2. 1 Vertebrate Evolution: A look at biomolecular evidence using gel electrophoresis. Part 1: Introduction. Available online at www.redwood.org/stewart

3. 2 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)

4. 3 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

5. 4 DNATAC CGA TCG TGA ACTTRANSCRIPTION mRNAAUG GCU AGC ACU UGATRANSLATION tRNAUAC CGA UCG UGA ACU amino acidMet - Ala - Ser -Thr - Stop DNA  RNA  Protein  Trait A. Biomolecules are the basis of traits

6. 5 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. A. Biomolecules are the basis of traits

7. 6 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

8. 7 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

9. 8 Type of Protein Function Example Transport Transport of other substances  Hemoglobin – iron containing protein of blood  Other proteins transport molecules across cell membranes

10. 9 Type of Protein Function Example Hormonal Coordination of activities  Insulin, a hormone secreted by the pancreas, helps regulate blood sugar

11. 10 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

12. 11 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

13. 12 Type of Protein Function Example Defensive Protection against disease  Antibodies combat bacteria and viruses

14. 13 Type of Protein Function Example Enzymatic Acceleration of chemical reactions  Digestive enzymes break down food

15. 14 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

16. 15 A functional protein is not just a polypeptide chain! Polypeptide chain (yarn) – not functional C. Levels of Protein Organization Protein Structure

17. 16 A functional protein is not just a polypeptide chain! Protein Structure Protein (sweater) –functional polypeptide chain C. Levels of Protein Organization

18. 17 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.

19. 18 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.

20. 19 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?

21. 20 Muscle contains proteins of many sizes ProteinkDaFunction titin3000center myosin in sarcomere dystrophin400anchoring to plasma membrane filamin270cross-link filaments into gel myosin heavy chain 210 slide filaments spectrin 265attach filaments to plasma membrane nebulin107regulate actin assembly a-actinin100bundle filaments gelosin90fragment filaments fimbrin68bundle filaments actin 42 form filaments tropomyosin35strengthen filaments myosin light chain 27 slide filaments troponin (T, I, C)30, 19, 17 mediate regulation of contraction thymosin5sequester actin monomers 1. What do you compare?

22. 21 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

23. 22 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?

24. 23 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

25. 24 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?

26. 25 Negatively charged proteins move to positive electrode Smaller proteins move faster Proteins separate by size Simulation A, B,Simulation AB B. How does a PAGE gel work? s-s SDS, ß-Me, heat proteins with SDS - + 2. Run the gel Vert. 1 Marker Vert. 2

27. 26 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? 3. Analyzing Results B. How does a PAGE gel work? End Part 1

28. 27 Vertebrate Evolution: A look at biomolecular evidence using gel electrophoresis. Part 2: Analysis. Available online at www.redwood.org/stewart

29. 28 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

30. 29 Molecular Weight Analysis kDa mm 203 8.5 135 12.0 86 18.5 19 41.5 33 34.0 8 44.5 41 28.0

31. 30 Vertebrate Protein Gel Analysis Marker Tunicate Perch Frog Turtle Pigeon Pig

32. 31 Fish Protein Gel Analysis Marker Tunicate Perch Frog Turtle Pigeon 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?

33. 32 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

34. 33 Vertebrate Cladogram