Bioinformatics at the DNALC!
In April 2003 it was announced that the final draft sequence of the human genome was
complete. This monumental achievement is fueling tremendous research efforts to
understand the information our DNA sequence encodes. Scientists have begun to identify
genes, define the proteins these genes may produce, and understand how these proteins
function. To achieve these goals, biologists are integrating computer-based tools into their
research routines. This new field, called bioinformatics, allows scientists to make sense of
huge amounts of sequence data and to "mine" genomes for meaning.
Students visiting the DNALC will have the unprecedented opportunity to work with the same
computer tools and data that genome scientists use. The six computer-based modules listed
below integrate enticing content with hands-on computer exercises. Students will analyze
human, plant, bacterial, and viral genomes; compare DNA sequences across species; study
the evolution of modern humans; understand how variations in DNA sequence contribute to
disease; view three-dimensional structures of proteins; and learn about new strategies for
developing therapeutic drugs.
All classes are two and a half hours in length, and will be conducted in our state-of-the-art
Biomedia computer lab.
Sickle Cell Anemia – A Disease of Diverse Populations
This computer-based lab will explore the molecular biology of sickle cell anemia from DNA
sequence, to protein structure, and ultimately to disorder. Students will compare sickle cell
gene sequences from patients around the world to elucidate the multiple origins of the
disease. Computer simulations will address questions about why the sickle cell mutation
continues to persist in several areas of the world. Students will also learn about current and
emerging therapies to improve the lives of individuals with this disorder.
RESERVATION DETAILS
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Bioinformatics labs are restricted to students in grades 10, 11, and 12.
Each Curriculum Study school is limited to four reservations – one free! – during
academic year 2003-04. Non-Curriculum Study schools are limited to three
reservations.
The group lab rate is $13 per student with a minimum fee of $260.
Unless other arrangements have been made in advance, all Bioinformatics labs begin
promptly at 9:30 AM.
Classes cancelled less than one month prior to their scheduled date will not be
permitted additional computer lab visits.
Reserve by phone; contact Amanda McBrien at (516) 367-5175.
Sickle-Cell Anemia – A Beneficial Genetic Disorder?
A Disease Persists – Does Looking at the Distribution Help Understand?
Map: distribution of sickle-cell anemia
Maps: distribution of malaria and anopheles spec.
How DNA Encodes Life
DNA  RNA  Protein
Schematic representations in Genome  Genome Mining  Gene Features
Animations on transcription and translation in Code
What Is a Genetic Disorder?
Example: Sickle Cell Anemia
Caused by faulty protein, due to mutated gene
What’s a mutation
SS is lethal, SA benign.
Why does it persist? Map: coincidence of malaria and HBS because HBS provides some
resistance for people infected with malaria.
How can it be detected? Electrophoresis of proteins: HBS runs faster than HBB  AA: 1 large
band; AS: 2 bands, 1 large and 1 small; SS: 1 small band
Proteins – How/Why They Work
Example: Hemoglobin
Goal: To find the hemoglobin beta in protein in databases, to examine its structure, and to learn
how to handle a protein structure viewer.
Physiology: cells need glucose and oxygen to make energy – how do they get oxygen?
Red blood cells: circulating; loading oxygen in lungs; unloading oxygen in tissue
Why red? Iron.
Hemoglobin molecule: consists of four pairwise identical subunits: hemoglobin alpha (HBA) and
hemoglobin beta (HBB). Each carries a ring-shaped molecule (porphyrin-ring) which harbors an
iron atom to form heme. The iron is the atom that captures the oxygen, the porphyrine anchors
iron in the protein, the protein directs the iron to bind and to release the oxygen.
What are proteins? Chains of amino acids.
How do they work? Because they have a very specific 3D-structure AND because they have very
specific amino acids in very specific places.
What’s with the amino acids?
Let’s look at four:
Glu
Pro
Val
A big one!!!
The characteristics of the amino acids in proteins direct proteins’ folding AND reaction capacity.
Hemoglobin: Usually, iron would just be by itself. However, correctly folded hemoglobinchains are able to harbor porphyrin-rings which, in turn, are capable of holding iron atoms (one
per chain).
Usually, iron binds oxygen very strongly. However, correctly placed amino acids in the
surrounding hemoglobin-chain regulate the ability of iron to hold on to oxygen.
Let’s look at cool images of proteins.
Go to NCBI (http://www.ncbi.nlm.nih.gov/)
Find the words Search Entrez for
Change Entrez to Nucleotide
Into the search window type HBB homo sapiens mRNA; hit Go.
Click on NM_000518
Click on Links (right side of the page)
Click on Protein
Click on NP_000509
How many amino acids does the protein have? 147 aa
Click on BLink
Click on 3D structures
Find the listing for accession 1KOYB; click on the blue circle for this entry.
Click Open
Maximize the graphic window (Cn3D 4.1); move the Sequence/Alignment Viewer window to the
lower edge of the screen; close the Cn3D Message Log window.
HOW TO ZOOM IN AND OUT
Left- click on the black background and press z to zoom in.
Click on the black background and press x to zoom out one step.
Hit x again.
HOW TO ROTATE THE MOLECULE
To rotate the molecule grab a corner of the molecule with your left mouse button, move your
mouse.
HOW TO MOVE THE MOLECULE
To move the molecule hold Shift down, grab the molecule with your left mouse button, move
your mouse right or left, up or down.
What different things can you identify in this view? Protein, porphyrin rings, iron, some organic
and anorganic molecules.
HOW TO CHANGE WHAT’s HIGHLIGHTED
Go to Style
Click Rendering Shortcuts.
Click Worms.
Go to Style.
Click Coloring Shortcuts.
Click ….
What different types of structures can you identify in this view? Beta sheets and alpha helices.
What’s the protein structure? Hemoglobin beta chain
What protein is HBB a part of? hemoglobin
How many different iron-bearing structures (porphyrin rings) can you identify? 4
What are they and what function do they serve? Heme-groups; oxygen-transport
Four subunits, two alpha and two beta chains.
HIDE/SHOW
Go to Show/Hide, click on everything that’s not highlighted, click Apply, click Done.
What happened? The entire hemoglobin molecule became visible.
CHANGE RENDERING
Go to Style, Coloring Shortcuts, click Molecule
What happened? The four different subunits are shown.
Go to Style, Rendering Shortcuts, click Worms.
What happened? The two different protein substructures alpha-helices and beta sheets became
visible.
How did they get to these images? They are determined by x-ray crystallography.
Purify and concentrate the protein.
Crystallize the protein.
Project x-rays through protein and onto film.
Develop film.
Analyze the patterns of shades and white spaces on the film.
Put the puzzle together to see where the atoms are that produced the shades – Voila! You see a
protein.
Not guess-work but thorough analysis!!!
A Mapquest For The Human Genome
Goal: To find out where in the human genome hemoglobin genes are located and what the
characteristics of the hemoglobin beta gene (HBB) are. Is HBB an ORF gene or a spliced
gene?
Go to NCBI (http://www.ncbi.nlm.nih.gov/)
Find the words Map Viewer; click on them.
The genomes of how many different organisms can be accessed through Map Viewer? 19
organisms
Find Homo sapiens (human); click on it.
How many chromosomes does the human genome consist of? 23, 24, or 25 chromosomes.
Justify your response.
How many chromosomes are on display? 25 chromosomes
Why this number? The human genome consists of 25 separate DNA entities.
Find Search for; type hemoglobin into the search window; click Find
On what chromosome(s) can you locate entries containing the word hemoglobin? 6, 7, 8, 11, 16,
and X
Check the list underneath the image and find out on which chromosome hemoglobin beta, HBB
is located. Chromosome 11.
Click on the link HBB.
Use the ruler next to the gene and determine the length of the gene. (Hint: the numbers on the
ruler mark nucleotide positions on the chromosome. Use your computers calculator to determine
the length of the HBB gene.) 1,600 bp
What three different structures can you identify in the cartoon representing the HBB gene? Filled
blue box, empty blue box, blue line
What do you think the blue boxes represent? Coding sequence that’s translated into protein
What the blue lines? introns
The Coding Sequence
Goal: To isolate and examine the DNA sequence encoding the hemoglobin beta protein.
Go to NCBI (http://www.ncbi.nlm.nih.gov/)
Find the words Search Entrez for
Following the word for type the words hemoglobin homo sapiens
Click the button Go
How many nucleotide entries did Entrez locate? 9,686
How many protein entries did Entrez locate? 535
How many protein structures did Entrez locate? 107
Find and click Nucleotide: sequence database (GenBank)
Does the listing only show entries for humans? If no, which else? No, rat, worm, mustard weed,
a bacterium
On the page listing the hits find Homo sapiens hemoglobin, beta (HBB), mRNA
Click on NM_000518
Study the entry
How many basepairs (bp) long is the nucleotide sequence displayed? 626bp
At what nucleotide position is the start codon located? That is the position where the coding
sequence of the mRNA (CDS) begins. 51
Where does the coding sequence end? 494
How many nucletoides long is the coding sequence? (The result must be a multiple of 3!) 444
Which of the three possible different stop codons TAA, TAG, TGA terminates the CDS? TAA
How many aminoacids (aa) is the protein long? 147
What kind of nucleotide polymer is the sequence represent? mRNA
How do you explain that the sequence does not contain U’s? To simplify the work with
nucleotide sequences databases only use A, C, G, and T, even though RNA does not contain T’s
but U’s. This is ok, however, because all you need to know whether a molecule is DNA or
RNA. If it’s RNA you could just replace all T’s with U’s to get a realistic representation of the
RNA molecule.
In order to work with the sequence, transfer it to a database called Sequence Server.
Highlight and copy the entire nucleotide sequence from nucleotide 1 through 626.
Go to the DNALC BioServers at http://www.bioservers.org/bioserver/
Under SequenceServer click Enter.
Close the pop-up Manual.
Click CREATE SEQUENCE.
Paste the sequence into the Sequence window.
Give the sequence some name (e.g. HBB mRNA) and write it into the Name window.
Click OK.
This is the RNA sequence, it’s 626 bp long.
The Gene
Goal: To find the HBB gene in human genomic DNA.
Identify the gene in the gene sequence (link) by aligning it with the mRNA sequence.
Highlight and copy the HBB mRNA sequence from SequenceServer.
Open http://pbil.univ-lyon1.fr/sim4.php
Paste the sequence into the first window cDNA Sequence.
Copy and highlight the genomic DNA sequence (link) and paste it into the lower window
Genomic Sequence.
Click Submit.
Visualize the alignment with LalnView.
Which represents the genomic sequence, Seq1 or Seq2? Which the RNA (cDNA)? Seq1 is
cDNA, Seq2 is gDNA
Which are the introns and exons? Exons are black boxes, introns are empty boxes
At which position in the RNA did the coding sequence (CDS) begin? Where was the start codon?
Nucleotide 51 Would that be at the beginning of a black box or somewhere within? 51
nucleotides into it So, do exons beging with start codons or do the begin before an actual start
codon? Exons begin before the start codon, a start codon is usually located within an exon.
The Mutation
Goal: To identify the DNA mutation and amino acid change leading to the HBS protein
Mutations in DNA:
Align the mRNA/coding sequence for HBB and HBS to find out where they differ.
Go to http://www.bioservers.org/bioserver/
Under SequenceServer click Enter.
Click Manage Groups.
In the upper right-hand corner find Sequence Sources, change Classes to Public.
Find HBB and Sickle Cell Anemia.
Check the box at the LEFT margin, click OK at the bottom of the page.
Go to the word None, click on the downward arrow at the right to open pull-down menu.
Click HBS CDS, homo sapiens.
Go to COMPARE and set the box to the right of it to Align: CLUSTAL W (it may be set already)
Click COMPARE to align the two sequences – WAIT!
Maximize the result window.
How many differences can you find between the two sequences? 4
What are the differences between the two sequences? T/C, A/T, T/A, T/A
Which nucleotides are different? (Hint: count the A in the start codon ATG as 1) 9, 20, 172, 387
Which amino acid positions in the protein would these differences effect? 3, 7, 58, 129
Mutations in proteins:
Get the amino acid sequences for betaglobin and sickle-cell globin by translating the DNAs.
Find HBB cDNA, homo sapiens, click Open.
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,
In a new browser window open Gene Boy (http://www.dnai.org/geneboy/)
Click Your Sequence.
Paste the sequence into the workspace.
Click Save Sequence (your sequence should have 576 nucleotides).
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.
Repeat this process for the sickle cell mRNA (HBS CDS, homo sapiens) with the following
modifications:
use 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.
Now you should have both amino acid sequences in the Word-file, both preceded by a line that
starts out with a “>” followed by the sequence name. Save the file as “betaglobin mutation”
Look at the two sequence – why do you think the one for HBB is longer than the one for HBS?
Because the nucleotide sequence used t o generate the HBS protein contained the coding
sequence exclusively – from start codon to stop codon. The HBB nucleotide sequence contained
132 nucleotides beyond the stop codon. Stops are identified with a star “*”. Examine where the
stops within the HBB amino acid sequence are located to identify the end of the HBB protein. It
should end with the same amino acids as the HBS protein.
Align the two amino acid sequences to identify how they differ:
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 and wait until the result is displayed.
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.
How many differences can you find between the two amino acid sequences? 1
Counting Met (M) as 1 what are the positions of the differences? Amino Acid 7
What are the differences? Glutamate in HBB vs. valine in HBS
How many differences did you find on the DNA level? 4
Where did you expect these differences to be located in the protein? 3, 7, 58, 129
Did any of your expectations turn out right? Yes, position 7 is different.
How about the others? DNA variation did not lead to aa variation due to redundancy of genetic
code.
Effect of mutation on protein structure
The mutation obviously has a significant effect on the function of the protein. Let’s see how the
exchange from E in HBB to V in HBS affects the protein structure.
1) What is the difference between glutamate (E) and valine (V)?
Go to http://info.bio.cmu.edu/Courses/BiochemMols/AAViewer/AAVFrameset.htm
Set left window to valine
What is the chemical formula for the Valine sidegroup? -CH-(CH3)2
CH3 is not charged, it is not ionic. It’s electrochemically neutral.
Is the sidegroup for Valine charged (polar) or not (non-polar)? No, its non-polar, not charged 
hydrophobic; rejects water
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.
What is the chemical formula for the Glutamate sidegroup? –CH2-CH2-COOH
COOH releases an electron to become COO-; it is a charged molecule.
So, is the sidegroup for Glutamate charged (polar) or not (non-polar)? Its polar, charged 
hydrophilic; accepts water
Open a new browser window and go back to the protein view
Let’s see how the differences between glutamate and valine affect the protein structure
The Sickle-Cell Protein
Goal: To determine the differences in the structures of hemoglobin beta (HBB) and sickle-cell
hemoglobin (HBS)
Open this file (link) to view the HBB structure and this file (link) for the HBS structure.
Arrange the two images side by side; align the Sequence windows for each structure underneath
the respective image screen and adjust the sizes so that they fit side-by-side as well. Close the
Message Log windows.
Identify in the sequence the amino acid which is different between the two proteins.
In both sequences click on this amino acid - this will highlight the amino acid within the protein,
too.
Moving the proteins about, can you identify any differences in the protein structures?
Zoom into the image and center on the highlighted amino acid.
Under Style Rendering Shortcuts choose Toggle Sidechains – can you identify the differences in
the sidegroup structures for Valine (V) in HBS and Glutamate (E) in HBB?
View an alignment of the HBB and HBS Proteins
Go to NCBI (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.
How different are the two proteins? Not at all.
Identify and highlight in both sequences the amino acid that’s different.
Can you see a difference now? Nope
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? Sure
Change the highlighting from position 6 to position 5 (Proline; P).
Can you see a difference now? Nope.
The Effect of the Mutation
The change from glutamate to valine in position 7 of the hemoglobin beta chain does not affect the
capability of the molecule to transport oxygen.
The introduction of valine in place of glutamate introduces a hydrophobic molecule into the position that
was previously occupied by a hydrophilic molecule. This generates a site that tries to minimize contact
with water (or hydrophilic molecules) – by means of connecting to other hydrophobic amino acids. If the
protein is loaded with oxygen the hemoglobin protein is twisted in a fashion that turns the valine to the
inside of the protein, covering it from the surrounding hydrated environment – everything is as in the
regular hemoglobine molecule. Release of oxygen, however, leads to a change in the 3D-structure exposing the valine to the surrounding hydrated environment. This generates a sort of “sticky-ness”, a
region which is available to connect with other hydrophobic (or at least neutral) amino acids in order to
form a local niche from which water is excluded. And Jennie is going to show you how that causes sicklecell hemoglobin to malfunction and cause discomfort and disease.
The Disease
RESOURCES
WWW Resources:
The Mistery of the Crooked Cell at the University of North Carolin at
http://www.unc.edu/cell/files/extensions/mystery/mystery.html
The Sickle Cell Information Center at http://www.scinfo.org/toc.htm
Sickle Cell Disease Association of America (SCDAA) at http://www.sicklecelldisease.org/
The American Sickle Cell Disease Association (ASCDA) at http://www.ascaa.org/
National Heart, Lung, and Blood Institute at http://123819272.net/Diseases/Sca/SCA_WhatIs.html
NIH’s Medline at http://www.nlm.nih.gov/medlineplus/sicklecellanemia.html
NCBI’s Online Mendelian Inhertiance in Man (OMIM) at
http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=603903
NCBI’s Genes and Disease resources at
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowSection&rid=gnd.section.98
The Humane Genome Sequencing Project at
http://www.ornl.gov/TechResources/Human_Genome/posters/chromosome/sca.html
Howard University’s Center for Sickle Cell Disease at http://www.huhosp.org/sicklecell/
Harvard University’s Information Center for Sickle Cell and Thalassemia Disorders at
http://sickle.bwh.harvard.edu/
WHO’s Malaria Information at http://www.who.int/tdr/diseases/malaria/diseaseinfo.htm
CDC’s Malaria Information at http://www.cdc.gov/travel/malinfo.htm
The Museum of South Africa’s Information on Malaria, Anopheles, and Plasmodium at
http://www.museums.org.za/bio/apicomplexa/plasmodium.htm