THE DISAPPEARED
http://www.uni.uiuc.edu/~dstone/disappeared_home.html
and
http://workbench.sdsc.edu
TUTORIAL
INTRODUCTION:
A. Background information
Mitochondria control cellular respiration and provide chemical energy for eukaryotic
(nucleated) cells. Mitochondria contain DNA and are able to divide according to the
increased energy needs of the cell. Mitochondrial DNA (mtDNA) differs from Nuclear
DNA in that it is circular in structure and contains on the order of three hundred million
fewer base pairs. Geneticists have discovered that mtDNA mutations compromise
cellular function and can be lethal (http://www.umdf.org/index_mainframe.html). During
fertilization the sperm's mitochondria are detached along with the flagella (tail) and do
not enter the egg.
Mitochondria
Flagellum
DNA packet
Hence, the only mitochondria contained in the fertilized egg are inherited from the
mother.
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The maternal inheritance of mtDNA became pivotal in the pairing of grandparents and
grandchildren after the political atrocities of the 1970's in Argentina. The Abuelas
(Grandmothers) of the Plaza de Mayo began searching for the identity of any surviving
yet missing grandchildren. In 1980 the Grandmothers approached the American
Association for the Advancement of Science in Washington, D.C. asking for their
assistance. After hearing of the plight of the missing children and grandchildren, Drs.
Marie-Clare King and Ana Maria Di Lonardo began their involvement with the
Disappeared. DNA testing seemed to be the best method of proving that children now
alive, yet living as adopted children since the 1970's. However, with the absence of the
parental DNA, lineage to the grandparents could not be determined. Knowing that
mtDNA is inherited maternally; a grandparentage test was developed. Children who
were unsure of their parentage could contact the Abuelas of the Plaza de Mayo and if the
child met the specified qualifications mtDNA comparisons with the National Genetic
Data Bank could be undertaken. To date over 100 young adults have been matched to
their maternal grandparents. For clarification see the figure below:
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Two regions on the mtDNA are used for sampling, Hypervariable Regions I and II. See
illustration below.
As described, the noncoding or control regions are those used to determine maternal
lineage. Dr, Brian Skyes (http://www.oxfordancestors.com) even offers online
information providing mtDNA testing to determine genetic linkage to the seven
daughters of Eve. The genetic mtDNA mapping creates a family tree depicting mutations
through branches and nodes on the tree. (Review maternal Inheritance figure above.)
B. Tutorial Sequence
This tutorial will simulate the technology used to determine the lineage of The
Disappeared. After logging on to Biology WorkBench, the students will be presented
with six children who have met the criteria of the Abuelas of the Plaza de Mayo and have
had their mtDNA entered into the gene bank. The students will use BLASTN to first
match their sequences with those in the Genbank Primate Sequences. They will then
compare the gene sequences through CLUSTLW and determine the maternal relative. A
second scenario will have students look into matching techniques when insertions in the
HVI region have occurred. Insertions and deletions are common in this area causing the
researcher to further study possible outcomes. Students will manipulate the data
sequences through SIXFRAME so that total alignment is possible. Rooted and Unrooted
trees as well as mathematical outcomes will determine correct maternal lineage.
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PART ONE: Opening an Account (Note: If you have already opened account,
you may skip this section.)
Go to the Biology Workbench homepage at http://workbench.sdsc.edu to open an account.
Click on the link Set up a free account (required, but painless).
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Once you are in the account page you will need to supply a user name and a password.
Please use your last name as your user name and then use a password that YOU will
remember from session to session.
When you have filled in your user name and password, click on REGISTER. You will
be taken to the Biology WorkBench homepage. Scroll down to the bottom of the
page. Change the background color to Rose. This will make it easier to see the colors
used when aligning the protein sequences. Then click on Session Tools.
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PART TWO: Resume Session
We will resume a session, one that has been set up for you. If you are unable to finish the
session in one class period, when you return to Biology WorkBench you can use
Resume Session and click forward to get to your imported data that has been saved
from the pervious session. We will be using the Session Tools and Nucleic Tools
for this tutorial.
Click on Resume Session and on the circle in front of The Disappeared. Then
click on Run. You will come to a similar window and click on Nucleic Tools. Scroll
down the Nucleic Tools choices and highlight BLASTN. BLASTN compare Gabriel's
mtDNA sequence to those in the database that we will search. Click on Run.
You will now have to choose the correct database to search. Scroll down and highlight
GenBank Primates Sequences Part I.
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Scroll back up to the top of the page and click on Submit. It will take a while to import
all the matching sequences.
Once the sequence importing is done, check the first sequence. Note that the scores are
over 400 indicating a good match. Also note the E value is 0.0 indicating an exact match,
one that did not happen by chance.
PART THREE: Aligning and analyzing sequences
Further investigation will have to be done to determine if it is an exact match. Scroll
down click on Import Sequences. This will bring you back to the Nucleic Tools
homepage. On the top box, scroll down and highlight CLUSTALW. This tool aligns
mtDNA sequences and finds common regions among them.
Click the boxes next to your imported mtDNA choice and Gabriel. Click on Run.
Again, it does not matter which run you click.
.
You are now on a "Check" page. This verifies that you have entered the information that
you want aligned. If you did not check all the appropriate boxes, you can go "Back" and
re-do your steps. Click on Submit.
The alignment may take a minute or two. When finished, scroll down the page. The
sequence alignments are color-coded. Blue indicates that there is a fully conserved
alignment. The black indicates no consensus.
Scroll down and look at the alignments. They appear to be an exact match. Go to the
bottom of the page and look at the aligning sequence numbers.
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Here is proof of an exact match. Number 1045416 must be Gabriel's grandparents.
Part C. Analyzing and Manipulating mtDNA Sequences
Return to the Nucleic Tools homepage. Delete the sequence you imported by
highlighting Delete Nucleic Seuqences and then highlighting the box next to the
desired sequence. Then click on Run.
Now choose Juanita and run a BLASTN on her. Rmember to choose the GenBank
Primate Seuqences on the intervining pages.
Once the data is present, highlight the top three choices. Then click on Import
Sequences. This takes you back to the Nucleic Tools homepage.
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Highlight the boxes next to the three newly imported sequences plus Juanita. Select
ClustralW and click on Run.
On the "check" page choose the Rooted and Unrooted Trees and click on Submit.
When ready, scroll down and look at the data. There are two areas that need attention on
the alignments. The first is:
You will note that there are a series of N nucleotides appear. This means that either a G
or an A could occur at this position. If an Y were present, it means that either a T or C
could occur at this position. Usually if an N or Y is present, the computer was unable to
properly 'read' the gel from the original DNA test.
You may also notice a section that looks like this:
Here in # 1045383 and 1045375 Guanine has replaced Adenine. This is a point mutation
that identifies these two mtDNA strands differ from the others.
In this segment, there is a series of unmatched amino acids. This area represents an
insertion of "extra" or "multiple" amino acids. These insertions do not seem to harm the
functioning of the mtDNA and are very common in humans. In this case, the #1045425
that has extra amino acids or an insertion belongs to Juanita.
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Scroll down to the Rooted and Unrooted Trees. These trees give a visual display of
the mathematical relationships of the mtDNA sequences in the form of a phylogenetic
tree. This visualization depicts similarities among the imported nucleic sequences. The
phylogenetic tree is binary and each parent branch splits into two daughter branches at
the node. The length of the branch represents an evolutionary distance between pairs of
sequences in mathematical relationship to all the imported sequences. The tree may be
drawn as a Rooted Tree in which it is assumed that there is a specific common ancestor.
Unrooted Trees only show evolutionary relationships between the imported sequences
and therefore no common ancestry. One must remember, however, that we are looking
at one specific part of the organism, i.e., the mtDNA, and not the entire organism.
Below are the examples of the Rooted and Unrooted Trees created through this tutorial.
You may wish to think of the Rooted Tree as a vertical slice of the tree, while the
Unrooted Tree is a horizontal slice thorough the tree.
Unrooted Tree
Rooted Tree
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Here, numbers 1045425 do not show an exact match even though they should. Let's find
out why.
Go back to Nucleic Tools homepage and choose Edit Nucleic Sequences.
Highlight the box shown below.
Click on Run.
Scroll down to the Sequence Box. We are going to delete the insertion in this
sequence and then should be able to match it back up to her grandmother's mtDNA.
Delete this sequence GGAGCTCTCC. It is on the sixth line down and begins with
the first double GG. Label this Juanita Edited. Go back to the top of the page and hit
Save. You will be returned to the Nucleic Tools page. Now highlight the edited Juanita
and the other three imported sequences. Run a ClustalW on them.
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The sequential and mathematical alignment shows a perfect match for Juanita.
Scroll back up and look at the Rooted and Unrooted Trees. Their visual display of
the mathematical alignments indicates a perfect match for #1045425. Thus proving that
#1045425 are actually Junita's grandparent.
Rooted Tree
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Unrooted Tree
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References
1. Sykes, Brian. The Seven Daughters of Eve, W.W. Norton & Company, New York,
2001.
2. Gibas, Cynthia and Jambeck, Per. Developing Bioinformatics Computer Skills,
O'Reilly, 2001.
3. Stone, David, Project Disappeared Web Quest,
http://www.uni.uiuc.edu/~dstone/disappeared_home.html
4. Wilson, Allan, Cann, Rebecca, and Mark Stoneking, "Mitochondrial DNA and human
evolution" Nature. January 1987.
5. Isenberg, Alice R. and Moore, Jodi M. "Mitochondrial DNA Analysis at the FBI
Laboratory", Forensic Science Communications, Volume 1, Number 2, July, 1999.
http://www.fbi.gov/hq/lab/fsc/backissu/july1999/dnalist.htm
6. United Mitochondrial Disease Foundation (http://www.umdf.org/index_mainframe.html)
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