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Mystery of the Crooked Cell:Investigate Sickle Cell Anemia
Using Gel Electrophoresis
Module developed at BostonUniversity School of Medicine
Presented by Dr. Dan Murray
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Outline
Sickle Cell Anemia
Central Dogma of Biology
Genetic Code
Hemoglobin
Electrophoresis
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Sickle Cell Anemia
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Sickle Cell Anemia
Genetic Disease
Heterozygous individuals carriers
Homozygous individuals diseased
Hemoglobin
Found in red blood cells
Carries oxygen to tissues
SCA Results from Defective Hemoglobin
Hemoglobins stick together
Red blood cells damaged
Complications from low oxygen supply to tissues
Pain, organ damage, strokes, increased infections, etc.
Incidence highest among Africans and Indians
Heterozygotes protected from Malaria
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Central Dogmaof Biology
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Central Dogma of Biology
Transcription:Conversion ofinformation fromDNA to mRNA
Translation:Conversion ofinformation fromRNA to protein
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The Genetic Code
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The Genetic Code
start
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Met
5 3mRNA sequence
N-terminus C-terminu
Protein chains always beginwith Met
Protein sequence
The Genetic Code
AUG AAC AAU GCG CCG GAG GAA GCG GAG
53 orientationcorresponds toN-term C-term orientation
Met---AsnMet---Asn---AsnMet---Asn---Asn---AlaMet---Asn---Asn---Ala---Pro---Glu---Glu---Ala---Glu
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Hemoglobin
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Hemoglobin
Multi-subunit protein (tetramer)
2 and 2 subunits
Heme
One per subunit
Has an iron atom
Carries O2
In red blood cells
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Sickle Cell Hemoglobin
GUG CAC CUG ACU CCU GAG GAG AAGval his leu thr pro glu glu lys1 2 3 4 5 6 7 8
GUG CAC CUG ACU CCU GUG GAG AAGval his leu thr pro val glu lys1 2 3 4 5 6 7 8
Mutation(in DNA)
Normal mRNA
Normal protein
Mutant mRNA
Mutant protein
Glutamate (glu), a negatively charged amino acid,
is replaced by valine (val), which has no charge.
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Sickle Cell Hemoglobin
Significant change
in structure caused
by the single mutation
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A Possible Cure for
Sickle Cell Anemia?During fetal development, a differentgene (gamma) produces hemoglobin
Expression of gamma gene stopsnaturally during development
Research efforts focused on stopping
silencing of gamma geneWould provide sickle cell patients withgood hemoglobin
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Electrophoresis
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Gel Electrophoresis
Method for separating molecules (DNA,
proteins, etc.) on the basis of physical orchemical properties such as:
(1) size
(2) shape
(3) electrical charge
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Electrophoresis of DNA
Gels are made of agarose or polyacrylamide
DNA samples loaded, voltage applied
Negatively charged DNA migrates toward + electrode
Smaller DNA fragments migrate faster
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Electrophoresis of Proteins
More complex than DNA electrophoresis
Different proteins have different charges
Proteins vary widely in shape
Polyacrylamide is usually the gel
medium
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Protein ElectrophoresisNon-Denaturing conditions
Non-denaturing (native): no pretreatment ofproteins before electrophoresis
Proteins retain normal shape
Proteins retain normal charge Proteins separated on basis of charge, size, and shape
Name Charge Mass Shape
Protein Q
Protein R
+2 30kD
4 42kD
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Non-Denaturing Electrophoresis of
Normal and Mutant Hemoglobin
Charge, Size, Shape
Q. Which of the above properties will be different fornormal Hemoglobin (HgA) and mutantHemoglobin (HgS)?
A. Charge: Yes, HgA has one more than HgS.Size: No, HgA and HgS are the same size.
Shape: Yes, the shapes are different.
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Migration Rates of Normal and
Mutant Hemoglobin
Which Hg migrates faster during electrophoresis?
NA NA
Normal
(HgA)
Mutant
(HgS) Reason
Charge
Size
Shape
HgA has one more than HgS
Amino acids Val andGlu about same size
HgA more compact
than HgS
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Protein Structure
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Protein Structure
1 = Primary Structure
2 = Secondary Structure3 = Tertiary Structure
4 = Quaternary Structure
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Primary Structure
Definition - Sequence of amino acids in aprotein
Example Primary structure of the enzymelysozyme:
1 2 3 4 5 126 127 128 129
Lys-Val-Phe-Gly-Arg...Gly-Cys-Arg-Leu
Note: By convention, amino acid sequences are
written starting with the amino terminus.
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Secondary Structure
Definition Regular patterns of relativelysmall segments of a protein held togethermainly by H-bonds
-helix -structure
http://www.ultranet.com/~jkimball/BiologyPages/S/SecondaryStructure.html
Examples:
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Tertiary Structure
Definition Overall 3-D shape of a protein.Two basic types are globularand fibrous.
Globular (Pepsin) Fibrous(Collagen)
http://www.ultranet.com/~jkimball/BiologyPhttp://dwb.unl.edu/Teacher/NSF/C10/C10Links/
main.chem.ohiou.edu/~wathen/chem302/ rotein.htmla es/S/Secondar Structure.html
Examples:
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Quaternary Structure
Definition
Overall 3-D shape of a multi-subunitprotein
Rabbit muscle
glycogenphosphorylase
Example:
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All Levels of Structure
htt ://sosnick.uchica o.edu/ rec uastru.html
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Protein Electrophoresis
Denaturing conditions
Proteins treated with SDS (anionic detergent) beforeelectrophoresis (SDS-PAGE)
SDS molecules bind to the Protein
Proteins lose normal shape
Proteins all have same charge/mass ratio
Proteins are separated on basis of size only
SDS treatment
Charge Mass
+3 30kD
4 42kD
Charge Mass
300 30kD
420 42kD