Nothing Fishy About Evolution: Explore biochemical evidence for evolution Module based on a kit from Bio-Rad Laboratories, Inc. Adapted from a presentation by Stan Hitomi Monte Vista High School, Danville, CA. Kirk Brown Tracy High School, Tracy, CA.

Outline Overview From DNA to Protein Taxonomy and Phylogenetic Trees Electrophoresis / SDS-PAGE Analysis of Fish Proteins Extension Activity

Overview

Question addressed in this module: Can we tell how closely related species are by analyzing their molecules?

Hands-on Evolution Lab Get four different fish! (grocery store, canal, pond, ocean; fresh or frozen is OK) Isolate total protein from fish muscle Use polyacrylamide electrophoresis to separate proteins by size Analyze protein profiles from a variety of fish Compare biochemical and phylogenetic relationships

From DNA to Protein

Making Proteins DNA: TAC CGA TCG TGA ACT mRNA: AUG GCU AGC ACU UGA Transcription mRNA: AUG GCU AGC ACU UGA Translation Protein: Met-Ala-Ser-Thr-Stop

Effect of Mutation on Protein DNA: TAC CGA TCG TGA ACT C Transcription mRNA: AUG GCU AGC ACU UGA G Translation Protein: Met-Ala-Ser-Thr-Stop Gly

Structural Effects of Mutation on Proteins Range of possible effects Change one amino acid Change many amino acids Shorten a protein Lengthen a protein Remove a protein Add a protein

Functional Effects of Mutation on Proteins Range of possible effects Abolish function Slightly alter function Generate new function No effect on function

Levels of Protein Structure

Taxonomy and Phylogenetic Trees

Traditional Systematics and Taxonomy Classification Kingdom Phylum Class Order Family Genus Species Traditional classification based upon traits: morphological behavioral

Phylogenetic Tree

Electrophoresis / SDS-PAGE

Electrophoresis Mixture of molecules (e.g., DNA or protein) migrates through a gel matrix Separation of molecules can be based on: Size Shape Charge Gel made of agarose or polyacrylamide - +

Why Use Polyacrylamide Gels to Separate Proteins? Smaller pore size than agarose Proteins much smaller than DNA average protein = 30-50 kD “average” DNA = >2000 kD

Vertical Electrophoresis Polyacrylamide gels are run vertically Gels must solidify in the absence of oxygen Therefore, gels poured between glass plates Forces use of comb which makes vertical wells

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) CH2 CH3 SDS detergent solubilizes proteins negative charge added to proteins SDS

Why heat the samples? Heating the samples helps denature proteins and protein complexes, allowing the separation of individual proteins by size s-s - + SDS, heat proteins with SDS

How does SDS-PAGE work? - + Proteins (negatively charged due to SDS) move to positive electrode Proteins separate by size Smaller proteins move faster - + largest large small smallest

Analysis of Fish Proteins

Experiment: Day 1

Experiment: Day 2

Experiment: Day 3

Protein Size Size measured in kilodaltons (kDa) Dalton = mass of hydrogen atom = 1 atomic mass unit Average amino acid = 110 daltons

Selected Muscle Proteins Protein kDa Function titin 3000 center myosin in sarcomere dystrophin 400 anchoring to plasma membrane filamin 270 cross-link filaments into gel myosin heavy chain 210 slide 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 actin 42 form filaments tropomyosin 35 strengthen filaments myosin light chain 27 slide filaments troponin (T, I, C) 30, 19, 17 mediate regulation of contraction thymosin 5 sequester actin monomers

Actin and Myosin Actin Myosin 5% of total protein Two heavy subunits 20% of vertebrate muscle mass Two light subunits 375 amino acids Breaks down ATP during muscle contraction Forms filaments

Gel Analysis Lane 1. Kaleidoscope Markers 2. Shark 3. Salmon 4. Trout 5. Catfish 6. Sturgeon 7. Actin and Myosin Standard

Gel Analysis Sturgeon Salmon Catfish Shark Trout Compare similarities and differences of different lanes to see if correlates well with the fish evolutionary tree

Extension Activity

Molecular Weight of Kaleidoscope Standards kDa mm 203 8.5 135 12.0 86 18.5 19 41.5 33 34.0 8 44.5 41 28.0 Size of proteins in Kaleidoscope standard is known Plot Distance Migrated (mm) vs. Size (kDa) on semi- log graph paper

Molecular Weight of Unknowns Measure distance migrated for selected unknown proteins on gel Determine size of unknowns from the graph

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