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Bio. 421
Molecular Cell Biology
Spring 2001
Instructor: Dr. Mary Ogilvie
Office: S203F
Phone #: 321-3437
email: mogilvie@cbu.edu
Office Hrs: Mon. 1:00 - 4:00 Wed. 2:00 - 5:00
Tues. 2:00 - 5:00 Fri. 2:00 3:00
Book: The World of the Cell, 4th ed.: Becker, Kleinsmith, Hardin
Grade will be based on:
4 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.
3 Quizzes ------------------------------------- 20 pts each
1 journal presentation ------------------ 50 Pts.
Total ------------------------------------------- 560 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.
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.
2. To appreciate the evolutionary relationships that exist between
prokaryotic and eukaryotic cells.
3. To understand how the cell must integrate the function of each
organelle to function as a whole.
4. 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 gain a greater knowledge of the roles that genes play in
the structure and physiology of a cell.
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Date Chapter Pages Topic
Jan. 10 Sci. Am. Article "The Birth of Complex Cells"
Jan. 12 " " " " " "
Jan. 15 Holiday: Dr. Martin Luther King's B 'day
Jan. 17 1 3 - 4 Important measurements
3 47 - 55 Macromolecules of cells
Jan. 19 7 164171, 182-195 Membrane Proteins
Jan. 22 7 192 - 193 Membrane Carbohydrates
7 172 177 Membrane Lipids
Jan. 24 7 177 - 181 Membrane Fluidity
Jan. 26 4 79 Surface area/volume ratio
12 332 - 336 Subcellular Fractionation,
Jan. 29 12 329 - 339 Endomembrane System, SER
Jan. 31 12 331 RER
20 689 - 693 Translational Translocation
Feb. 2 12 344 - 348 Golgi and Protein sorting
Feb. 5 12 359 - 363 Lysosomes
366 - 369 Peroxisomes
Feb. 7 Exam I
Feb. 9 12 351 - 359 Receptor-mediated Endocytosis
Feb. 12 16 521 526 Nuclear
envelope
Feb. 14 Student Papers #1 & #2
Feb. 16 16 526 - 528 Nucleolus and Nucleoplasm
Feb. 19 Platelet morphology
Feb. 21 Platelet aggregation
Student Paper #3
Feb. 23 14 405 - 412 Mitochondriondrial morphology
Feb. 26 14 437 440 Mitochondrial proteins
20 692 696 Post-translational translocation
Feb. 28 Exam II
Mar, 2 22 766 - 771 Cytoskeletal Actin Microfilaments
March 3 - 11 Spring Break
Mar. 12 22 771 - 777 Cytoskeletal Microtubules
Mar. 14 22 777 Intermed. Filaments
11 299 - 311 The Extracellular Matrix
Mar. 16 11 " " TheExtracellular Matrix
Mar. 19 10 294 295 Platelet Intracellular signalling 10
269 - 275 G-proteins, DAG & IP3
Mar. 21 10 280 284 Receptor Tyr. kinases
10 276 279 Calcium/Calmodulin
Mar. 23 Student Papers #4 & #5
March 26 10 280 Nitric oxide
March 28 17 564 - 567 The Cell cycle
Mar. 30 17 567 - 573 Cell cycle checkpoints
Apr. 2 17 567 - 573 Regulation of the Cell
Cycle
Apr. 4 17 573 577 Cellular Transformation
Apr. 6 Exam III
Apr. 9 17 577 582 Cancer-causing mutations
Apr. 11 17 " " " "
Apr. 13 Good Friday, No classes
Apr. 16 17 544 - 545 Polymerase chain reaction
Apr. 18 16 509 - 514 Interspersed repeats
Apr. 20 16 509 - 514 Tandem DNA repeats
April 23 17 549 551 Telomeres
April 25 Exam IV
April 27 21 The Reg. of Gene Expression
Apr. 30 The Reg. of Gene Expression
May 2 - 8 Final Exams
Paper Summary and Presentation
Each presentation will be given by two students. Each pair
of students will be given a review article on one of the topics
below. You must find two more related sources. Presentations must
incorporate information from all 3 articles.
Presentations may be no more than 20 min. Points will be subtracted
if talks run over20 min. Each student should talk for approximately
10 min. Time will be given for students (and moi) to ask questions
of the speakers.
Power Point must be used to show diagrams, tables, pictures or
pertinent text. Avoid using small fonts and putting too much on
each slide.
A typed outline of the talk must be given to each student in the
class, on the day of the presentation.
I will collect your outline and the two other articles on the
day of the talk.
**************************************************************************************************
Date Topic Names
Feb. 14 #1____________________________
Feb. 16 2____________________________
Feb. 21 #3____________________________
Mar. 24 #4____________________________
#5____________________________
Mar. 27 #6____________________________
April 19 #7____________________________
#8____________________________
Cell Presentation Evaluation Sheet
Names of Speakers ___________________
_________________
Topic____________________
(8) 1. Transparencies/Visual Aids ________
Comments:
(10) 2. Clarity________
Comments:
(2) 3. Handled questions________
Comments:
(5) 4. Outline________
Comments:
Total Pts = ___________
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Home Page
___________________________________________________________________________
Lecture Outline
Cell Biology
Outline #1
Chapter 1
Measurements (p.3)
1. nanometer
2. micrometer
3. Angstom = 0.1nm
4. Have some idea of the size of cells and organelles
5. Dalton
Chapter 2
Chemistry of the Cell
I. Polymers
1. What are they?
2. Formed by condensation
3. Broken down by hydrolysis
II. Self-Assembly
1. Ribonuclease A
a) Denaturation
b) Renaturation
2. Molecular Chaperones
a) Assisted self-assembly
b) Heat shock proteins
3. Heirarchical Assembly
III. Important Bonds for Biopolymers
1. Covalent
2. Ionic
3. Hydrogen
4. Hydrophobic interactions
IV. Macromolecules of Cell
A. Proteins
1. Informational molecules
2. Be familiar with general properties of Amino Acids
3. Primary Sequence
4. Secondary Structure
a) Alpha Helix
b) Beta Pleated Sheet
5. Tertiary Structure
-Important bonds?
6. Quaternary Structure
- Important bonds?
B. Nucleic Acids
C. Polysaccharides
1. Storage Molecules
2. Glycosidic bonds
D. Lipids
Chapter 7
Membranes
I. Membrane Functions
1. Delineation and Compartmentalization
2. Localizationand 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
-Multipass protein
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
b) Peripheral form shell under membrane
i.Spectrin
ii. Ankyrin
iii. Actin
b) RBC membrane different from other cells:
i. Not as heterogeneous
ii.More extensive attachment to cytoskeleton
C. Membrane-associated carbo.
1.Form glycocalyx
-Function?
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) Asymmetry of glycoproteins in membrane
i. Galactose oxidase oxidizes galactose
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. In RBCs:
a) 55% phospholipid
b) 25% cholesterol
3.Triglycerides
a) Glycerol + Fatty acids
- Saturated and unsatur.
b) Memb. Phospholipids p. 72
i. Asymmetry in membrane
ii. Flip flop very slow in artificial membrane
iii. Faster in natural membrane....Why?
4. 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. = Higher transition temp.
2. Unsat. F.A.
i. Lower transition temp.
ii. Double bonds make kinks in membrane
-Less stability
3. Homeoviscous adaptation
i. E.coli = expressionof desaturase in low temp.
ii.Arctic fish have increase unsat. FA
4. Homeotherms
-Hibernating animals increase unsat. FA
5. Liposomes
i. Lateral diffusion studied
ii. Rate of movement?
6.FRAP
a) Use a fluor. tag on lipids
b) Bleach with laser
c) Calculate diffusion coefficient
c) Why a different diffusion rate in liposomes and cell membranes?
D. Protein Mobility in Membrane
1. Lymphocytes + lectin + Fluor. tag
-Stimulate mitosis
2. Why are patching and capping possible?
3.Aggregation of lectin receptors
-Aggregation of Tyrosine protein kinases
V. Membrane Asymmetry
A. Carbohydrates
-Always face outwards
B. Proteins = No flip-flop
C. Lipids
1. Which lipids are in which leaflets?
2. Which lipids are equally 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.Characteristics of Prokaryotes
1. Nucleoid
2. No membrane bounded organelles
3. No cytoskeleton
4. No exocytosis or endocytosis
5. Cell wall for support
6. Reproduction via Fission
7. Very little or no RNA processing
- Euk. = Removal of intravening sequences from HnRNA
8. Often Translation occurs before Transcription complete
III.Cytoplasm vs. Cytosol
-What's the difference?
Chapter 9
I. Subcellular Fractionation
A. Factors Determining Rate of Movement
*What are they?
B. Preparation of Sample?
C. Differential Centrifugation
1. Svedberg units
-Sedimentation rate
2. Centrifuge 1st to remove large cellular debris
3. Remove supernatant and spin again
a) at increased speed
b) increase time
D. Density Gradient Centrifug.
*READ p. 231
E. Equilibrium Density Gradient Centrifugation
*READ p. 232
II. Endomembrane System
A. Components
1. SER, RER
2. Golgi
3. Lysosomes
4. Nuclear envelope
5. Plasma membrane
6. Vesicles
B. Endoplasmic reticulum
1. Compare to plasma membrane
2. Microsomes---what are they?
3. RER vs. SER
Differences in appearance?
C. Smooth E.R.
1.Hydroxylation of Phe -->Tyr
a) Series of reactions
b) NADPH + H+ passes electrons to electron carriers in SER
c) P450 final electron carrier
-Passes electrons to Phe
d) Mono-oxygenases pass oxygen to Phe
e) PKU = Missing mono-oxygenase
i.Can't eliminate Phe
ii. Mental retardation by 12 mo. if no change in diet
2. Detoxification
a) Toxins made more hydrophilic by hydroxylation
-Easier to eliminate
b) Drug detoxification
i. Barbiturates (phenobarb. in rats)
ii. See more SER
iii. See more mixed function oxidases
iv. Rebound effect...
c) Increases metabolism of other drugs
3. Glycogen metabolism
a) Role in glycogenolysis
i.Activate via Epinephrine or glucagon
ii. Actitivate cascade
b) Glucose-6-phosphatase in SER membrane
i. Removal of phosphate
-Why necessary?
ii. Increase or decrease in blood sugar?
4. Phospholipid synthesis and Arrangement
a) Amphipathic biosynthetic enzymes
b) Synthesis first in cytoplasmic leaflet
i. Addition of FA to glycerol
ii. Addition of head group
c) Flippases
i. What do these do?
ii. Phospholipid head group specific
iii. How might these account for membrane lipid asymmetry?
D. Movement of P-lipids from ER
1. Vesicles bud and fuse
-Endomembrane system
2. Phospholipid Exchange Proteins
a) Important for mitochondria, chloroplast, peroxisome
b) Water-soluble carriers of specific phospholipids
-Pick up P-lipid from one membrane and releases 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 18)
a) Signal peptide on nascent protein
b) Signal Recognition particle (SRP)
i. Attaches to:
- signal peptide
- SRP receptor on ER
- Ribosome
ii.Temporarily stops translation
-Why important?
c) SRP and SRP receptor bind GTP
i.Translation starts
ii. Translocation of signal protein to pore protein
d) What does GTP hydrolysis do?
e) Signal peptidase
i. cleaves signal peptide
Characteristics?
ii. After translation complete
f) Folding in lumen and 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
b) Departure of pore protein --> diffusion
c) How are multi-pass proteins inserted?
(Chapter 9)
5. Core glycosylation
a) High mannose core
-Sugars found in core?
b) Added as an entire unit to Asn
-N-linked
c) Removal of 3 glucose and one mannose
E. Golgi
1. Structure
a) Stacks of 4-8 cisternae
b) Cis
i.Transition vesicles from RER to Cis
-Coatomer aids in budding
ii. Mannose-6-phosphate receptor found here
iii. KDEL receptor
c) Medial = shuttle vesicles between cisternae
d) Trans
i.Secretory vesicles from trans to membrane
ii. Clathrin aids in budding
Rd. Sci. Am. Article
2. Terminal Glycosylation
a) Non-Lysosomal Proteins
i.a-Mannosidase-1
ii. 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
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
G. Lysosome Read pp. 97-98 (De Duve)
1. What kinds of acid hydrolases?
2. Targeting of Lysosomal enzymes
a) GlcNac Phosphotransferase
i. in cis Golgi
ii. Phosphorylation of lysosomal proteins
-due to recognition domain on protein
b) alpha-Mannosidase-1
i. Substrate = non-lysosomal proteins
ii.Phophorylation of protein inhibits a-Mannosidase
c) Mannose-6-phos. receptor
i. In Cis Golgi
ii. Accumulation of acid hydrolases into vesicles
-Vesicles bud
iii. Fusion of vesicles into primary lysosomes
iv. Proton pump
- Decreasing pH
- Dissociation of proteins from receptor
v. Phophatases cleave phosphate from hydrolases
-Activation of acid hydrolases
vi. M-6-P receptor recycled to 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
5. Digestion by Lysosomes
a) Phagocytosis
i. Phagosome
ii. 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
I. Peroxisome
1. Contains catalase and several oxidases
2. Degradation of Fatty acids
a) By oxidases
b) b-oxidation
c) Generation of H2O2
i. Harmful to certain enzymes
ii.Broken down by catalase
3. Degradation of amino acids
3. Not part of endomembrane system
4. 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
i.Gene for luciferase from firefly
ii. Transfect into tobacco cells
iii. Result? Conclusions?
II. 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
5. Endosome
a) ATPase proton pump decreases pH
b) Uncoupling of ligand and receptor
c) Often recycling of receptors to plasma membrane
6. Often fusion of Endo. with Lysosome
- Degradation of ligand
B. Transferrin
1. Carries iron to cell
2. Picks up iron mostly in intestinal blood supply
3. Apotransferrin
a) No Fe
b) High affinity for receptor at pH ~6
4. Ferrotransferrin
a) Fe3+ bound
b) High affinity for receptor at pH 7
4. Ligand recycled in this case
5. Experiment using 125I and 59Fe
-Interpret graphs
C. Epidermal Growth Factor (EGF)
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
D. Clathrin Coat
1. Pentagons and hexagons of Triskelions
-Numbers of these depends on size of vesicle
2. Requires GTP and ARF for assembly on membrane
3. Clathrin Light chain
a) Aids in disassembly
b) Binding to Calcium as vesicle moves into cell
i.Triskelion more susceptible to ATPase
ii. ATP hydrolysis necess. for disassembly
Chapter 12
Mitochondrion
A. General Info.
1. Lgst organelle in nucleus
-Size?
2. Different #s in different cells
3. Found in parts of cell where intense metabolic activity occurs
a) Muscle myofilaments
b) Microtubule tracks in axon
-Fast axonal transport
B. General Structure
1. Outer membrane
-Freely permeable to small molecules and ions
2. Intermembrane space
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. hasmany more proteins than outer
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. F0F1 Complexes on Cristae
1. Be familiar with the Chemiosmotic model
2. Be familiar with experiment to determine function of F0F1
a) F1 = ATP synthase
i. 3 a subunits
ii. 3 b subunits = catalytic
b) Stalk
i. g, d, e polypeptides
ii. Required for assembly of complex
c) F0 = Proton transporter
i. a, b, c polypeptides
ii. b binds d of stalk
iii. Hydrophobic complex
Chapter 14
Structural Basis of Cellular Info.
I. The Nuclear Envelope
A. Double membrane
1. Inner membrane
2. Outer membrane
3. Perinuclear space
4. Nuclear pore
B. Nuclear Pore Complex
1. Structure
a) >100 polypeptides
b) Rings of 8 subunits
c) Central granule = transporter
-~9nm channel
d) Fibers extend inward and outward into cytosol
2. Passive Transport
a) Aqueous channels
b) Eight, 9nm channels around periphery
c) Small ions and mol. pass thru freely
-What's the limit on protein size?
d) Experiments with colloidal gold give dimensions of pores
3. Active Transport (ATP-dependent)
a) Nuclear localization signals on proteins
b) Steps in transporting NLS polypeptides into nucleus
i. Binding to possible receptor in cytosol
ii. Movement to pore
-via fibers?
iii. Binding and transport thru transporter
-ATP-dep.
II. Structural Fibers in the Nucleus
A. Nuclear Matrix
1. May not exist
2. Possible functions:
a) Maintenance of cell shape
b) Organization of chromosomes
c) Tracks for mRNA
B. Nuclear Lamina
1. Lamins
2. Possible functions
a) Support of nuc. envelopw
b) Organization of chromosomes
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
d) Behavior during mitosis?
4. Size of nucleolus correlated with level of cellular activity
Platelets
I. Origin and Structure
A.Derived from bone marrow Megakaryocyte
1. Fragmentation
2. Loss of nucleus and most organelles
B. Discoid when inactive
C. Subcellular Structures
1. Mitochondria
2. Alpha granules
a) Thrombospondin (TSP)
b) Fibrinogeen (Fg)
c) Platelet-derived growth factor
-Mitogen
3. Dense granules
a) Serotonin
-smooth muscle vasoconstrictor
b) ADP
II. Platelet Activation
A. Injury ----->Collagen exposure
1. Platelet Shape Change
a) Form filipodia
b) WHY?
2. Adhesion
a) Activation of GP IIb-IIIa
b) Binding of von Willebrand factor
i.To IIb-IIIa
ii. To subendothelium
3. Platelet secretion and aggregation
B. Coagulation Cascade
1. Release of Tissue Factor
2. TF + Calcium + Factor VII
-Forms thromboplastin
3. Factor X activated---> Prothrombinase
4. Prothrombin ---> Thrombin
5. Fg ----> Insoluble fibrin
-Clot = fibrin + RBCs, WBCs, platelets, serum
C. Thrombin and Collagen are Agonists
1. Activation of IIb-IIIa
a) Fg binding
i.Cross-linking of platelets
ii. Via RGDS
b) Primary Aggregation
2. Secretion of platelet granules constituents
a) TSP release
b) Binds platelet and Fg
c) Secondary aggregation
D. Aggregometer
1. Use ADP as agonist
2. See primary wave of aggregation
-Reversible
3. See secondary wave of aggregation
-Irreversible
4. Add peptides to platelets
a) RGDS = Inhibition
b) KGDS = Decreased inhibition
c) RADS = No Inhib.
d) RGES = No Inhib.
e) RGDV = Full inhibition
5. Conclusions?
Chapters 4 and 20
II. Cytoskeleton
A. Actin Microfilaments (MF)
1. Strucure
a) G-Actin ---->F-actin ----> rt. handed double helix (7 nm diameter)
b) Inherent polarity for directionality
c) ATP
ii.Increases G-actin affinity
iii. Hydrolysis favors disassembly
2. Actin-binding Proteins
a) Fragmin = severs and caps at one end
b) Profilin
i. Releases G-actin from thymosin
ii. May also help actin release ADP
3. Functions
a) Cleavage furrow
b) Locomotion of cells
c) Cell cortex ----> shape and strength
i. Gel-like properties
ii. Due to Filamin which cross-links adjacent MF
iii. Cytochalasin B
(A) Disrupts MF
- How?
(B) Fibroblasts in culture lose shape
Chapters 4 and 20
Cytoskeleton
A. Actin Microfilaments (Last test)
B. Microtubules
1. Structure
a) Diameter = 25 nm
b) Tubulin subunits
i. Dimer of a and b
ii. Both bind GTP or GDP
c) Tubulin ---->13 Protofilaments ----> Microtubule
d) Polarity
i.End with GDP less stable
ii. GTP hydrolysis promotes disassembly
e) Elongation by addition of individual tubulin molecules
2. Spindle fibers at Anaphase
a) Polar micrtubules
i.Elongate
ii. Slide past each other at equator
iii. May be due to cross-linking proteins
-Act like molecular motors
b) Kinetochore microtubules
i.Shorten at kinetochore end
ii. Chromatids move to poles
3. Axoneme of flagella and cilia
a) 9 + 2 array
b) Sliding Microtubule model
i. Dynein (type of MAMP)
(A) ATPase
(B) Between outer doublets
(C) Attaches and detaches along B tubule
ii. Bending of axoneme
4. Organelle Movement
a) Kinesin (MAMP)
i. Head binds Microtub.
-Where ATPase activity is
ii.Tail binds organelle
iii. Moves toward + end
b) Fast Axonal Transport
i. Transport of neurotrans. to axon knobs
-Use microtubules
ii. Mitochondria associated for ATP
iii. Colchicine inhibits
-Attaches to tubulin dimer which is added to microtub.
C. Intermediate Filaments (IF)
1. Structure
a) Diameter = 8 - 12nm
b) 6 different classes of proteins
-Single family of related genes
c) Cell-type specific
i.IF typing with immunofluorescence
ii. Why important?
d) Most stable and least soluble
-May serve as scaffold for rest of cytoskeleton
2. Function of IF:
a) Positioning of nucleus?
b) Tension-bearing
i. Found in areas subjected to mechanical stress
ii. Ex) Tonofilaments in desmosomes
Chapter 10
Junctions and Extracellular Matrix
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
II. Extracellular Matrix (ECM)
A. General characteristics
1. Few cells
2. Protein fibers
a) collagen
b) elastin
3. Gel matrix
-Proteoglycans
4. Adhesive proteins
a) Fibronectins
b) Laminin
B. Protein Fibers
1. Collagens
a) Secreted by fibroblasts
b) Lg. family of closely related proteins
-14 different types
c) Be generally familiar with collagen structure
-Fibers covalently cross-linked at lysines
d) Different types due to different a-chains
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
3. Skin, lung, intestine
4. 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) High in carbohydrate
i.1 - 200 carbo. chains per polypeptide
ii. Up to 95% carbo. by wt.
3. Glycosaminoglycans
a) Main carbohydrate components
b) Repeating disaccharides
4. Ex) Aggrecan
a) A major component of cartilage
b) Over 100 GAGs
c) Mol. wt. = 3 x 106
D. Adhesive Glycoproteins
1. Adhesive for:
a) Proteoglycan + collagen
b) Proteoglycan + membrane
c) Collagen + membrane
2. Two most common:
a) Fibronectin
b) Laminin
3. Fibronectins
a) Two polypeptides
-Covalently linked
b) Binding domains for various MOLECULES
c) Family of similar but not identical glycoproteins
i.Encoded by same gene with at least 50 exons
ii.Alternative splicing from single HnRNA
-Purpose?
-Get 20 different mRNAs
iii.Examples:
- Plasma FN
- FN filaments
d) Cell migration requires Fn
i.Migration of mesodermal cells in gastrula
ii. RGDS inhibits
iii. Fn Ab inhibits
e) Cell + ECM adhesion mediated by Fn
f) Maintenance of cell shape due to adhesion to substrate
g) Splicing pattern of embryonic Fn different from adult
4. Laminins
a) Prominent in basal lamina
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 a-chain
3. Different from other ligand receptors
i. Low affinity
ii. 10-100x more of them per cell
4. Many recognise RGD
5. Most indirectly bind actin microfilaments
6. Fibronectin (FN) Receptor
a) Isolation by affinity chromatography on FN column
-Elute with RGD
b) Binds Talin on cyto. side of membrane
c) Phosphorylation of talin decreases affinity for FN receptor
i.Cells round up as occurs during Mitosis
ii. Essential for cell migration
d) Dephosphorylation of talin increases affinity for FN receptor
i.Cells become spindled ii. Reattach to ECM
Chapter 23
Cell Signalling
I. Purpose of Complex Protein Interactioins
A. Amplification of signal
B. Fast termination
II. General DAG and IP3 Pathway (found in almost all cells)
A. Ligand binds receptor
1. Receptor changes conformation
2. G-protein activated
3. Phospholipase C activated
a) Cleavage of PIP2
b) Generate DAG and IP3
B. IP3
1. Diffuses to calceosomes and binds receptor
2. Ca++ channel proteins open
-Increase in cytosolic Ca++
3. Termination of signal
a) IP3 breaks down quickly
-Why important?
b) Ca++ release stops
-followed by calcium reuptake
4. Mimicked by Ca++ ionophore
B. DAG
1. Remains in membrane
2. PKC
a) DAG lowers threshold for activation by Ca++
b) Phosphorylation of Ser and Thr on proteins
3. Examples of PKC activation
a) Phosphorylation of ion channels in neurons
b) Gene activation by mitogens
i. Phosphorylates MAP kinase (activated)
ii. MAPK enters nucleus and phosphorylates transcription factors
- genes for cell growth activated
3. Phorbol esters mimic DAG
a) Tumor promoters
b) Activate PKC
i. Is PKC involved in transformation?
ii. Fibroblasts in culture
with up-regulation of PKC
iii. Cells grow unattached to substrate (like cancer cells)
III. Signalling in Platelets (NOT IN BOOK)
A. General Response to ADP
1. Shape change
-Due to phosphorylation of cytoskeleton?
2. Primary aggregation
a) GP IIb/IIIa activation
i. Inside-out mechanism
ii. Phosphorylation of cytoskeleton
iii. dissociation between microfilaments & GP IIb/IIIa
- may cause conformational change
b) Fg cross-linking via GP IIb-IIIa
B. Signal Transduction Leading to Secretion
1. ADP + receptor
2. Activation of PLA2 due to:
a) Release Ca++
b) Activ. of Na+/H+ antiport
c) G-protein
3. PLA2 hydrolyzes arachadonic acid (AA)
-from membrane phopholipids
4. Cyclooxygenase converts AA to PGE2
a) Aspirin irreversibly inhibits
b) Secondary aggregation inhibited for ~ 10 days
-Why 10 days?
-Why irreversible?
c) See below for PGE2 effect on endothelium
5. Thromboxane synthase
i. PGE2 conversion to Thromboxane A2 (TxA2)
ii. Diffusion of TxA2 out of platelet
6. TxA2 binds membrane receptor
a) Conformational change in receptor
b) Activation of via G-protein
c) Activation of PLC
7. PLC hydrolyzes PIP2
a) Production of IP3
i. Enters cytosol
ii. Release of Ca++
iii. Secretion = Exocytosis of TSP ---> secondary aggregation
b) Production of DAG
8. Both DAG and IP3 necessary for full aggregation
-Phorbol esters + calcium ionophores required for full aggregation
C. Endothelium and PGE2
1. Due to release of ACh
-Parasympathetic impulse to blood vessel endothelium
2. Prostacyclin cyclase
i. Production of prostacyclin I2
ii. Diffusion to smooth muscle
3. Blood vessel dilation
IV. Intracellular Ca++ in Cell Signalling
A. Free Ca++
1. ~1mM in resting cell
2. Increase to ~6mM in activated cell
3. Examples of cell activation due to Ca++?
B. Calmodulin
1. Binds 4 Ca++
2. Activation mostly of kinases
3. Activates Calcium/ATPase pump
-regulation of calcium levels
4. Ca++ /CaM Kinase II
a) In neurons releasing catecholamines
b) Voltage-gated channels cause influx of Ca++
i. Activates secretion of catecholamines
ii. Binds CaM which activates CaM Kinase II
-Phosphorylation of tyrosine hydroxylase
-rate-limiting enzyme in catecholamine synthesis
V. Nitric Oxide (Not in book)
A. Produced by what cells?
B. Synthesis
1. NO Synthase
2. Coupled with deamination of Arg.
3. Short half-life
*Acts locally
4. Converted to nitrates/nitrites
B. Erection of Penis
1. Acetylcholine release from autonomic nerves
-ennervating smooth muscle of blood vessel
2. Induces NO production by endothelial cells
3. NO diffuses to smooth muscle of blood vessels
a) Reacts with guanylyl cyclase
b) Production of cGMP in smooth muscle
c) Smooth muscle relaxation
4. Dilation of blood vessels
-Voila'!!!!
5. Nitroglycerin for angina
-conversion to NO
Chapter 15
Cell Cycle
I. Cell Cycle Times
A. Determine Time of Each
1. Generation time
a) Count under 'scope at intervals
b) Monitor with spectroph.
c) Average mammalian cell's generation time?
d) Time for entire cell cycle
2. S-Phase = DNA replication
a) 3H-thymidine added
b) Autoradiography
c) Find % labeled cells
-multiply by generation time
3. M-Phase = Mitosis
a) Find % of cells in M
-Visually identify
b) Multiply by generation time
4. G2 Phase = Preparation for mitosis
a) 3H-thymidine added
b) Autoradiography
b) Look for 1st label appearing in mitotic cells
-Find %
c) Multiply by generation time
5. G1 Phase
a) Protein and RNA synthesis in prep. for S-phase
b) G0 at end of phase = check point
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
c) 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
d) Many replicons
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
a) Controls cell's entry into M
b) Not in all cells
4. Metaphase checkpt.
-Will stop if chrom. not attched 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
C. Maturation Promoting Factor (MFP)
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
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) Characteristics of Cdc2
i. Protein kinase
ii. Always present at same concentration
iii. Only active when complexed with cyclins
iv. Belong to a family of cdks = "cyclin-dep. kinases"
-Different cdk's 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
iv. MPF activates proteases
-Digest Cyclins at Metaphase-Anaphase transition
6. Cyclin- Cdc2 Complex Activity
a) Chromosome condensation
-Phosphoryl. of Histone-1
b) Spindle assembly
i.Phosphoryl. of microtubule assoc. proteins
ii. MPF binds centrosome
c) Breakdown of nuclear envelope
i. Phosphorylation of lamins
ii. Dissociation of lamins from inner nuclear membrane
ii. Fragmentation of nuclear envelope
d) Activ. of Mitotic proteases
i. Degradation of cyclin at end of mitosis
-Important for exit from M
ii.Cdc2 Recycled
iii. Enzymes inactivated as MPF destroyed
D. Cdk-cyclins at Other Checkpts.
1. Different types of Cdk and Cyclins
2. Diff. combinations of Cdk and Cyclins at diff. stages
E. M Checkpt.
Frog egg extract:
1. Demonstrated that cyclin breakdown doesn't "trigger" Anaphase
2. Used non-degrad. cyclin B
3. Results:
a) Mitosis did not proceed to completion
-Since cyclin not destroyed
b) Sister chromatid separation occurred
i. Mitotic proteases degraded proteins between chromatids
ii.This is the "trigger"
F. Regulation of Cdc2-cyclin
1. Effect of inhibiting kinase
a) Phosphorylation of Cdc2
b) G2 arrest
2. Effect of activating kinase
a) Phosphorylation of Cdc2
-At different Tyrosine than inhibiting kinase
-Phosphate required for activity
3. Phosphatase
a) Removal of inhibitory phosphates
b) Positive Feedback loop
4. Autonomous Clock
a) External and internal environment affect cycle
b) Some molecules activate kinases others activate phosphatases
Chapter 25
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
4. Two major characteristics different from benign:
a) Invasiveness
i. Produce degradative enzymes
ii. Inability to produce contact junctions
-No contact inhib.
iii. Penetrate blood vessel
iv. Migration by invasion into capillary lumen
v. Small clumps break off
b) Metastasis
i. Tumor cells travel to distant sites in body
ii. Possible means of transport thru body?
iii. Vascular metastasis
- Angiogenin attracts endothelial cell growth
-Capillary growth around tumor
C. Tumor Classifications
1. Blastoma
2. Carcinoma
-Adenocarcinoma
3. Sarcoma
4. Lymphoma
5. Leukemia
6. Melanoma
D. Similarity to Rapidly dividing cells
1. High nucleus/cyto. ratio
2. Prominent nucleoli
3. Many mitotic divisions
4. Few specialized structures
E. Cell Culture Characteristics
1. Normal cells in culture:
a) Require 5 - 20% serum
- Why?
b) Must adhere to proteins on glass or plastic surface for growth
c) Limit to # of cell divisions
~60 in embryonic cells
d) Contact inhibition
2. Transformed Cells
a) Decreased growth factor requirements
-Often make their own
b) Loss of dependence on adhesion
c) Loss of contact inhib.
d) Increased mobility of membrane proteins
-Why?
e) Altered transcription
-Only 3% of mRNA is tumor cell-specific
f) Immortal
II. Causes of Transformation
A. Chromosomal Alterations
1. Philadelphia Chromosome a) 85% of people with Chronic Myelogenous Leukemia
b) Slow tumor progression
c) Eventually blast cell crisis-¦ death
d) Reciprocal translocation of chrom. 22 to chrom. 9
e) Somatic mutation (Only in granulocyte stem cells)
B. Oncogenic Viruses
1. DNA Viruses
a) Oncogenes often part of viral genome
b) Incorp. into host genome
c) Viral products constitutively expressed --->tumor
d) May initiate chrom. breaks
e) Human papilloma virus
-Cervical cancer
f) Hepatitis B
- liver cancer
2. RNA viruses
a) Retroviruses are the most oncogenic
i.Replication by reverse transcriptase
ii.Integration
iii.Pick up proto-oncogenes
-incorp. into viral genome
iv. Mutation to oncogene
v. Infect next cell --> becomes tumor
b) At least 20 assoc. with tumor development
3. Proto/Oncogene Nomenclature
a) Overt = italicized, 3 letters
b) Proto-oncogene = "c" myc
c) Evolutionarily conserved indicates importance
d) Wide distribution in eukaryotes
4. Types of Oncogene alterations:
a) Expression Effects i. Quantitative = overproduction of gene product
ii. Regulatory site affected by chromosomal rearrangement
iii. Burkitt's Lymphoma -c-myc translocation to sites of immunoglobulin genes
-Too much myc leads to overexpression of transcription factors
-> mitosis
b) Gene mutation
i. Qualitative=Altered function of protein
ii.Deletion or point mutation
iii. Ha-ras gene ¦ bladder cancer
-Harvey sarcome virus
-Transform. due to a single pt. mutation
-Ras = Modified G-protein
-Constitutively activated
¦Mitosis
-How was this determined?
C. Environmental Carcinogens
1. Chemicals
a) Procarcinogen converted to ultimate carcinogen
- Due to Mixed function oxidases in SER
b) Ultimate carcinogens bind DNA bases covalently
i. Disturb H-bonding between DNA strands
ii. Alter base-pairing during replication
iii. Affect interaction of DNA with DNA-binding proteins
c) Ames test
i. Correlates mutagenicity with carcinogenicity
ii. Mutant Salmonella which is His(-)
iii. Grow on His(-) media
- Plus Mixed function oxidases
-Plus known carcinogens
iv. Look for revertants and calculate mutation frequency
v. Ames conclusions?
d) Chemical modification of base can lead to pt. Mutation
i. Improper base-pairing during replication
ii. Substitution of one nucleotide for another
2. Radiation
a) Ionizing Radiation
i. Ionizing radiation and x-ray
ii. Cause blunt end breaks in DNA
b) Ultraviolet radiation causes Thymine dimers
i.Breaks C-C double bond in thymines
ii. Formation of covalent bond b/ neighboring thymines
D. Repair Mechanisms
1. Base Excision repair
a) Often for chemical mutagens
b) Remove base with one enzyme
c) Remove sugar and phos. with other enzymes
d) Replace with correct nucleotide
2. Nucleotide Excision Repair
a) For repair of thymine dimers
b) Remove several entire nucleotides in strand
c) Replace nucleotides using other strand as template
3. Repair mechanisms very accurate
4. Error-Prone Repair
a) Wrong ends may be joined after blunt end breaks
b) Replication before repair complete
c)Inducible repair in bacteria
i.SOS Repair
ii. Only induced if normal repair systems are saturated
iii. High mistake rate
d) Inducible systems in eukaryotes too
-Not well characterized
e) Xeroderma pigmentosum
i. Autosomal recessive
ii. No repair of UV damage
iii. Increased skin cancers and cataracts
III. Growth Factors
A. Examples
1. Epidermal Growth Factor
2. Platelet-derived Growth Factor
B. Requirements for G.F. in culture
1. Cells stop growing at saturation density
2. Cell density proportional to amt. of serum supplied
3. Replace serum with EGF and Transferrin ---> growth returns
C. Functions of GF
1. Embryogenesis = Growth
2. Adults = Repair
D. Mechanisms of G.F. Activtiy
1. Receptors have Tyr. kinase activity after ligand binding
a) Autophosphorylation
b) Phosphorylation of target proteins
i. Kinase cascade activation
ii. MAPK activated (enters nucleus)
- Activation of transcription factors
- Gene expression
- Mitosis
2. Receptor coupled to G-protein (Ex- c-ras)
a) Ligand binding-> ras activation
b) PLC activated
c) PLC catalyzes PIP2
d) Production of :
i. DAG = Activation of protein kinase C
-Activ. of MAPK
ii. IP3= Release of Ca++ ¦Activ. of Ca++/CAM
- Activ. of MAPK
E. G.F. and Neoplastic Growth
1. Autocrine secretion
a) Cells transformed by simian sarcoma virus
b) Produce own G.F. similar to PDGF
c)Competitive binding assay with SSV-conditioned media
i.Add labeled PDGF
-Inhibition of labeled PDGF binding to cells
ii. Add Ab to PDGF
-No growth of normal fibroblasts
d) Sequenced sis oncogene
-Very similar to gene for b-subunit of PDGF
2. G.F. Receptor Altered
a) erb-B oncogene in erythroblastosis virus
b) Constitutive Tyr. kinase activity
c) Gene similar to EGF receptor
i.Modified cyto -plasmic domain
-Constitutive activation
ii. Same trans -membrane domain
iii. Very little exoplasmic domain
-No binding of EGF possible
READ: "Signal Transduction Pathways as Drug Targets"
IV. Tumor Suppressor Genes
A. Recessive phenotype of tumor
1. Fusion of normal and cancer cell --->Hybrid
2. Normal pheno. of hybrid due to chrom. with p53
3. Loss of chromosomes associated with development of tumor
-Specifically loss of tumor suppressor genes
B. p53 Gene
1. Most commonly mutated gene in human tumors
2. Guardian of the Genome
3. Halts cell cycle for repair
a) Halt at Go so cell will have time to repair
b) Activates 21kD gene
c) 21kD protein prevents activ. of cdk + cyclins
4. Stimulates DNA repair
5. Triggers apoptosis if too much damage
a) May generate ROS
b) Loss of Mitochondrial membrane potential
c) Release of Apoptosis Initiating Factors
d) AIF activate caspases
-Proteases for terminal apoptosis events
6. Often found in combination with other mutations
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.
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?
b) Selfish DNA?
c) Telomeres = protect from harm of shrinkage
8. At least 50 human genes with triple 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
d) Loss of nervous funtion
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. 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 premut. from mom
ii. Somatic cells have expanded mutation
iii. Expansion must appear after germ cell line differentiates
D. Causes of Problems?
a) Aberrant replication
i. Meitic division or early embryo
ii. Overactive replic. mechanism at repeats
b) Error in DNA repair
i. Confused repair system
-Overactive repair system at repeats
c) No transcription or faulty transcription
-Super tight interaction between DNA & histones
III. Interspersed Repeats
A. Characteristics
1. Repeats scattered
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 oprigins
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 lg fragment on gel
ii. Both Tpa25 alleles
-One smaller fragment on gel
c) Heterozygotes
i.48% in Caucasian population
ii.Only one insert present
iii. One lg. and one smaller band
Chapter 15
Cell Cycle, DNA Replic..
Not responsible for:
I. The PCR Revolution (pp. 468-469)
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 Gilbert Progeria
1. Children look old
-Short life span
2. Short telomeres at birth in fibroblasts
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 a
a) Synthesizes lagging strand end (5' ----->3')
b) One unit is a primase for synthesis of primer
c) Other unit is a DNA Polym.
E. Cancer
1. Almost all tumor cells reactivate telomerase gene
2. May use activity to detect early cancers
3. Potential therapy = repress genes which neg. regulate telomerase
Chapter 19
Regulation of Gene Expression
I. Transcription Factors
A. Xenopus experiments
1. Gene from Herpes virus transfected into Xenopus
2. Mutations induced outside of gene
3. Several areas necessary for transcription
a) Promoter = site of RNA Polymerase binding
b) 4 other distinct sites near genes
c) DNA sequences which bound transcription factors (TF)
II. Anatomy of a Eukaryotic Gene
A. Coding region
1. Exons = transcribed and translated
2. Introns = transcribed but not translated
B. Inr (Initiator sequence)
1. Upstream
2. Start pt. of transcription
C. Promoter (site of RNA Polym. binding)
1. -25 bp (upstream)
2. TATA box
3. TFIID binds here
4. Other proteins bind via protein-protein interaction
-Including RNA Polymerase II
5. Initiates trans. at only low rate
D. Proximal Control Elements
1. Within ~100 bp of Promoter
2. Number and identities vary with gene
3. Two most common:
a) CAAT box
i. binds a TF
ii. Found near many genes
-80bp from Inr
b) GC box
i. binds a different TF
ii. Found near many genes
-100bp from Inr
4. Greatly enhance rate of transcription
-Probably due to interaction with Polymerase and associated proteins
E. Distal Control Elements
1. May be 10,000 - 20,000 bp from gene
2. Enhancers
a) Increase transcription
b) Some GC boxes act this way
c) Bind TF called activators
3. Experiments with enhancers
a) Manipulate position of enhancer
i. Move farther away ----> still works
ii. Move to downstream position ----> still works
b) Manipulate orientation
-invert = still works
4. Mode of action:
a) DNA looping
b) Binding of activator TFs to TFIID
c) Then Polym. II and assoc. proteins bind
d) Trans. inititated
III. Transcription Factors
A. Characteristics
1. Usually 5 distinct T.F. binding regions/gene
2. Many have a lack of specificity for DNA sequences
a) TFs from large complexes
b) Often interchangeable parts
-Allows greater amount of specificity
3. At least two domains/T.F.
a) DNA binding domain
b) Recognition domain
-Binds other proteins
4. 1/3 = one of a kind TFs
5. 2/3 = belong to recognizable groups
B. Types of T.F.'s
1. Zinc Finger Proteins
a) Identified in Xenopus in 1985
b) Domains found in a number of TFs
b) Part of TFIIIA
i. Assoc. with 5S rRNA gene
ii. One of at least 3 T.F. for 5S gene
iii. Had to eliminate Zn++ to isolate protein
-TFIIIa assoc. with 7 - 11 Zn++
iv. DNA Footprinting
-How was this done?
-Find several regions of DNA protected from DNase
2. Structure of TFIIIA
a) 1st 3/4 of protein had 9 similar domains
i. Zn++ fingers
ii. ~30 amino acids
iii. Cys. and His bind Zn++
b) Intervening AA loop out to contact DNA
c) How is some degree of specificity obtained by Zinc fingers?
3. Leucine Zipper Proteins
a) Dimers (many TFs are)
i. Leu.-rich regions of two polypeptide bind
ii. hydrophobic interactions
ii. Homodimers and heterodimers
-Add to specificity of DNA binding
b) DNA binding regions
i. Rich in basic amino acids
ii. Grip DNA like a clothespin
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