Transcript Structure

Sicklecell Anemia - A Disease of
Diverse Populations
Jennie Aizenman
Scott Bronson
Uwe Hilgert
Sickle Cell Anemia
Red blood cells shaped like sickles.
A genetic disorder: HBBA, HBBS
Severe if both copies of gene are affected.
Benign if only one copy of gene is affected.
Why does disease persist?
HH – healthy
HS – benign
SS – severe
Malaria prevalence
HBS Allele
Mosquito (Anopheles species)
Genes and Life
How DNA Encodes Life
DNA  RNA  Protein
DNA - What are genes?
Transcription – Copying the code
Translation – Reading the code
Code Table - The Code
What Are Proteins?
Polymers of amino acids
20 different amino acids
Consist of core (-NH-CO-) and side group.
20 side groups  20 characteristics
• Go to http://info.bio.cmu.edu/Courses/Bioche
mMols/AAViewer/AAVFrameset.htm
• Set left molecule to alanine.
• Set right molecule to tryptophane.
• How do the two amino acids differ?
• Examine glutamate, proline, valine.
How Do Proteins Work?
Proteins: Structure, Catalysts, Sensors, …
Proteins are polymers (long chains) of amino acids.
Sequence  Structure  Function
Let’s look at a protein together.
Bioinformatics
DNA and protein sequences are stored in
databases. Bioinformatics provides the tools
to handle and analyze these data.
Bioinformatics is the science of unraveling
the information in biological molecules
utilizing computer technology.
Bioinformatics: Structures in Databases
NCBI: National Center for Biotechnology Information
Open browser, go to http://www.ncbi.nlm.nih.gov/
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Find Search Entrez for.
Change Entrez to Structure.
Type hemoglobin (after for).
Click Go.
Find entry 1KOY, click it, click View 3D Structure.
Hemoglobin
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Oxygen is picked up by iron (Fe)
which is held by porphyrine (ring),
which is presented by protein chain,
of which 4 aggregate in Hb molecule,
so that together (cooperatively) they influence
irons affinity to oxygen
Allosteric Effect - Cooperativity
http://www.rcsb.org/pdb/molecules/pdb41_2.h
tml
Hemoglobin’s 576 amino acids determine its
ability to bind and release oxygen and serve
as oxygen transport vehicle in red blood cells.
Find the Genes
GenBank database maintained by the National
Center for Biotechnology Information (NCBI)
Go to http://www.ncbi.nlm.nih.gov/
Which organisms can you find mentioned at the
NCBI homepage?
What other things catch your attention?
“Roadmaps” for Genomes
Map Viewer: tool to locate genes
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Go to http://www.ncbi.nlm.nih.gov/.
Find Map Viewer, click on it.
Which genomes can you access?
Click homo sapiens.
Answer the questions on your worksheet.
Search the Map
Find hemoglobin gene(s)
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Find Search for.
Into the search window type hemoglobin.
Click Find.
Answer the questions in the worksheet.
Find the Hemoglobin Beta Gene (HBB)
• Click on HBB.
• Answer the questions on the worksheet.
Find the HBB Nucleotide Sequence
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Go to http://www.ncbi.nlm.nih.gov/.
Find Search Entrez for.
Delete Entrez.
Behind for write hemoglobin homo sapiens.
Click Go.
Answer the questions in the worksheet.
Find and Examine the Nucleotide Sequence
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Click Nucleotide: sequence database (GenBank).
Click Homo sapiens hemoglobin, beta (HBB) mRNA.
Click on NM_000518.
Answer the questions on the worksheet.
Work With the Nucleotide Sequence
Transfer RNA sequence to database
• Highlight and copy nucleotide 1 through 626.
• Go to http://www.bioservers.org/bioserver/
• Under SequenceServer click Enter.
• Click CREATE SEQUENCE.
• Paste the sequence into the big window.
• Write HBB mRNA into the Name window.
• Click OK.
• This is the RNA sequence, it’s 626 bp long.
Find the Gene in the Human DNA Sequence
• How?
• What’s the difference between mRNA and a gene?
• So, how does having mRNA help you find its gene
in DNA?
Align mRNA with DNA to Identify Gene Structure
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Copy HBB cDNA from SequenceServer.
Open a second browser window.
Go to SIM 4: http://pbil.univ-lyon1.fr/sim4.php.
Paste the sequence into the cDNA window.
In SequenceServer, change HBB cDNA to
HBB gene.
• Transfer sequence into the lower window of
the SIM4 tool.
• Click Submit.
• Save result onto desktop and visualize the
alignment with LalnView.
How Can Genes Cause Disease?
What is a genetic disorder?
 An inheritable disorder.
 Discuss healthy vs. carrier vs. disease.
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Beta globin gene: HbbA (normal gene/protein)
Sickle cell gene: HbbS (mutated/faulty gene/protein)
Healthy people: HbbA HbbA
Sickle cell trait: HbbA HbbS (carrier, sickle cell disease)
Sickle cell anemia: HbbS HbbS
Find the Mutation in the Gene
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How? What’s a mutation?
Align the sequences in SequenceServer.
Go to http://www.bioservers.org/.
Change Classes to Public.
Find HBB and Sickle Cell Anemia.
On the left, check the box.
On the bottom, click OK.
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Find HBB cDNA, homo sapiens.
What does cDNA stand for?
Find the word None, click it.
Click HBS CDS, homo sapiens.
What does CDS stand for?
Find COMPARE.
Set box next to it to Align Clustal W.
Click COMPARE – WAIT!!
Find Mutations
• Mutations are changes in sequences.
• Find the differences between the HBB and
HBS nucleotide sequences.
• Answer the questions in the worksheet.
Find Mutations
• How?
• Identify what changes the nucleotide
differences cause within the protein.
• First translate the HBS and HBB coding DNA
into their respective amino acid sequences.
• How?
• Code Table - The Code
How do the DNA mutations affect the protein?
Translate DNA into amino acids.
• Click Open for HBB cDNA, homo sapiens.
• Move the cursor just before the A of the ATG
on the third line.
• Hit Return/Enter on your keyboard, this
moves the ATG to the fourth line.
• Highlight and copy the sequence from the
ATG to the end (don’t worry about the stop
codon …).
• Click Done.
How do the DNA mutations affect the protein?
Translate DNA into aminoacids.
• Open a new browser.
• Open Gene Boy http://www.dnai.org/geneboy/.
• Click Your Sequence.
• Paste the sequence into the workspace.
• Click Save Sequence (should yield 576 nucleotides).
How do the DNA mutations affect the protein?
Translate DNA into amino acids.
• On the Operations panel to the right click Transform
Sequence.
• Select Amino Acids..
• Highlight the sequence under Reading Frame RF1
and copy it.
• Open the Word program and paste the amino acid
sequence into it.
• Place a carriage return at the end of the sequence.
• Place a “>” sign in front of the sequence, followed
by the letters “HBB”.
• Type a carriage return.
How do the DNA mutations affect the protein?
• Repeat the process for the sickle cell mRNA
(HBS CDS, homo sapiens) with the following
modifications:
– Use sequence from HBS CDS, homo sapiens
instead of HBB cDNA, homo sapiens;
– copy the entire sequence;
– pasting this sequence into GeneBoy should yield
you 444 amino acids;
– write HBS before the sequence instead of HBB.
How do the DNA mutations affect the protein?
Align the amino acid sequences for HBB and HBS.
• Highlight and copy the content of the Word-file.
• Go to http://www.ebi.ac.uk/clustalw/.
• Find Enter or Paste a set of Sequences … and
paste the sequence into the box.
• Click Run.
• WAIT!
How do the DNA mutations affect the protein?
• The result window shows an alignment of the two
amino acid sequences.
• Underneath the alignment is a string of stars
denoting identical amino acids. Find the amino
acid differences between HBB and HBS. Ignore,
however, the end where only HBB shows amino
acids; this region is not part of the HBB protein.
The HBB as well as the HBS proteins end with
the amino acid sequence AHKYH.
• What are the differences between HBB and HBS?
Effect of the Glu  Val mutation on HBB Structure
How do valine and glutamate differ?
• http://info.bio.cmu.edu/Courses/BiochemMols
/AAViewer/AAVFrameset.htm.
• Set left window to valine.
• Turn the molecule so that the aminoacid-core
molecules, the red/blue “V”, is positioned on
top.
• Set the right window to glutamate; position
red/blue “V” on top.
• Compare and contrast the two structures.
• Answer the questions in the worksheet.
Effect of the Glu  Val mutation on HBB Structure
How do HBB and HBS differ?
• Open
http://www.dnalc.org/bioinformatics/sickle_cell_m
odule/hbb_cn3d.val.
• Open
http://www.dnalc.org/bioinformatics/sickle_cell_m
odule/hbbs_cn3d.val.
• Close the Message Log windows.
• Align the Cn3D windows side by side.
• Place each Sequence windows underneath its
structure.
Effect of the Glu  Val mutation on HBB Structure
How do HBB and HBS differ?
• Can you see any difference between HBB and HBS?
• Highlight the differences: E in HBB and V in HBS.
• Orient highlighted amino acids in similar positions.
• Can you identify differences now?
• Go to Style, Rendering Shortcuts, Worms.
• Zoom in on the highlighted amino acids.
• Go to Style, Rendering Shortcuts, Toggle
Sidechains.
• Go to Style, Coloring Shortcuts, Structure.
• What difference do you see between HBB and HBS?
Align the HBS and HBB Proteins
Go to NCBI at http://www.ncbi.nlm.nih.gov/.
• Find the words Search Entrez for.
• Change Entrez to Structure.
• Into the search window type HBS; hit Go.
• Click on 2HBS.
• Click on the term Chain B (find the blue bar …).
• Click on View 3D Structure.
• Click on Open.
• Maximize the Cn3D screen; align the Sequence
screen underneath; close the Message Log
screen.
Align the HBS and HBB Proteins
• How different are the two proteins?
• Identify and highlight in both sequences the
amino acid that’s different.
• Can you see a difference now?
• Go to Style, Rendering Shortcuts, click Toggle
Sidechains. Make sure the V and E in position 6
of both sequences are highlighted.
• Can you see a difference now?
• Change the highlighting from position 6 to
position 5 (Proline; P).
• Can you see a difference now?
Summary
Glutamic Acid, E
Valine, V
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Hydrophilic, Polar
Hydrophobic, Non-polar
Jennie’s slides
http://r2d2.dnalc.org/media/a/sickle_cell_big.swf
http://www.ygyh.org/sickle/causeb.htm
http://www.rcsb.org/pdb/molecules/pdb41_2.html
http://www.ygyh.org/sickle/have02.htm
http://www.ygyh.org/sickle/have03.htm
http://www.ygyh.org/sickle/have07.htm
Geographic Distribution of Hemoglobin S
Geographic Distribution of Malaria
Malaria
Caused by protozoa of the Plasmodium genus.
Overlap of malaria endemic areas (Plasmodium
falciparum) and areas where sickle cell mutation
occurs.
Sickle cell mutation confers protection from
Plasmodium falciparum.
Anopheles mosquito
Malaria infects 300 to 500 million people worldwide and accounts
for over 1 million deaths annually.