2. 1 Is There Something Fishy About Evolution? A look at biochemical evidence for evolution

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. Biochemicals 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. Biochemicals are the basis of traits

6. 5 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

7. 6 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.

8. 7 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.

9. 8 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?

10. 9 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?

11. 10 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

12. 11 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?

13. 12 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

14. 13 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?

15. 14 Negatively charged proteins move to positive electrode Smaller proteins move faster Proteins separate by size Simulation B. How does a PAGE gel work? s-s SDS, ß-Me, heat proteins with SDS - + 2. Run the gel

16. 15 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? 3. Analyzing Results To Phylogenetic Tree - Click Here Click here to view a gel B. How does a PAGE gel work?

17. 16 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

18. 17 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

19. 18 SALMON TROUT ANCHOVIES HERRINGS SARDINES CARP MINNO W CATFISH PIKE SMELT COD HAKE POLLOCK FLOUNDER SOLE HALIBUT TUNA MACKEREL SNAPPER PERCH WALLEYE BASS Metazoa Protostome ArthropodMollusk OYSTER CLAM MUSSEL SCALLO P OCTOPUS SQUID CRAB LOBSTER SHRIMP EchinodermChordate Deuterostome GAR OstheichthyesChondrichthyes STURGEON SHARK Reptili a AvesMammali a AmphibiaAgnatha Phylogenetic Tree

20. 19 Fish Protein Analysis Gel Marker Scallop Shark Halibut Tuna Trout Salmon