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Biochemical Evidence for Evolution: Analyzing Fish Proteins

Explore the use of biochemical evidence to determine evolutionary relationships. This lab experiment compares muscle proteins from related and unrelated fish using polyacrylamide gel electrophoresis (PAGE) to analyze and interpret results.

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Biochemical Evidence for Evolution: Analyzing Fish Proteins

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

  2. Classification Kingdom Phylum Class Order Family Genus Species Traditional classification based upon traits: structure function (behavior) I. Traditional Method for Classifying Organisms: Structure and Function

  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

  4. 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

  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

  6. 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. Primary Secondary

  7. Tertiary Quaternary 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.

  8. 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

  9. 1. What do you compare? • Muscle contains proteins of many sizes ProteinkDaFunction titin 3000 center myosin in sarcomere dystrophin 400 anchoring to plasma membrane filamin 270 cross-link filaments into gel myosin heavy chain 210slide filaments spectrin 265 attach filaments to plasma membrane nebulin 107 regulate actin assembly a-actinin 100 bundle filaments gelosin 90 fragment filaments fimbrin 68 bundle filaments actin42form filaments tropomyosin 35 strengthen filaments myosin light chain 27slide filaments troponin (T, I, C) 30, 19, 17 mediate regulation of contraction thymosin 5 sequester actin monomers

  10. 1. What do you compare? • Example proteins • 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

  11. 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)

  12. 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

  13. 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? 1. Prepare the Protein Samples

  14. 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 B. How does a PAGE gel work? 2. Run the gel

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

  16. 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 Gel Analysis

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

  18. Phylogenetic Tree TUNA MACKEREL SALMON TROUT CARP MINNOW SNAPPER PERCH WALLEYE BASS CATFISH COD HAKE POLLOCK SMELT ANCHOVIES HERRINGS SARDINES FLOUNDER SOLE HALIBUT PIKE STURGEON GAR SHARK Agnatha Chondrichthyes Ostheichthyes Amphibia Reptilia Aves Mammalia OYSTER CLAM MUSSEL SCALLOP OCTOPUS SQUID CRAB LOBSTER SHRIMP Mollusk Arthropod Echinoderm Chordate Deuterostome Protostome Metazoa

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

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