1. 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.

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

3. Overview

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

5. 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

6. From DNA to Protein

7. 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

8. 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

9. 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

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

11. Levels of Protein Structure

12. Taxonomy and Phylogenetic Trees

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

14. Phylogenetic Tree

15. Electrophoresis / SDS-PAGE

16. 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 - +

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

18. 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

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

20. 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

21. 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

22. Analysis of Fish Proteins

23. Experiment: Day 1

24. Experiment: Day 2

25. Experiment: Day 3

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

27. Selected Muscle Proteins
Protein kDa Function
titin center myosin in sarcomere
dystrophin anchoring to plasma membrane
filamin cross-link filaments into gel
myosin heavy chain 210 slide filaments
spectrin attach filaments to plasma membrane
nebulin regulate actin assembly
a-actinin bundle filaments
gelosin fragment filaments
fimbrin bundle filaments
actin 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

28. 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

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

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

31. Extension Activity

32. Molecular Weight of Kaleidoscope Standards
kDa mm
Size of proteins in Kaleidoscope standard is known
Plot Distance Migrated (mm) vs. Size (kDa) on semi- log graph paper

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

34. Biotechnology
Explorer Program
Serious About Science Education