Biology Skills Set
I. Exploring Oxygen and Hemoglobin
Evolution of a Gene Cluster
Because oxygen is not very soluble in the watery part of blood, animals have evolved a number of strategies for acquiring, holding, and releasing oxygen in the blood. Chief among these is hemoglobin, a protein consisting of four polypeptide chains, each of which is complexed with its own molecule of heme. At the center of the heme molecule is an atom of iron which is where an oxygen molecule temporarily associates. An adult’s hemoglobin molecule contains two alpha (α)chains and two beta (β) chains. Hemoglobin expressed at other life stages may have two gamma (γ) chains or two epsilon (ε) chains instead of β chains or have two zeta (ζ) chains, but instead two α chains. Vertebrate muscle tissues also contain a related molecule myoglobin to store oxygen locally. Oxygen has only one positive role, yet a very important one: to accept electrons and hydrogen ions produced during metabolism. To become familiar with some of the wonders of hemoglobin consider the follow questions. Many of them can be answered quite adequately at , although there is much more information there than will be needed in this course. If you use a resource other than Wikipedia, be sure to indicate what it was. Be aware that Wikipedia, like any source, may contain errors that haven’t been caught yet. Complete the table to show the number of kinds of polypeptides found in the hemaglobin of human beings at different stages of development:
Life Stage Embryo (Gower 2)
alpha (α)
zeta (ζ)
beta (β)
gamma (γ)
epsilon (ε)
Fetus Adult Name three organisms that you were surprised to learn make hemoglobin for some reason or another.
Name one way in which the behavior of fetal hemoglobin differs from hemoglobin in adults. Why would this be a good thing?
Identify one other role of hemoglobin besides binding and releasing oxygen.
The work you’ve done to this point was to establish a common set of background knowledge and skills. For the next part of our exploration of hemoglobin, animals that make it and their evolution we will work with some additional tools and information available online. The same tools are used by professional biologists at universities and other research institutions. Because only an Internet Browser is needed to access the tools you will be able to continue and extend your use of them from your home computer, putting the power of a supercomputer at your fingertips.
ߧ Originated in BioQuest workshop 2006.06.10 by Bucher, Pape-Lindstrom & Tran revised 2007.03.20 by Michael Bucher, CSM with NSF support
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II. Evolution of the Hemoglobin Gene Cluster
Now you are going to consider the hemoglobin polypeptide chains from an evolutionary perspective. To do this we are going to use a set of tools called the “BIOLOGY WORKBENCH,” a real tool for authentic research. Your lab group needs to choose an hypothesis to investigate. ■ Hypothesis One: ■ Hypothesis Two: an evolutionary tree based on amino acid sequences of the hemoglobin chains will accurately reflect the evolutionary relationships of the major vertebrate classes. an evolutionary tree based on amino acid sequences of the hemoglobin chains will accurately reflect the evolutionary relationships within the mammalian order, primates.
■ Hypothesis Three: your group’s own hypothesis regarding evolutionary relationships reflected by amino acid sequences of hemoglobin chains. Record your group’s hypothesis here:
Consider that what we determine is accurate and true about the evolutionary past depends upon the work of many people and a careful and valid interpretation of that work. We make as few assumptions as we can, understanding that untested—and especially untestable—assumptions undermine the confidence we can place in our conclusions.
ߧ Originated in BioQuest workshop 2006.06.10 by Bucher, Pape-Lindstrom & Tran revised 2007.03.20 by Michael Bucher, CSM with NSF support
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Look at the screen shot above. You should see something like when you go to the URL at the top. Click on “register” to get username: _______________ a free account. You will only need one account per lab group. At this point, please make sure to write down your user password: ______________ name and password before you hit: Click to Enter... .. You can get your own account for doing other searches, if you are interested in doing so, but you probably can’t get access to the results of a search done while logged onto a different account Scroll down and click on Session Tools. From the menu select Start New Session and then click the Run button. You will have to name the Session before you proceed; this will make it easier to return later to view, continue or print results from your work online. You might name the session for the protein that you are investigating (globins, globin a, globin b, cytochrome b, cytochrome c, keratin, etc.)
ߧ Originated in BioQuest workshop 2006.06.10 by Bucher, Pape-Lindstrom & Tran revised 2007.03.20 by Michael Bucher, CSM with NSF support
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Be careful of Delete. You may think you have produced a fiasco, but actually may have stumbled onto something useful and interesting. Once it’s deleted it’s gone.
Click on Protein Tools and scroll down to select Ndjinn – Multiple Database Search. Click Run.
ߧ Originated in BioQuest workshop 2006.06.10 by Bucher, Pape-Lindstrom & Tran revised 2007.03.20 by Michael Bucher, CSM with NSF support
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When you get to Ndjinn, enter a search term (remember what you learned about the names of the separate molecules that make up hemoglobin) and also check the box of the database that you wish to use. A good database to choose is “Swissprot” which is in a maroon-colored row in the alphabetized list. Setting “Hits per page” at 50 per page before you click on “Search” should give plenty of choices.
Now select about 7-9 proteins* that you think will help you address the hypothesis that you identified earlier in the session. (*Actually the same protein as found in different organisms.)
ߧ Originated in BioQuest workshop 2006.06.10 by Bucher, Pape-Lindstrom & Tran revised 2007.03.20 by Michael Bucher, CSM with NSF support
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What do you think of this selection? >—> It’s a good idea to choose carefully the proteins so you don’t end up comparing “apples to oranges.” Check the boxes of the relevant proteins and scroll to the bottom of the screen to click “Import Sequences.” Once you’ve imported the sequences you can confirm (or narrow) your selection when you choose a tool to align them. Check your final selection of proteins and choose CLUSTALW Multiple Sequence Alignment before clicking Run.
ߧ Originated in BioQuest workshop 2006.06.10 by Bucher, Pape-Lindstrom & Tran revised 2007.03.20 by Michael Bucher, CSM with NSF support
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Once in CLUSTALW you’ll see your selection of protein in a screen where you will choose for “Output Order:” Aligned and for “Guide tree display:” Rooted and Unrooted Trees before clicking the Submit button.
Your results should include something like the following. What do the letters mean? You did meet letters like this in a previous lab. (EvolSeq) And the colors? A look at the “Consensus key” below and the sequence alignment itself will probably give you some insight.
When you constructed family trees from the various spellings of the word “cytochrome” you made some assumptions or even rules for yourself about interpretting the changes. You followed similar rules to draw trees from the Distance Matrices generated by EvolSeq. CLUSTALW follows its own rules and produces the trees you should see if you scroll further down the Multiple Sequence Alignment output page.
ߧ Originated in BioQuest workshop 2006.06.10 by Bucher, Pape-Lindstrom & Tran revised 2007.03.20 by Michael Bucher, CSM with NSF support
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Here are examples of a rooted and and below an unrooted trees. Since you selected different proteins the trees generated from them by CLUSTALW may not look anything like these. What do you notice about what is implied in these trees about chicks? Can that be true? What else is going on here? Look carefully.
Examine your own trees, both the rooted vs. the unrooted. Discuss in your group which conveys the relationships most clearly and why. Sketch your graph in the space provided below. Interpret your tree diagram. Discuss with your group the major relationships illustrated within your tree. Write 2-3 sentences summarizing your results and interpretation.
Do the results from your tree agree with current consensus regarding the evolutionary relationships in the group you chose to study)? Use your text or a web resource if you are uninformed of evolutionary relationships within your research area. If your animals were all mammals, look at the Mammalian Supertree.Write 2-3 sentences explaining how your tree illustrates current understanding…or not. What would your group like to investigate next using these techniques? If you found anomalies in your tree, you may wish to further investigate this by importing additional protein sequences or eliminating others. If your tree is similar to current consensus regarding evolutionary relationships, think of another set of relationships to investigate. After discussion with your group, write an original hypothesis and describe how you would test it using information that already exists in online data bases. Write a paragraph describing this that begins with your hypothesis.
ߧ Originated in BioQuest workshop 2006.06.10 by Bucher, Pape-Lindstrom & Tran revised 2007.03.20 by Michael Bucher, CSM with NSF support
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