleins
-Ca++dependent
d) Adhesion plaque
e) Tonofilaments (intermediate filaments)
B. Adherens Junction
1. Similar to desmosome?
2. Different from desmosome
a) Often forms an adhesion belt just under tight junction
b) Plaques not as dense
c) Cytoskeletal link = actin microfilaments
3. May synchronize contractile movement of adjacent heart muscle cells
C. Hemidesmosome
1. Connect cell to basal lamina.
2. Similar to desmosome
3. Distribution of sheer forces throughout tissues
II. Tight Junction
A. Tissues where commonly found?
B. Separation of fluid-filled compartments
1. Just under microvilli
2. Experiment with lanthanum hydroxide
a) High mol. wt.
b) Inject into blood vessels around Pancreatic acinar cells
c) Evidence for T.J. as a barrier
-Found only on basolateral surface
C. Structure (Freeze-fracture)
1. Protoplasmic face (P-face)
i.Ridges = TJ Elements
ii. Destroyed by trypsin
2. Exoplasmic face = shallow grooves
3. Two rows of proteins interlock
-one row from each cell
D. Prevents mixing between Apical and Basolateral surface
1. Blocks lateral movement in membrane
2. Lipids in cytoplasmic leaflet blocked
-What evidence?
3. Integral membrane proteins separated
i. Apical
-Na+/glucose symport
ii. Basolateral
-Basolateral permease
-Na+/K+ ATPase
II. Gap Junction
1. Connexon links
a) Transmembrane connexins
b) 6 in each adjacent membrane
2. Close:
a) Increased Ca++
b) Increased pH
3. Molecules of up to 1200 Daltons pass freely
4. Functions
a) Electric coupling of heart muscle
-Intercalated discs
b) Electric coupling of invertebrate neurons
-need no Acetylcholine
c) cAMP coupling
d) Embryonic cooperativity
Covered on TEST Extracellular Matrix
I. General characteristics
1. Few cells
2. Protein fibers
a) collagen
b) elastin
3. Gel matrix = Proteoglycans
4. Adhesive glycoproteins
a) Fibronectins
b) Laminin
B. Protein Fibers
1. Collagens
a) Secreted by fibroblasts
b) Lg. family of closely related proteins
-15 different types
c) Be generally familiar with collagen structure
-Fibers covalently cross-linked at lysines
d) Different types due to different alpha-chains
i. 25 different types of alpha-chains
ii. Type I = skin, bones, tendons, ligaments
iii. Type II = Cartilage
e) Provide strength to tissue
2. Elastin
a) Looser network of proteins
-Less cross-linking
b) Provides resilience when tissue distorted
c) Interwoven with collagen
d) Found most in skin, lung, intestine
3. What happens when we age?
C. Proteoglycans
1. Hydrated gel
a) Resists compression
b) Allows diffusion of molecules from capillaries
2. Family of glycoproteins
a) Integral membrane or
b) Covalently linked to phospholipids
c) High in carbohydrate
i. 1 - 200 carbo. chains per polypeptide
ii. Up to 95% carbo.
3. Glycosaminoglycans
a) Main carbohydrate components
b) Repeating disaccharides
c) Examples?
4. Aggrecan
a) A major component of cartilage
b) Over 100 GAGs
c) Mol. wt. = 3 million
5. How are proteo. linked to other ECM components?
D. Adhesive Glycoproteins
1. Adhesive for:
a) Proteoglycan + collagen
b) membrane+ membrane
c) Collagen + membrane
2. Two most common:
a) Fibronectin
b) Laminin
3. Fibronectins
a) Two non-identical polypeptides, covalently linked
b) Binding domains for various molecules
-What are some of them?
c) Family of similar but not identical glycoproteins
i.Encoded by same gene with at least 50 exons
ii.Alternative splicing from single HnRNA
-Get 20 different mRNAs
-Purpose?
iii.Examples:
- Plasma FN (soluble)functions?
- FN filaments (insoluble)
d) Cell migration requires FN
i. Migration of mesodermal cells in gastrula
-High concentration of FN in the pathway
ii. RGDS inhibits
iii. FN Ab inhibits
e) Maintenance of spindled cell shape due to adhesion to substrate
4. Laminins
a) Prominent in basal lamina
-Underlies epithelium and endothelium
b) More restricted in occurrence than FN
E. Integrins
1. Receptors for adhesive glycoproteins
2. Structure
a) Transmembrane heterodimers
b) Some promiscuous
-GP IIb-IIIa
c) Some specific
i. FN receptor on some cells
ii. Specificity due to alpha-chain
3. Different from other ligand receptors
i. Low affinity
ii. 10-100x more of them per cell
4. Many recognize RGD
- How can Fn receptors be purified using RGD?
5. Indirectly bound to actin microfilaments
6. Fibronectin (FN) Receptor
a) Binds Talin on cyto. side of membrane
b) Talin + vinculin
c) Vinculin + actin MF
d) Mitosis
i. Kinase activation
ii. Phosphorylation of FN receptor decreases affinity for talin
iii. Lose link between ECM and cytoskel.
iv. Cells round up
Chapter 10 Signal Transduction
I. Purpose of Complex Protein Interactions
A. Amplification of signal
B. Fast termination
II. Signaling in Platelet
A. Inside-Out Mechanism (IP3 Pathway)
1. General idea:
a) Agonist binds to receptor
-Signal sent inside
b) Signal leads to IIb-IIIa activ.
-On outside
2. ADP receptor
a) May be a Calcium channel (Na+/ Ca+Exchanger)
-Increase in cyto. Calcium
b) Coupled to a G-protein
-Activation of Tyr. Protein kinases (TPKs)
3. Activ. of phospholipase C by:
a) Increase in Calcium
b) Phosphorylation by TPKs
4) Cleavage of PIP2 in membrane
a) Generate DAG and IP3
b) IP3
i. Diffuses to dense tubular system
ii. Binds receptor
iii. Increase in cyto. Calcium
-IIb-IIIa is Calcium- dependent
c) Diacylglycerol (DAG)
i. Remains in membrane
ii. PKC activated when recruited to membrane to bind DAG
-Phosphorylates Ser and Thr on proteins
-Substrates not known
iii. Required for IIb-IIIa activation
d) DAG and IP3 are synergistic
B. Outside-In Mechanism
Cyclooxygenase Pathway
1. General idea:
a) Triggered by primary aggregation
b) Oligomerization of IIb-IIIa
-by Fg binding
2. PLA2
a) Integral membrane protein
b) Hydrolyzes phospholipids
-Production of arachadonic acid (AA)
3. Cyclooxygenase converts AA to PGE2
a) Aspirin irreversibly inhibits COX2
i. Acetylation of COX2
b) Secondary aggregation inhibited for ~ 10 days
i. Why 10 days?
ii. Why irreversible?
TxA2 Pathway + IP3 Pathway
1. PGE2 conversion to Thromboxane A2 (TxA2)
a) By Thromboxane synthase
b) Diffusion of TxA2 out of platelet
2. TxA2 binds membrane R
a) Conform. change in R
b) Activation of G-protein
c) Activation of specific PLC
3. PLC hydrolyzes PIP2
a) Production of IP3
i. Enters cytosol
ii. Release of Calcium
iii. Exocytosis = secretion of TSP, etc. ---> secondary aggreg.
b) Production of DAG
i. PKC activation
ii. Phosphoryl of proteins
-Required substrate unknown
iii. Also necessary for secretion
III. Mimics of DAG and IP3
A. Phorbol esters mimic DAG
a) Tumor promoters
b) Activation of PKC leads to platelet activation
c) Is PKC involved in transformation?
i. Use phorbol esters to study cancer
-tumor promoters
ii. Fibroblasts in culture + phorbol esters
-up-regulation of PKC
-Cells grow unattached to substrate
B. Calcium Ionophore mimics IP3
1. Increase in cytosolic Calcium due plasma membrane channels
2. Platelet activation
C. Measure degree of platelet secretion with labeled serotonin
1. Neither produces 100% aggregation by itself
2. DAG and IP3 synergistic
IV. Endothelium
1. PGI2 due to release of ACh
-Parasympathetic impulse to blood vessel endothelium
2. Prostacyclin synthase in endothelium
a) Production of prostacyclin I2
-from PGE2
b) Diffusion to smooth muscle
i. Decreased calcium in muscle
ii. Muscle relaxation leads to blood vessel dilation
3. Inhibits platelet aggregation
-How?
V. Intracellular Calcium in Cell Signalling
A. Free Calcium
1. ~0.1mM in resting cell
2. Increase to ~1mM in activated cell
3. Examples of cell activation due to free Calcium:
a) Muscle contraction
b) Exocytosis
c) Glycogenolysis
B. Calmodulin
1. Small protein that binds 4 Calcium molecules
2. Causes activation
-mostly of kinases
3. Activates Calcium/ATPase pump
a) Plasma membrane
b) Regulation of calcium levels in cytosol
4. Calcium/CaM Kinase II
a) In axon terminus of neurons
b) Voltage-gated channels cause influx of Calcium
i. Rise in free calcium
ii. Triggers secretion of catecholamines
c) Calcium also binds CaM
i.. Calcium/CaM activates CaM Kinase II
ii. Phosphorylation of tyrosine hydroxylase leads to activation
-rate-limiting enzyme in catecholamine synthesis
VI. Nitric Oxide, the X-rated Molecule
A. Synthesis in endothelial cells:
1. ACh released by Parasympathetic nerves
2. ACh + R leads to G-protein activ.
3. PLC activation causes IP3 release
a) Calcium release
b) Calcium binds CaM
c) Ca /CaM binds and activates NO synthase
4. NO Synthase
a) Deamination of Arginine
b) Generates Citrulline + NO
B. Effect on Smooth Muscle
1. NO diffuses to smooth muscle
2. NO binds and activ. guanylate cyclase
3. Generation of cGMP
a) Activation of protein kinase G by cGMP
b) cGMP is rapidly converted to GMP so short-lived
4. PKG phosphorylates muscle proteins
a) Muscle relaxation
b) Blood vessel dilation leads to penile erection
C. Nitroglycerine
1. Given for angina
2. Converted to NO
3. Dilation of blood vessels to heart
D. Short half-life
1. Acts locally since only transient
2. Converted to nitrates/nitrites
VII. Receptor Tyrosine Kinases
1. Often act as dimers in membrane
2. Autophosphorylation of Tyrosines
-allows attachment of adaptor protein (GBR2)
3. SOS binds GBR2
a) SOS = Guanine nucleotide release protein
b) Activ. of small, monomeric G-protein = Ras
4. Ras releases GDP and binds GTP
a) Activation of Raf-1 (kinase)
b) Leads to a kinase cascade
5. Map kinase (MAPK) activated
a) Enters nucleus
b) Phosphoryl. of nuclear proteins, Jun and Ets
i. Jun binds Fos (another nuclear protein)
ii. Formation of AP-1
-transcription factor
c) Activation of early genes by Ets and AP-1
- Primarily myc and jun
6. Myc activates delayed genes:
a) cyclin and cdck
i. Phosphoryl, of Rb
ii. Rb dissociates from E2F
b) E2F binds genes that make cell progress into S-phase
Chapter 17 Cellular Aspects of Cancer
I. Neoplasms
A. Benign Tumors
1. Cells often look normal
2. Surrounded by fibrous capsule
3. When is this life-threatening?
B. Malignant Tumors
1. Monoclonal
2. Less differentiated
3. Phenotype different from normal cells
C.Malignant vs. Benign cells:
1. Malignant cells are Invasive
a) Produce degradative enzymes
i. Penetrate surrounding tissues
ii. Small clumps break off into blood vessels, etc.
b) No contact inhibition.
i. Grow over and under tissues
ii. Normal cells obey an anti-proliferative factor
-via TAPA on membrane surface
2. Malignant cells are capable of Metastasis
a) Tumor cells travel to distant sites in body
b) What are some possible means of transport thru body?
3. Vascular metastasis in malignant tumors
a) Angiogenin attracts endothelial cell growth
b) capillary growth around tumor
D. Tumor Classifications
1. Blastoma 4. Lymphoma 7. Adenocarcinoma
2. Carcinoma 5. Leukemia
3. Sarcoma 6. Melanoma
C. Cell Culture Characteristics
1. Normal cells in culture:
a) Require 5 - 20% fetal calf serum
- Why?
b) Adhere to proteins produced on glass or plastic surface
i. Must adhere for growth
ii. Spindled appearance
c) Limited # of cell divisions
~60 in embryonic cell
d) Contact inhibition
2. Transformed Cells
a) Decreased growth factor requirements
-Often make their own
b) Loss of dependence on adhesion for growth
-Spherical in appearance
c) Loss of contact inhibition.
d) Increased mobility of membrane proteins
-Why?
e) Altered transcription
-Only 3% of mRNA is tumor cell-specific
f) Immortal
II. Ras Pathway
A. Taken by most Growth Factors
-Malfunction in pathway may cause cancer
B. Six steps that can become cancerous:
1. GF binds receptor
2. Receptor tyrosine kinase activity
a) Usually dimerizes with a 2nd receptor
b) Autophosphorylation
3. Ras activation
a) Adaptor proteins bind receptors
-GRB2
b) Sos = guanine nucleotide exchange protein
i.Binds GRB2
ii. Passes GTP to Ras
c) Ras is now active
4. Activation of kinase cascade
a) Ras binds and activates Raf
b) Raf phosphorylates MEK
c) MEK phosphoryl. MAPK
5. MAPK enters nucleus
a) Phosphoryl. of Jun and Ets
b) Jun and Ets activate "early genes"
i. Encode transcription factors
-Myc, Jun, Fos
ii. Activation of "delayed genes"
-Encode E2F = Activate genes for moving cells into S-phase
6. Other delayed genes encode Cdk and cyclin
a) Phosphoryl.of Rb so it can't bind E2F
b) E2F no longer inhibited by Rb
III. Three Main Types of Genes Can Play a Role in Transformation
A. Oncogenes
B. Tumor suppressor Genes
C. DNA Repair Genes
IV. Oncogenes
A. Proto/Oncogene Nomenclature
a) Oncogene = italicized, 3 letters
b) Proto-oncogene = "c" myc
c) Wide distribution in eukaryotes
-More than 50 identified
B. Four Mechanisms for Transforming Proto to Oncogene
1. Point Mutation
a) Qualitative change in gene leading to altered function of protein
b) Ha-ras gene causes some bladder cancer
i. Harvey sarcome virus
ii. Transform. due to a single pt. Mutation
-GGC (Glycine) to GTC (Valine)
iii. How was this determined?
iv. Ras = Constitutively activated
2. Local DNA Rearrangements
a) Often due to Oncogenic viruses
i. Insert DNA into host genome
ii. Human papilloma virus= Assoc. with cervical cancer
iii. Hepatitis B = Assoc. with liver cancer
b) Virus may not carry oncogene
i. Viral DNA insertion into proto-onco.
ii. Causes either deletion of base-sequence exchanges
-between proto. and surrounding genes
3. Gene amplification
a) Increase # of copies of proto.
b) Overproduction of protein
-Quantitative change in gene
c) Some lung cancers
i. Amplification of c-myc
ii. Increased production of Myc
-over-activation of delayed genes
d) Example: Non functional p53 gene
i. No repair of strand break in chromatid
ii. Chromatid may have no telomere
iii. Replication leads to two chromatids without telomeres
iv. Telomeres fuse and then pull apart so that one chromatid ends up with both copies of gene
4. Chromosomal translocation Ex) Chronic myelogenous leukemia
a) Philadelphia chromosome in 85% of cases
b) Reciprocal translocation of chrom. 22 to chrom. 9
c) Breakpoint at abl = tyrosine kinase
d) Slow tumor progression at 1st
e) Eventually blast cell crisis and death
f) Somatic mutation
-Only in granulocyte stem cells
g) Each person's breakpoint is slightly different
i. Monoclonal
ii. Within cells from the same tumor, sequence of abl is the same
C. Most Oncogenes in GF Signalling Pathways
1. Growth Factors
a) Necessary for growth of cells
i. Grow 3T3 fibroblasts in 10% fetal calf serum
ii. Cells stop growing at saturation density
-Cell density proportional to amt. of serum supplied
iii. Cells grow again when:
-Add more fetal calf serum or..
-Replace serum with EGF and Transferrin (iron carrier)
b) Autocrine secretion
i. 3T3 cells transformed by simian sarcoma virus
ii. Produce own G.F. similar in activity to PDGF
iii What type of GF?.
iv. Competitive assay
-SSV-conditioned media + labeled PDGF + 3T3
-Result?
v. Characterize GF with inhibitory assay
- Add Ab against PDGF
-Result?
vi. Conclusion: GF is some form of PDGF
vi. Sequenced sis oncogene
-Very similar to gene for beta-subunit of PDGF
- Source of gene was from virus rather than host cell
2. Receptor Alterations
a) Receptor Tyr. kinase
b) erb-B oncogene in erythroblastosis virus
i. Cancer in chickens
ii. Erythroblast cell tumors
c) Sequence erb-B oncogene
i. Gene similar to EGF receptor
ii..Modified cyto -plasmic domain
-Constitutive activation
iii. Same trans-membrane domain
iv. Very little exoplasmic domain
-No binding of EGF possible or necessary
d) Constitutive Tyr. kinase activity
3. Altered G-proteins
a) Most GF work through Ras pathway
b) Oncogenes encoding mutant Ras are the most common oncogenes in human cancers
c) Ras retains GTP instead of hydrolyzing it
-Constitutively on
4. Altered Protein Kinase Genes
a) Several cancer viruses have raf oncogenes
-Constitutively on
b) Other kinases in pathway are also potentially oncogenic
5. Altered Genes for Transcription factors
a) Continuous production of TF
b) Continuous activation of genes for cell proliferation
c) Burkitt's Lymphoma
i. Due to Epstein Barr virus leading to somatic mutation
ii. Children in western Africa
iii. Translocation of piece of chrom. 8 to 2, 14 or 22
-Chrom. 8 = c-myc
-Chrom. 2, 14, 22 = sites of immunoglobulin (Ig) genes
iv. c-myc now controlled by Ig gene regulatory sites
-always "on" in lymphocytes
v. Overexpression of Myc
6. Altered Cdk-Cyclin Expression
a) cdk4 = cdk oncogene
-Amplified in certain sarcomas
b) CYCD1 = cyclin oncogene
i. Overexpressed in several types of cancer
ii. Ex) Some breast cancer
c) Constitutive phosphoryl. of Rb so it comes off E2F
-E2F continually activates S-phase genes
V. Tumor Suppressor Genes (p53 Gene)
1. Most commonly mutated gene in human tumors
2. Guardian of the Genome
a) Expose cells to UV to cause DNA mutations
i. Cells have increased levels of p53
ii. Damaged DNA triggers a decrease in degradation of p53
b) p53 activates:
i. Cell cycle arrest
ii. Cell death
iii. DNA repair mechanisms
3. Cell cycle arrest
a) p53 acts as a T. F. for p21 gene
-Binds as a tetramer to control element for p21
b) p21 is a cdk inhibitor
i. Suppresses passage of cell thru G0
ii. Gives cell time for DNA repair
4. Cell death through Apoptosis
a) Occurs if:
i. DNA can't be repaired
ii. Cell has passed G0
b) p53 generates reactive oxygen species (ROS) in Mito.
i. Release of cytochrome c
ii. Release of Apop. Inducing factors (AIF)
c) Cytochrome c and AIF activate caspases
d) Caspases cause:
i. Cell shrinkage
ii. Collapse of cytoskeleton
iii. Nuclear envelope breakdown
iv. Chromatin condensation
v. Degradation of DNA
5. Li-Fraumeni Syndrome
a) Inherit only one copy of p53
b) Why does cancer tend to arise when have only one good copy?
-Hint: p53 is tetrameric as a TF
6. Most p53 mutations not inherited
a) Mostly due to environmental exposure to:
i. Chemicals
ii. Radiation
b) Benzopyrene
i. In cigarette smoke
ii. Causes a point mutation in p53 gene
c) liver cancer
i. Protein made by Hepatitis B virus
ii. Binds p53 protein
-Prevents binding of p53 to DNA
VI. Defective DNA Repair Genes
A. Abnormal Nucleotides
1. Deamination
a) Due to chemicals
b) Cytosine to Uracil
-Uracil not a normal DNA base
c) During replication, polymerase places A at site normally occupied by G
2. Thymine dimers
a) Due to UV
b) Neighboring thymines are covalenly bound
c) Not directly mutagenic so what's the problem?
B. Repair Mechanisms for Minor damage
1. Base Excision repair triggered by minor distortions in DNA helix
a) Corrects single, damaged bases
b) Remove base with DNA glycosylase
c) Remove sugar and phos. with other enzymes
d) Replace with correct nucleotide
2. Nucleotide Excision Repair
a) For repair of thymine dimers, for example
b) Detects distortion on DNA helix
c) NER Endonuclease
-Makes nicks on either side of error
d) Helicase unwinds
-DNA fragment released
e) DNA Polymerase fills gap
f) DNA Ligase = seals DNA
g) Xeroderma pigmentosum
i. Defective nucleo. excision repair
ii. System requires 7 polypeptides
-Won't repair damage if any polypeptide missing
iii. Skin cancer due to minimal UV exposure
-Also Increase in cataracts
iv. Autosomal recessive
3. Mismatch Proofreading
a) Targets errors made during replication
b) Detects distortion
c) MutS binds at error
d) MutL scans strand for nick
e) Nucleotides removed from nick to point of error
f) Correct nucleo. replaced
g) Hereditary Nonpolyposis Colorectal Cancer
i. 15% of colorectal cancer
ii. Mutation in gene for MutS
C. Repair Mechanisms for Major Damage
1. Radiation damage often causes blunt end breaks
a) Wrong ends may be joined
b) Translocation
2. Replication occurs before repair complete
-Replication of unrepaired lesions
3.Inducible repair in bacteria
a) SOS Repair = Error-prone
b) Only induced if normal repair systems are saturated
c) High mistake rate
-mostly due to random insertions
d) Inducible systems in eukaryotes too
-Not well characterized
Chapter 14 Structural Basis of Cellular Info.
I. Repeated Sequences
A. Discovery
1. Cut DNA with RE
2. End up with fragments much shorter than expected
3. normally RE site every 3000 b.p.
4. Due to repeated sequences
B. Types
1. Tandem repeats
2. Interspersed repeats
II. Tandem Repeats
A. Characteristics
1. 10- 15% of mammalian genomes
2. Mostly 10 b.p. or less
3. Multiple tandem repeats
4. Usually not transcribed
6. Location
a) Centromere
b) Telomere
c) May occupy entire arm of a chromosome
7. Possible functions:
a) Protection of genes from degradation?
- Telomeres protect from harm of shrinkage
b) Selfish DNA?
8. At least 50 human genes with tandem repeats
B. Huntington's disease (HD)
a) Progressive neurological dysfunction
b) Gene sequenced
i. Called huntington gene
-function of gene product not known
ii. Tandem repeats of CAG within coding region of gene
-Normal = 11 ? 34 repeats
- HD = 100x
c) Extra repeats may interfere with unpackaging of gene
-No transcription
C. Fragile X (FX) Syndrome
a) Most common cause of inherited retardation
b) X-linked (at tip of long arm of X)
i. Males much more often affected
ii. Milder retardation in females
-due to random X-inactivation
c) FMR-1 gene
i. Sequenced but don't know function of gene product
-binds RNA in cytosol
ii. Repeats in non-coding region
iii. >200 Repeats ? Methylation of CpG island? no gene expression
d) CGG repeats
i. Normal = 30 - 50
ii. Premutation = 50 - 200
-No symptoms
-Mothers with >80 repeats often have FX sons
iii. >200 = FX
iv. Repeats tend to grow from generation to generation
e) Germ cells of FX males
i. Have only premutation from mom
ii. Somatic cells have full mutation
iii. Expansion must appear after germ cell line differentiates
D. Causes of Increased Tandem Repeats?
1. Aberrant replication?
a) When?
i. Meiotic division of germ cell precursor?
ii. Early embryo?
b) Overactive replication mechanism at repeats
2. Error in DNA repair?
a) Confused repair system
b) Overactive repair system at repeats
III. Interspersed Repeats
A. Characteristics
1. Repeats scattered throughout genome
2. 100 - 1000 b.p. per repeat
3. 1000's of copies throughout genome
-Similar but not identical
4. 24 - 40% of mammalian DNA
B. Alu Family Repeats
1. Why named this?
2. 5% human DNA
3. ~300 b.p. sequence
6. Similar to transposons
a) Retropositioning by reverse transcriptase
i. Makes DNA copies of mRNA
ii. Insertion elsewhere in genome
b) Probably why so many copies throughout genome
7. Function unknown
-Selfish DNA?
8. Used to study human origins
a) Find human-specific Alus
b) Trace point mutations in mitochondrial Alus
-Inherited thru female
c) Conclusion: Eve originated in Africa 200,000 years ago
8. Alu sequence similar to RNA component of SRP
-Function unknown
9. Will sometimes jump into an exon
-At least 3 genetic diseases associated with Alu jumps
10. Tpa 25
a) Insert in "tissue plasminogen factor" gene
i. In intron
ii. Does not harm Tpa protein
b) Fairly recent insertion
c) Obtain DNA from epithelial cells (cheek)
i. Use PCR to amplify Tpa gene
ii. Cut with Alu
iii. Run on gel
b) Homozygotes
i. No Tpa25 insert
-One larger (slow moving) band on gel
ii. Both Tpa25 alleles
-One smaller (faster moving) band on gel
c) Heterozygotes
i. 48% in Caucasian population
ii. Only one insert present in one allele
iii. One larger and one smaller band
Chapter 17
I. The PCR Revolution
A. Purpose = Amplification of a specfic DNA sequence
B. Reaction mixture
1. Single stranded DNA oligonucleotides
a) 15 - 20 nucleo.
b) Act as primers
2. DNA sequence for amplif.
a) Double stranded
b) Obtained by restriction digestion of DNA fragments
c) Do fingerprinting on gel
3. Four deoxyribonucleoside triphosphates
4. Polymerase
a) Taq = Synthesis of DNA in 5' ---> 3' direction
-Recognises 3' OH of primer
b) From bacterium (Thermus aquaticus)
-Live in hot springs
c) Optimal temp. = 72oC
d) Stable at higher temp.
5. Magnesium
C. PCR Process in Thermocycler
1. Step 1
a) Heat to 95oC
b) Separation of DNA strands
2. Step 2
a) Cool to 50oC
b) Allows primers to bind DNA fragments
3. Step 3
a) Raise temp. to 72oC
b) DNA synthesis by Taq
D. Result
1. Double DNA mol. with each cycle
a) 2n
b) "n" is the number of cycles
3. Usually 25 - 30 cycles
4. With each cycle more of the DNA will be of desired length
How so?
II. DNA Synthesis of Telomeres
A. Lagging strand (Discontinuous replication)
1. Syn. in Okazaki fragments since moving away from fork
2. DNA Polymerase requires RNA Primer
3. End up with shorter strand at end of chromosome
a) At 5' end of each lagging strand
b) Primer excision causes gap
-Cannot be filled in by normal DNA Polymerases
B. Solutions
1. Tandemly repeated DNA at Telomeres
a) Humans = TTAGGG
-10,000 b.p. at birth
b) May be long enough to last thru lifetime
c) May contribute to determining life span
d) Found in all vertebrates
2. Telomerases
a) In germ cells
b) Sperm cell DNA lengthens with age
c) Ribonucleoprotein enzyme
i. Reverse transcriptase
ii. RNA template
C. Hutchison Gilford Progeria
1. Children look old
-Short life span
2. Short telomeres at birth in fibroblasts
3. Exceedingly rare genetic disease
D. Telomerase Mechanism
1.RNA of Telomerase used as template
a) Contains 9 nucleotides
b) Complementary to tandem repeats
2. Reverse transcriptase lengthens Leading strand
a) 5' ---> 3' direction
b) Addition of 6 nucleotides
c) Repeats process several times
3. DNA Polymerase__
Chapter 17 Cell Cycle
I. Cell Cycle Times
A. Determine Time of Each
1. Generation time
a) Count under 'scope at intervals
b) Monitor with spectrophotometer.
2. S-Phase = DNA replication
a) 3H-thymidine added
-as a pulse
b) Autoradiography
c) Determine fraction of labeled cells
-multiply by generation time
3. M-Phase = Mitosis
a) Determine fraction in M
-Visually identify
b) Multiply by generation time
4. G2 Phase = Preparation for mitosis
a) 3H-thymidine added
b) Autoradiography
c) Look for 1st label appearing in mitotic cells
-Find fraction
d) Multiply by generation time
5. G1 Phase
a) Protein and RNA synthesis in prep. for S-phase
b) G0 at end of G2 = checkpt.
c) Subtract all other phases from generation time
B. Variations in Cell Cycle
1. Due mostly to G1 Phase
2. Non-dividing cells
a) Examples?
b) Stuck in G0
3. Non-dividing ---> Dividing
-Examples?
4. Continuously dividing
a) No stop at G0
b) Examples?
5. Xenopus laevis
a) Adult = 20 hr. cycle
b) Early embryonic cleavage
-Cell cycle < 1hr.
c) S ---> M ---> S
i. No G1 or G2
ii. No need for protein synthesis since stored in egg
II. Regulation of Cell Cycle
A. Checkpoints
1. How is cell artifically arrested?
2. G1 checkpoint (most important)
a) Can restrict cell growth here
b) Past this is pt.of no return
c) Controls cell's entry into S
3. G2 checkpoint
-Controls cell's entry into M
4. Spindle Assembly checkpt.
-Will stop if chrom. not attached to spindle
B. Early Evidence for Cell Cycle Regulation
1. Fusion of Hamster Cells = Heterokaryons
2. S + G1 = Chrom. duplication in G1
3. M + any phase = chromosome condensation in other cell
4. M + G1 = Chromatid condensation in G1
5. Conclusion = must be some trans-acting substance reg. cell cycle
-Substance made in one cell affects the other cell
C. Maturation Promoting Factor (MPF) History
1.Oocyte
a) Primary oocyte arrest in G2
-During embryo. develop.
b) Hormones stim. Meiosis at puberty
c) Activate protein synthesis
-Synthesis of MPF
2. Transfer cytoplasm from mature egg to primary oocyte
a) Primary oocyte begins meiosis
-Trans-acting
b) Due to hypothetical substance
i.MPF
ii. Found in all eukaryotes
3. MPF = two proteins eventually identified
a) Cdc2
b) Cyclin
4. Cdc2 protein identified
a) Yeast experiments (temp.-sensitive mutants)
i. 35 ---- > 37oC, yeast stuck in G2
ii. 20 --- > 23oC no problem
iii. Mutant gene encodes mutant protein
iv. Increased temp. disrupted shape of Cdc2
b) Cdc2 = "Cell division cycle"
c) Protein kinase
i. Always present at same concentration
ii. Only active when complexed with cyclins
iii. Belongs to a family of cdks = "cyclin-dep. kinases"
-Different cdk's expressed at different times in cell cycle
5. Mitotic Cyclin
a) Other 1/2 of MPF
b) Concentrations oscillate throughout cell cycle
i. Increase from G1 through early mitosis
ii. Peak mid-way at Metaphase
iii.Sudden decrease after Metaphase
-Due to digestion by proteases
-MPF activates proteases thru phosphorylation
6. Cyclin- Cdc2 Complex Activity for Mitosis
a) Chromosome condensation
i. Phosphoryl. of Histone-H1
ii. Phosphoryl. of condensin complex
b) Spindle assembly
i.Phosphoryl. of microtubule assoc. proteins
ii. MPF binds centrosome at mitosis
c) Breakdown of nuclear envelope
i. Phosphorylation of nuclear lamins
ii. Depolymerization of lamins
iii. Fragmentation of nuclear envelope
-forms tiny membrane vesicles
iv. Phosphatases necess. for reforming envelope
d) Activ. of Mitotic proteases
i. Degradation of cyclin at end of mitosis
-Important for exit from M
ii.Cdc2 recycled
iii. Proteases inactivated as MPF destroyed
E. Regulation of Mitotic Cdc2-cyclin
1. Effect of inhibiting kinase
a) Phosphoryl. of Cdc2
b) G2 arrest
2. Effect of activating kinase
a) Phosphorylation of Cdc2
b) ATP required for activity
3. Phosphatase
a) Removal of inhibitory phosphates on MPF
b) Positive Feedback loop
-MPF activates phosphatases
4. Autonomous Clock
a) External and internal environment affect cycle
b) Some molecules activate kinases others activate phosphatases
i. Ex) Starvation ---- > limited aerobic respiration
ii. Not much ATP for activating kinase
iii. Not much ATP for phosphorylation of phosphatase
-Cell division stops
F. G1 checkpt.
1. External growth factor
2. Signal = Ras pathway
-Production of G1 Cdk-cyclin
3. Phophorylation of Rb protein
4. Rb removed from E2F
a) E2F activates transcription
b) Synthesis of enzymes, etc. for S phase
Did not cover in 2002
G. Spindle Assembly Checkpt.
1. MPF phosphoryl. of Anaphase Promoting Complex
-APC
2. APC joins proteins to ubiquitin for degradation
a) Mitotic cyclin
i. Use non-degradable cyclin-B
ii. Cell does not exit Mitosis
b) Anaphase inhibitors
i. Prevent Anaphase
ii. maintain adhesion b/ sister chromatids
3. If chromatids unattached to spindle:
a) Mad2 released from kintetochore
b) Inhibits APC
c) Mitosis stops
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