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Vertebrate Evolution:Vertebrate Evolution:A look at biomolecular evidence using gel A look at biomolecular evidence using gel

electrophoresis. electrophoresis.

Part 1:Part 1: Introduction.Introduction.

Available online at Available online at www.redwood.org/stewartwww.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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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. Biomolecules are the basis of traits

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End Product of Transcription and Translation:

ProteinsBefore you begin a lab to use

bio-molecular evidence to determine the evolutionary

relationships of organisms, let’s take a closer look at proteins.

A. Biomolecules are the basis of traits

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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 Function Example

Transport Transport of other substances

Hemoglobin – iron containing protein of blood

Other proteins transport molecules across cell membranes

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Type of Protein Function Example

Hormonal Coordination of activities

Insulin, a hormone secreted by the pancreas, helps regulate blood sugar

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Type of Protein Function Example

Receptor

Receptors built into the membrane of nerve cell detect chemical signals release by other nerve class

Response of cell to chemical stimuli

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Type of Protein Function Example

Contractile Movement

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 Function Example

Defensive Protection against disease

Antibodies combat bacteria and viruses

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Type of Protein Function Example

Enzymatic 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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A functional protein is not just a polypeptide chain!

Polypeptide chain (yarn) – not functional

C. Levels of Protein Organization

Protein Structure

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A functional protein is not just a polypeptide chain!

Protein Structure

Protein (sweater) –functional polypeptide chain

C. Levels of Protein Organization

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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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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

III. Vertebrate 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 A, B,

B. How does a PAGE gel work?

s-sSDS, ß-Me, heat

proteins with SDS

-

+

2. Run the gel

Vert. 1

Marker

Vert. 2

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• 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?DO THEY MATCH?

3. Analyzing Results

B. How does a PAGE gel work?

End Part 1

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Vertebrate Evolution:Vertebrate Evolution:A look at biomolecular evidence using gel A look at biomolecular evidence using gel

electrophoresis. electrophoresis.

Part 2:Part 2: Analysis.Analysis.

Available online at Available online at www.redwood.org/stewartwww.redwood.org/stewart

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Gel Analysis

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

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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Vertebrate Protein Gel AnalysisMarker

Tunicate

PerchFrog

TurtlePigeon

Pig

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Fish Protein Gel Analysis

MarkerTunicat

e

PerchFrog

TurtlePigeon

Pig: arguably, most complex vertebrate (top right of cladogram)Myosin (210

kDa) = about 2000 am. acids

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*.

Measure and record distances in mm

* Do bands line up?

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Node: Specific trait (or # of proteins in common).

Organisms branching to right HAVE this trait.

Organisms branching to left DO NOT have this trait.

Family tree that branches systematically at points (nodes) representing specific traits possessed by some groups, but not others.

Branches: Organisms

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Vertebrate Cladogram

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.