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

Is There Something
Fishy About Evolution?
A look at biochemical evidence for evolution
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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. Biochemicals 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. Biochemicals 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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B. Biochemical 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
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
42
form filaments
tropomyosin
35
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. Fish Protein Analysis Lab
B. the Experiment
• Purpose: Compare muscle proteins from
related and unrelated fish
• 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
• Smaller proteins move faster
• Proteins separate by
size
• Simulation
s-s
SDS, ß-Me,
heat
proteins with
SDS
-
+
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B. How does a PAGE gel work?
3. Analyzing Results
• Compare banding patterns among the fish identify similarities and differences among
Click here to view a gel
them.
• Illustrate the relationships among the fish.
• Compare illustration based on biomolecular
evidence to an illustration based on traditional
classification
» DO THEY MATCH?
To Phylogenetic Tree - Click Here
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Gel Analysis
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2
3 4
5 6
15% SDS-PAGE
• Lane 1: Marker
• Lane 2: Shark
• Lane 3: Salmon
• Lane 4: Trout
• Lane 5: Catfish
• Lane 6: Sturgeon
• 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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Phylogenetic Tree
CARP
MINNO
W
SALMON
TROUT
CATFISH
SMELT
ANCHOVIES
HERRINGS
SARDINES
PIKE
COD
HAKE
POLLOCK
SNAPPER
PERCH
WALLEYE
BASS
TUNA
MACKEREL
FLOUNDER
SOLE
HALIBUT
STURGEON
GAR
SHARK
Agnatha
OYSTER
CLAM
MUSSEL
Chondrichthyes
OCTOPUS
SQUID
CRAB
LOBSTER
SHRIMP
Mollusk
Arthropod
SCALLOP
Protostome
Metazoa
Ostheichthyes
Echinoderm
Amphibia
Reptili
a
Aves
Mammali
a
Chordate
Deuterostome
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Fish Protein Analysis Gel
Marker
Salmon
Shark
Tuna
Scallop
Halibut
Trout
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