Transcript - Cal State LA - Instructional Web Server

Sickle Cell Anemia
An example of why:
a change in protein can lead to disease
a change in DNA can lead to a change in protein
Ground Rules for Class
Discussions and Workshops
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Be on time.
Speak so that everyone from front to back
can hear you.
Listen when others are speaking.
If it’s review for you, use you intellect to
hear it in a new way.
Write down your answers or consolidate to
print.
Central Dogma
DNA
RNA
Protein
What do you already know about
hemoglobin?
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What is the function of hemoglobin?
What class of biomolecules does hemoglobin
belong to?
What are the symptoms of sickle cell
anemia?
Is sickle cell anemia hereditary?
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What does that tell us?
Symptoms of Sickle Cell Anemia
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pain episodes
strokes
increased infections
leg ulcers
bone damage
yellow eyes or
jaundice
early gallstones
lung blockage
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kidney damage and
loss of body water
in urine
painful erections in
men (priapism)
blood blockage in
the spleen or liver
(sequestration)
eye damage
low red blood cell
counts (anemia)
delayed growth
Proteins
synthesized from amino acids
Circle; Triangle; Square; Bond; Amino terminal; Carboxy terminal
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4 classes of structure.
Website for Amino acid
interactive Workshop
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Amino acids – everyone open to this page
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http://www.biomed.curtin.edu.au/biochem/tutori
als/AAs/AA.html
Power of the R Groups
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Note the one letter and 3 letter abbreviations for your amino acid(s).
Identify the atoms in red, blue, white, gray, and other colors
Find the carboxy group, amino group, beta carbon, R group
Categorize the amino acids – and be able to say why – some fit in more
than one category!
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Aromatic
Aliphatic, unbranched
Aliphatic, branched
Polar
Positively charged (basic)
Negatively charged (acidic)
Small
Has a sulfur atom in the R group
Hmm?
Which
Which
Which
Which
Which
are hydrophobic vs. hydrophilic?
would attract each other if brought together?
would repel?
would likely fold to the interior in an aqueous environment?
would likely fold to the exterior in a lipid environment?
Amino acid characteristics
Beta sheet
Branched R groups
Val, Thr, Ile
Helix disrupters with
close H bond participants:
Ser, Asp, Asn
Alpha helix is the “default”
Ala, Glu, Leu
Turns (not shown):
Gly, Asp, Pro
The Nucleic Acids: DNA and RNA
DNA
synthesized from deoxynucleotide triphosphates
(dNTPs)
RNA
synthesized from nucleotide triphosphates
(NTPs)
OH
OH
dNTP
5’ and 3’
OH
NTP
Website for interactive workshop
for DNA analysis
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DNA sequence
Write the primary sequence of the DNA
displayed in 3B from the 5’ to the 3’ end of
both strands
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DNA  RNA 
PROTEIN
Replication
Central Dogma
DNA
Transcription
RNA
Translation
Protein
DNA  RNA 
PROTEIN
Transcription
DNA  RNA  PROTEIN
RNA processing
RNA, but NOT mRNA
RNA, but NOT mRNA
Mature mRNA
Central Dogma
DNA
Transcription
RNA
Translation
Protein
Translation
DNA  RNA  PROTEIN
Translation
5’ UTR
Etc.
DNA
RNA (with ribosomes)
Translation exercise
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Translate the following sequence using the codon table:
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Perform same procedure on the sequence below using a
software program:
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ATG GTG CAC CTG ACT CCT GAG GAG AAG TCT GCC GTT ACT
ATG GTG CAC CTG ACT CCT GTG GAG AAG TCT GCC GTT ACT
http://us.expasy.org/tools/dna.html
How many nucleotides have changed in the codon in
boldface?
What is the amino acid difference in the two sequences?
What is the quality of that difference with respect to R
groups?
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The early evidence that sickle cell anemia is caused by an amino acid change
in hemoglobin. Tryptic digest: the protease trypsin cleaves C terminal to
lysine and arginine.
Summary
DNA (mutated = changed)
RNA (mutated)
Protein (possibly mutated)
Remember: Mutation is not always “bad”! For example:
Mutation → Evolution → An additional normal genome
Multiple sequence alignment for
cytochrome C – mutation and
conservation
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Human protein accession number AAA35732
(see next slide)
Dog protein accession number XP_532493
Yeast protein number from structure
database
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1YCC
CLUSTAL W PROGRAM
FASTA format for a protein sequence in single letter code
Hemoglobin HBB1
>gi|4504349|ref|NP_000509.1| beta globin [Homo sapiens]
MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPD
AVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLL
GNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVAN ALAHKYH
How to prepare the sequences
for the MSA on ClustalW
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For Human and Dog
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For Yeast
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Go to NCBI
Select to search the protein database from the dropdown menu
Enter the Accession Number (previous slide) and GO
Click on the link
Change the display to a FASTA file
Copy the FASTA output for both species into a single text file.
Make sure the header is separate from the sequence.
Clink on the link, find the FASTA format and copy into the same file
Copy or upload the file into ClustalW
Workshop due as email to me by
9AM Wednesday, 6/24.
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Give the answers to questions/challenges from slides within today’s
PowerPoint.
Print out your ClustalW results and add a short paragraph discussing
how Clustal W gives you a clue as to which part(s) of the Cytochrome
C protein you would hypothesize are most important to its function
(which is/are the same in all 3 organisms). Start your paragraph as a
hypothesis as to which parts are most important, and write your
discussion as a defense of your hypothesis.
What is the chromosomal location of the gene that causes sickle cell
anemia?
What is the name of the gene?
State the nucleotide change and amino acid change that leads to sickle
cell anemia (there may be more than one change that gives rise to the
disease)
If sickle cell anemia is so devastating, why has it lasted in the
population for such a long time? Give a molecular, mechanistic,
evolutionary explanation (you may have to do a little research to get
this). What does the sickled molecule do that the normal molecule
can’t?