Transcript PowerPoint Presentation - Are All Fish Related? A look at

Vertebrate Evolution:
A look at biomolecular evidence using gel
electrophoresis.
Part 1: Introduction.
Available online at
www.redwood.org/stewart
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I. Traditional Method for Classifying
Organisms: Structure and Function
• Classification
– Kingdom
– Phylum
– Class
– Order
– Family
– Genus
– Species
• Traditional
classification based
upon traits:
– structure
– function (behavior)
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II. Using biomolecular evidence to
determine evolutionary relationships.
A. Biomolecules are the basis of
traits
• Traits represent organisms':
- Structure
- Function
• Proteins determine structure and function
• DNA codes for proteins that confer traits
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A. Biomolecules are the basis of
traits
DNA  RNA  Protein  Trait
DNA
TAC CGA TCG TGA ACT
TRANSCRIPTION
mRNA
AUG GCU AGC ACU UGA
TRANSLATION
tRNA
UAC CGA UCG UGA ACU
amino acid
Met - Ala - Ser -Thr - Stop
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A. Biomolecules are the basis of
traits
End Product of Transcription and
Translation:
Proteins
Before you begin a lab to use
bio-molecular evidence to
determine the evolutionary
relationships of organisms, let’s
take a closer look at proteins.
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Type of Protein
Function
Example
Structural Protein Support
 Keratin is the protein of hair, horns, feathers
 Collagen and elastin provide a fibrous
framework in animal connective tissue
Insects and spiders use silk fibers to make their
cocoons and webs
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Type of Protein
Function
Example
Storage Storage of Amino Acids
Ovalbumin is the protein of egg white, used for
developing embryos
Casein – milk protein for developing babies
Plants have storage protein in their seeds
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Type of Protein
Transport
Function
Example
Transport of other substances
 Hemoglobin – iron containing protein of
blood
Other proteins transport molecules across cell
membranes
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Type of Protein
Hormonal
Function
Example
Coordination of activities
Insulin, a hormone secreted by the pancreas,
helps regulate blood sugar
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Type of Protein
Function
Example
Receptor
Response of cell to chemical stimuli
Receptors built into the membrane of nerve cell
detect chemical signals release by other nerve
class
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Type of Protein
Function
Contractile
Movement
Example
Actin and myosin are responsible for
movement of muscles
Other protein are responsible for cilia and
flagella of organelles
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Type of Protein
Defensive
Function
Example
Protection against disease
Antibodies combat bacteria and viruses
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Type of Protein
Enzymatic
Function
Example
Acceleration of chemical reactions
Digestive enzymes break down food
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B. Biomolecular
Differences
• Changes in DNA  changes in protein,
these changes result in:
- different functions
- unique traits
- positive (for survival), negative
(for selection), or no effects
• Genetic diversity provides pool for natural
selection = evolution
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C. Levels of Protein Organization
A functional
protein is
not just a
polypeptide
chain!
Protein Structure
Polypeptide chain (yarn) – not functional
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C. Levels of Protein Organization
A functional
protein is
not just a
polypeptide
chain!
Protein Structure
Protein (sweater) –functional polypeptide chain16
C. Levels of Protein Organization
1. Primary Structure -
Primary
Proteins begin as a straight
chain of amino acids.
Secondary
2. Secondary Structure The chains begin to bend
and twist like a corkscrew
or a flat folded sheet.
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C. Levels of Protein Organization
3. Tertiary Structure -
Tertiary
The twisted chain folds even
more and bonds form, holding
the 3-dimensional shape.
4. Quaternary structure -
Quaternary
Several amino acid chains in
the tertiary structure come
together. This is a functional
protein.
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D. Comparing Protein Size
1. What do you compare?
• Dalton (Da) = mass of hydrogen molecule
= 1.66 x 10 -24 gram
• Avg. amino acid = 110 Da
• Protein size measured in kilodaltons (kDa)
• Avg. protein = 1000 amino acids =
100,000 daltons = 100 kDa
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1. What do you compare?
• Muscle contains proteins of many sizes
Protein
titin
dystrophin
filamin
kDa
3000
270
Function
center myosin in sarcomere
400
anchoring to plasma membrane
cross-link filaments into gel
myosin heavy chain
210
slide filaments
spectrin
nebulin
a-actinin
gelosin
fimbrin
265
107
100
90
68
attach filaments to plasma membrane
regulate actin assembly
bundle filaments
fragment filaments
bundle filaments
actin
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form filaments
tropomyosin
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strengthen filaments
myosin light chain
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slide filaments
troponin (T, I, C)
thymosin
30, 19, 17
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mediate regulation of contraction
sequester actin monomers
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1. What do you compare?
• Actin:
•
•
•
•
• Example proteins
5% of total protein
20% of vertebrate muscle mass
375 amino acids = 42 kDa
Forms filaments
• Myosin:
• Tetramer of two heavy subunits (220 kDa)
and two light subunits (20 kDa)
• Breaks down ATP for muscle contraction
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D. Comparing Protein Size
2. How compare?
• Break protein complexes into individual
protein chains (using chemicals)
• Denature proteins so they lose their
shape and gain a charge (using
detergent and heat)
• Separate proteins based on size (using
gel electrophoresis)
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III. Vertebrate Protein Analysis Lab
A. the Experiment
• Purpose: Compare muscle proteins from
related and unrelated vertebrates to determine
evolutionary relationships.
• Procedure:
- Extract proteins from tissue
- Denature proteins
- Separate proteins by size using
polyacrylamide gel electrophoresis (PAGE)
- Stain proteins to see banding patterns
- Analyze and interpret results
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B. How does a PAGE gel work?
1. Prepare the Protein Samples
• Put muscle in buffer which includes:
- SDS detergent (Sodium Dodecyl
Sulfate) to solubilize and denature
proteins and negative charge to
proteins
- Reductants (beta-mercaptoethanol,
DTT) break disulfide bonds
• Heat muscle/buffer mixture to
denature proteins
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B. How does a PAGE gel work?
2. Run the gel
• Negatively charged proteins
move to positive electrode
Vert. 1 Vert. 2
• Smaller proteins move faster
• Proteins separate by
size
• Simulation A, B,
s-s
SDS, ß-Me,
heat
proteins with
SDS
Marker
-
+
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B. How does a PAGE gel work?
3. Analyzing Results
• Compare banding patterns among the
vertebrates - identify similarities and
differences among them.
• Illustrate the relationships among the
vertebrates .
• Compare illustration based on biomolecular
evidence to an illustration based on traditional
classification
» DO THEY MATCH?
End Part 1
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Vertebrate Evolution:
A look at biomolecular evidence using gel
electrophoresis.
Part 2: Analysis.
Available online at
www.redwood.org/stewart
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Gel Analysis
1
7
2
3 4
5 6
15% SDS-PAGE
• Lane 1: Tunicate
• Lane 2: Fish
• Lane 3: Amphibian
• Lane 4: Reptile
• Lane 5: Bird
• Lane 6: Mammal
• Lane 7: Actin/myosin
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Molecular Weight Analysis
mm
203
135
86
8.5
12.0
18.5
41
28.0
33
34.0
250
200
kDa
kDa
150
100
50
0
0
19
41.5
8
44.5
20
40
60
mm from well
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Vertebrate Protein Gel Analysis
Marker
Pig
Perch
Turtle
Tunicate
Frog
Pigeon
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Fish Protein Gel Analysis
Myosin (210
kDa) = about
2000 am. acids
Measure
and
record
distances
in mm
Marker
Pig: arguably, most
Perch
Turtle
Tunicate
Frog
Pigeoncomplex vertebrate (top
right of cladogram)
* Do bands
line up?
To make your vertebrate cladogram, compare each vertebrate to the pig by:
1. measuring distance protein bands traveled from wells,
2. recording (to scale) on paper (IMPORTANT: relative position of bands),
3. counting number of proteins each vertebrate has in common with pig*.
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Family tree that branches systematically at
points (nodes) representing specific
traits possessed by some groups, but
not others.
Branches:
Organisms
Organisms
branching to
left DO NOT
have this trait.
Organisms
branching to right
HAVE this trait.
Node: Specific trait (or #
of proteins in common).
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Vertebrate Cladogram
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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