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Nothing Fishy About Evolution:Nothing Fishy About Evolution:Explore biochemical evidence

for evolution

Adapted from a presentation by

Stan HitomiMonte Vista High School, Danville, CA.

Kirk BrownTracy High School, Tracy, CA.

Module based on a kit from Bio-Rad Laboratories, Inc.

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OutlineOutline

• Overview

• From DNA to Protein

• Taxonomy and Phylogenetic Trees

• Electrophoresis / SDS-PAGE

• Analysis of Fish Proteins

• Extension Activity

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OverviewOverview

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Question addressed in this module:Question addressed in this module:

Can we tell how closely related species are by analyzing their

molecules?

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Hands-on Evolution LabHands-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

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From DNA to ProteinFrom DNA to Protein

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Making ProteinsMaking Proteins

DNADNA: TAC CGA TCG TGA ACT

mRNAmRNA: AUG GCU AGC ACU UGA

ProteinProtein: Met-Ala-Ser-Thr-Stop

TranscriptionTranscription

TranslationTranslation

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Effect of Mutation Effect of Mutation on Proteinon Protein

DNADNA: TAC CGA TCG TGA ACT

mRNAmRNA: AUG GCU AGC ACU UGA

ProteinProtein: Met-Ala-Ser-Thr-Stop

TranscriptionTranscription

TranslationTranslation

C

G

Gly

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StructuralStructural Effects of Mutation Effects of Mutation on Proteinson 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

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FunctionalFunctional Effects of Mutation Effects of Mutation on Proteinson Proteins

• Range of possible effects– Abolish function– Slightly alter function– Generate new function– No effect on function

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Levels of Protein StructureLevels of Protein Structure

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Taxonomy and Taxonomy and Phylogenetic TreesPhylogenetic Trees

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Traditional Systematics and Traditional Systematics and TaxonomyTaxonomy

• Classification

– Kingdom

– Phylum

– Class

– Order

– Family

– Genus

– Species

• Traditional classification based upon traits:

– morphological

– behavioral

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Phylogenetic Phylogenetic TreeTree

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Electrophoresis / Electrophoresis / SDS-PAGESDS-PAGE

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ElectrophoresisElectrophoresis

• 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

-+

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Why Use Polyacrylamide Why Use Polyacrylamide Gels to Separate Proteins?Gels to Separate Proteins?

• Smaller pore size than agarose

• Proteins much smaller than DNA– average protein = 30-50 kD

– “average” DNA = >2000 kD

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Vertical ElectrophoresisVertical 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

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SDS-Polyacrylamide Gel SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)Electrophoresis (SDS-PAGE)

SDS detergent –solubilizes proteins

–negative charge added to proteins

O

S

O

O

O

-

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

SDS

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Why heat the samples?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

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• Proteins (negatively charged due to SDS) move to positive electrode

• Proteins separate by size• Smaller proteins move faster

How does SDS-PAGE work?How does SDS-PAGE work?

-

+smalles

t

largest

largesmall

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Analysis of Fish ProteinsAnalysis of Fish Proteins

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Experiment: Day 1

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Experiment: Day 2

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Experiment: Day 3

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Protein SizeProtein Size

• Size measured in kilodaltons (kDa)

• Dalton = mass of hydrogen atom

= 1 atomic mass unit

• Average amino acid = 110 daltons

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Selected Muscle ProteinsSelected Muscle ProteinsProtein kDa Functiontitin 3000 center myosin in sarcomere

dystrophin 400 anchoring to plasma membrane

filamin 270 cross-link filaments into gel

myosin heavy chain 210 slide filamentsspectrin 265 attach filaments to plasma membrane

nebulin 107 regulate actin assembly

-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 filamentstroponin (T, I, C) 30, 19, 17 mediate regulation of contraction

thymosin 5 sequester actin monomers

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Actin and MyosinActin and MyosinActin 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 Forms filaments

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Gel

An

alys

is

Lane 1. Kaleidoscope Markers 2. Shark 3. Salmon 4. Trout 5. Catfish

6. Sturgeon 7. Actin and Myosin Standard

30S

hark

Sal

mon

Tro

utC

atfis

hS

turg

eon

Gel Analysis

Compare similarities and differences of different lanes to see if correlates well with the fish evolutionary tree

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Extension ActivityExtension Activity

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Molecular Molecular Weight of Weight of

Kaleidoscope Kaleidoscope StandardsStandards

kDa mm203 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

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Molecular Weight of UnknownsMolecular Weight of Unknowns

•Measure distance migrated for selected unknown proteins on gel

•Determine size of unknowns from the graph

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