Transcript Are All Fish Related? A look at biochemical evidence for

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.
1
Outline
• Overview
• From DNA to Protein
• Taxonomy and Phylogenetic Trees
• Electrophoresis / SDS-PAGE
• Analysis of Fish Proteins
• Extension Activity
2
Overview
3
Question addressed in this module:
Can we tell how closely related
species are by analyzing their
molecules?
4
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
5
From DNA to Protein
6
Making Proteins
DNA:
TAC CGA TCG TGA ACT
Transcription
mRNA:
AUG GCU AGC ACU UGA
Translation
Protein:
Met-Ala-Ser-Thr-Stop
7
Effect of Mutation
on Protein
DNA:
TAC CGA TCG
C
TGA ACT
Transcription
mRNA:
AUG GCU G
AGC ACU UGA
Translation
Protein:
Met-Ala-Ser-Thr-Stop
Gly
8
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
9
Functional Effects of Mutation
on Proteins
• Range of possible effects
– Abolish function
– Slightly alter function
– Generate new function
– No effect on function
10
Levels of Protein Structure
11
Taxonomy and
Phylogenetic Trees
12
Traditional Systematics and
Taxonomy
• Classification
– Kingdom
– Phylum
– Class
– Order
– Family
– Genus
– Species
• Traditional
classification based
upon traits:
– morphological
– behavioral
13
Phylogenetic
Tree
14
Electrophoresis /
SDS-PAGE
15
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
16
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
17
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
18
SDS-Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
CH3
CH2
CH2
SDS detergent
CH2
CH2
–solubilizes proteins
–negative charge added to
proteins
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
O
S
O
-
O
SDS
19
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
+
20
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
-
+
21
Analysis of Fish Proteins
22
Experiment: Day 1
23
Experiment: Day 2
24
Experiment: Day 3
25
Protein Size
• Size measured in kilodaltons (kDa)
• Dalton = mass of hydrogen atom
= 1 atomic mass unit
• Average amino acid = 110 daltons
26
Selected Muscle Proteins
Protein
kDa
Function
titin
dystrophin
filamin
3000
400
270
center myosin in sarcomere
anchoring to plasma membrane
cross-link filaments into gel
myosin heavy chain 210
slide filaments
spectrin
nebulin
a-actinin
gelosin
fimbrin
265
107
100
90
68
attach filaments to plasma membrane
regulate actin assembly
bundle filaments
fragment filaments
bundle filaments
actin
42
form filaments
tropomyosin
35
strengthen filaments
myosin light chain 27
slide filaments
troponin (T, I, C)
thymosin
mediate regulation of contraction
sequester actin monomers
30, 19, 17
5
27
Actin and Myosin
Actin
Myosin
5% of total protein
Two heavy subunits
20% of vertebrate muscle Two light subunits
mass
375 amino acids
Breaks down ATP during muscle
contraction
Forms filaments
Forms filaments
28
Gel Analysis
Lane
1. Kaleidoscope Markers
2. Shark
3. Salmon
4. Trout
5. Catfish
6. Sturgeon
7. Actin and Myosin Standard
29
Shark
Salmon
Trout
Catfish
Sturgeon
Gel Analysis
Compare similarities
and differences of
different lanes to see if
correlates well with the
fish evolutionary tree
30
Extension Activity
31
Molecular
Weight of
Kaleidoscope
Standards
• Size of proteins in
Kaleidoscope
standard is known
• Plot Distance
Migrated (mm) vs.
Size (kDa) on semilog graph paper
kDa
mm
203
135
86
8.5
12.0
18.5
41
28.0
33
34.0
19
41.5
8
44.5
32
Molecular Weight of Unknowns
•Measure distance
migrated for
selected unknown
proteins on gel
•Determine size of
unknowns from
the graph
33
Biotechnology
Explorer Program
Serious About Science Education
34