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Is There Something Is There Something Fishy About Evolution?Fishy About Evolution?

A look at biochemical evidence for evolutionA 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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DNA TAC CGA TCG TGA ACTTRANSCRIPTIONTRANSCRIPTION

mRNA AUG GCU AGC ACU UGATRANSLATIONTRANSLATION

tRNA UAC CGA UCG UGA ACU

amino acid Met - Ala - Ser -Thr - Stop

DNA DNA RNA RNA Protein Protein Trait Trait

A. Biochemicals are the basis of traits

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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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Primary

Secondary

C. Levels of Protein Organization

1. Primary Structure -

Proteins begin as a straight chain of amino acids.

2. Secondary Structure -

The chains begin to bend and twist like a corkscrew or a flat folded sheet.

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Quaternary

Tertiary

C. Levels of Protein Organization

3. Tertiary Structure -

The twisted chain folds even more and bonds form, holding the 3-dimensional shape.

4. Quaternary structure -

Several amino acid chains in the tertiary structure come together. This is a functional protein.

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• 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

D. Comparing Protein Size

1. What do you compare?

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• Muscle contains proteins of many sizes

Protein kDa Functiontitin 3000 center myosin in sarcomere dystrophin 400 anchoring to plasma membranefilamin 270 cross-link filaments into gel

myosin heavy chain 210 slide filamentsspectrin 265 attach filaments to plasma

membranenebulin 107 regulate actin assembly a-actinin 100 bundle filaments gelosin 90 fragment filamentsfimbrin 68 bundle filaments

actin 42 form filaments tropomyosin 35 strengthen filaments

myosin light chain 27 slide filamentstroponin (T, I, C) 30, 19, 17 mediate regulation of

contractionthymosin 5 sequester actin monomers

1. What do you compare?

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• Actin:•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

1. What do you compare?• Example proteins

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D. Comparing Protein Size

• 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)

2. How compare?

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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

III. Fish Protein Analysis Lab

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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

B. How does a PAGE gel work?

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• Negatively charged proteins move to positive electrode • Smaller proteins move faster • Proteins separate by

size

• Simulation

B. How does a PAGE gel work?

s-sSDS, ß-Me, heat

proteins with SDS

-

+

2. Run the gel

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• Compare banding patterns among the fish - identify similarities and differences among them.

• Illustrate the relationships among the fish.

• Compare illustration based on biomolecular evidence to an illustration based on traditional classification

» DO THEY MATCH?DO THEY MATCH?

3. Analyzing Results

To Phylogenetic Tree - Click Here

Click here to view a gel

B. How does a PAGE gel work?

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Gel Analysis

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

1 2 3 4 5 6 7

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Molecular Weight Analysis

kDa mm 203 8.5

135 12.086 18.5

19 41.5

33 34.0

8 44.5

41 28.0

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SALMONTROUT

ANCHOVIES HERRINGS SARDINES

CARPMINNOW

CATFISH

PIKE

SMELTCODHAKEPOLLOCK

FLOUNDERSOLE

HALIBUT

TUNAMACKERELSNAPPER

PERCHWALLEYEBASS

Metazoa

Protostome

ArthropodMollusk

OYSTERCLAMMUSSEL

SCALLOPOCTOPUSSQUID

CRABLOBSTERSHRIMP

Echinoderm Chordate

Deuterostome

GAR

OstheichthyesChondrichthyes

STURGEON

SHARK

Reptilia Aves Mammalia

AmphibiaAgnatha

Phylogenetic Tree

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Fish Protein Analysis GelMarker

ScallopShark

HalibutTuna

TroutSalmon