MITOCHONDRIAL DNA
and
TUTORIAL
INTRODUCTION:
A. Background information
Mitochondria are cellular structures found in the cytoplasm of the cell whose main purpose is twofold:
q Complete the final stages of aerobic respiration by releasing energy from nutrients taken into the cell
q Create adenosine triphosphate or ATP, chemical energy that drives the chemical reactions in the cell.
Scientists theorize that mitochondria were prokaryotic (non-nucleated) cells that developed a symbiotic relationship with eukaryotic (nucleated) cells. The mitochondria entered the eukaryotic cells and were given protection from the environment. In return, the mitochondria supplied energy to its host. The more energy needed by the cell, the more mitochondria are present. Because mitochondria contain their own DNA, they can divide to match increasing energy needs within the cell independently of the nucleus. Thus high-energy muscle and liver cells may contain as many as 2,500 mitochondria while epidermal cells may only contain 500.
Mitochondrial DNA (mtDNA) differs from nuclear DNA in that it's helical structure is circular and contains only sixteen and a half thousand bases as opposed to the three thousand million bases on the ribbons of the double helix. (See figure above.) Further, mtDNA is only inherited from the mother as described in the information provided through the Project Disappeared (insert final website) in the GeneticTesting Methodologies section. Because of mtDNA's unique transference from mother to child, genetic histories and changes in populations over time can be traced. A paper entitled 'Mitochondrial DNA and human evolution' published by Allan Wilson, Rebecca Cann and Mark Stoneking in the January, 1987 edition of Nature presented a human evolutionary tree based upon genetic mutations in mtDNA. (Insert figure below.)
B. Tutorial Sequence
This tutorial will introduce the user to the Biology Workbench. After opening an account and logging on, one will use the Ndjinn-Multiple Database Search to specify the type of organic molecule to be studied. One will then choose the proper database from which the mtDNA sequences will be imported. In this tutorial, GenBank Primate Sequences database will be used. The imported sequences will be aligned and compared through CLUSTALW. Data set outcomes may be copied, saved or printed. Data imported during the session will be automatically saved and is available for future sessions.
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).
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.
PART TWO: Starting a New Session
On this page that you have several choices. We will begin a new session, one that you have not worked on before. If you have done other sessions they should be recorded here. 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 Start New Session and then click on Run. You will then come to the window shown below.
Here you will name your session "HVI Human mitochondria". Now click on Start New Session.
Your new session appears below the Default Session. This means that your work is being logged and will be available to you the next time you log in.
You have now named your session and have successfully logged in so that your work will be "saved" for future use.
Since we are going to be looking at sequences of DNA, we will use the Nucleic Tools. If we were to investigate proteins, we would use Protein tools. Click on the Nucleic Tools. This brings you to the Nucleic Tools homepage. Notice that it is "empty" meaning that no DNA sequences have been imported. This will change when you import sequences from a database.
To import mitochondria DNA sequences, we need to choose a search engine much like you choose a search engine for an Internet search. For this session we will use Ndjinn - Multiple Database Search.
Highlight Ndjinn - Multiple Database Search and click Run. It does not matter which Run you click.
Now type in the material for which you are conducting the search. In this case we are going to look at the hyper variable region #1 on human mtDNA. HV regions I and II are the commonly used regions for comparison. Type in HVI (that is a capital I not a 1) Human mitochondria and then change the number of hits to "Show all Hits".
You now need to choose which type of gene sequences that you want to analyze. We are going to use the GenBank Primate Sequence Database. Scroll down the page until you find that database. And highlight the box next to it.
Scroll back to the top of the page and click on Search. You will notice that the computer is busy importing sequences and may take a minute or two to complete the task.
You will see a window that looks like this:
(Note that the highlighting will not be there when the page opens to you. You will highlight the appropriate items next.)
Highlight the following gene types:
1045416
1045422
1045423
1045430
1045431
1045434
Highlight the data that you will import by holding down the control button and clicking on the left mouse button.
When those are highlighted, click on Import Sequences.
PART THREE: Aligning and analyzing sequences.
You are now brought to this window:
On the top box, scroll down and highlight CLUSTALW. This tool aligns mtDNA sequences and finds common regions among them. The sequences are aligned one on top of the other and are color coded to help see commonalties and differences. Click all the boxes next to your DNA choices and the 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. Make one change on the information boxes below the Submit button. Change Guide tree display from Text Only to Rooted and Unrooted Trees.
Once you have done the change recommended, click on Submit.
The alignment may take a minute or two. When finished, scroll down the page. The page is color-coded. Blue indicates that there is a fully conserved alignment. The black indicates no consensus.
There are two areas that need attention on the alignments. The first is:
You will note that there are a series of N nucleotides that appear in black. 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 this occurs, the computer was unable to properly 'read' the gel from the original DNA test.
You may also notice a section that looks like this:
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. There may also be sections of deletions, which may be evident in #1045416.
Scroll down to the Rooted and Unrooted Trees. These trees give a visual display of the mathematical relationships of the mtDNA sequences. The unrooted tree is a view from the "top" and the rooted tree is from the "side".
Unrooted Tree
Rooted Tree
Scroll down past the trees and you get a mathematical comparison of the sequences. This is a comparison of "likeness" from the first sequence to each of the sequences in order.
In the first box are the sequences in the order in which they were imported. The second box compares sequence 1 to sequence 2, then sequence 3 and continues until all of the first set is compared. It then takes the second sequence imported and compares it with sequence 3,4,5, and 6. Sequence 3 is compare to 4,5 and 6. For very large data sets, this type of comparison is useful. We only need to look at the first five comparisons. They appeare to have a relatively high comparison rate..
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