Introduction
There are several ways to compare organisms and look for homologous traits. One way is through their morphological (or physical) characteristics. Physical characteristics that organisms share based on common ancestry are called anatomical homologies. If these similarities in structure are not due to shared ancestors and instead due to convergent evolution (or facing similar adaptive pressures), we call them anatomical analogies.
Another more recent way that evolutionary biologists compare organisms to look for close evolutionary relationships is through molecular homologies, similarities in proteins or DNA (sequence and function) due to common ancestry. Because it isn’t realistic for us to compare entire genomes of organisms, we are going to focus just on one protein to look for molecular homologies.
This activity will allow you to compare five different animals using a specific protein called beta-hemoglobin.
And guess what? By the end of this, you will be familiar with using the sequencing tool called UniProt, which is used in a variety of molecular biology settings and allows scientists from all over the world to enter and compare sequencing information with specific focus on sequence and function of proteins. This is an essential research-related tool and skill!
Find Animals to Compare
- Begin by going to UniProt database at http://www.uniprot.org/
- In the search box in the top toolbar type the phrase hemoglobin beta and click “search”. The computer will retrieve many entries and display them on multiple pages.
- Go back to your search box and add the word “chicken” to your search. You should now see a chicken entry show up who has had its hemoglobin beta protein sequenced.
- Choose 1 result from the database, trying to find one that shows hemoglobin subunit beta (beta chain, not gamma or alpha) and do not have numbers after the entry name (ex: choose one with HBB, not HBB1).
- Write down the animal’s common name in your notebook, along with the entry name (Ex: HBB_BMC)
- Choose “Add to Basket” in the top toolbar. You should see the number “1” pop up next to the Basket in the top right corner of the page.
- Repeat step 3 for FOUR more animals you are interested, making sure you end up with 5 total animals in your basket. You can use any 5 animals you are interested in.
Make Comparisons
- Now you should have 5 animals in your basket. Click on “Basket.”
- Select 2 animals to compare by clicking on the box in front of their names. Click on “”
- This will make a data request to compare the protein functions for the two animals you have selected. The program may take a minute or two to run.
- The protein (chain of amino acids) sequence is located under the “Alignment” heading in the middle of the page. Find the amino-acid sequence.
- The amino acids are indicated by single-letter symbols. This shows you the actual alignment of the two sequences and allows you to compare the sequence and function of the amino acid across the two organisms.
An * (asterisk) indicates positions which have a single, fully conserved residue.
Same amino acid group, same function
A : (colon) indicates conservation between groups of strongly similar properties
Different amino acid group, similar function
A . (period) indicates conservation between groups of weakly similar properties
Different amino acid group, less similar function
A – (dash) indicates there is no amino acid in that location
No amino acid in sequence to compare amino acid group or function
- Look farther down on the page to the “Result Information” heading and find the “Identity.” Record the value in your notes.
- This value is the percent of amino acids that are similar between the two proteins. If all the amino acids were the same, the percent identity would be 100%. The different amino acids between the organisms are the results of mutations that have accumulated in the DNA over time.
- More differences would yield a lower percent identity and indicate that the organisms are less closely related. Few differences would yield a higher percent identity and indicate that the organisms are more closely related.
- Repeat Steps 2-5 until you have the “percent identity” value for all side-by-side comparisons between all of your organisms. On the table below, write the % for each alignment, and identify the organisms by common name.
Build a Tree!
Use the data in your table above to build and draw a phylogenetic tree, which is a visual representation of how you think organisms are related to one another. For a brief overview of trees, check out this link to Understanding Evolution (Phylogenetic Trees). Remember that the two species with the highest percent identity will be paired together on the tree. Distances in the tree will increase as the percent identity goes down and additional organisms are added. Make a sketch of your final tree.
{Note: You can use the draw tools above in Word (Go to Draw tab for tools), along with inserting a TEXT box (Go to insert – find text box) to add in the names of your organisms.}
Compare Your Tree to the Uniprot Tree!
- Now go back and click on the box in front of all five organisms. Click on the “Align” button to run a comparison of all 5 organisms at the same time. This process may take longer to compute than the side by side comparisons.
- A phylogenetic tree will appear in the results. Compare this tree to your phylogenetic sketch. Don’t worry about the lengths of the lines on the graph but check to see if the relative position of one organism relative to the others is the same. How does the uniprot tree compare to your tree? Make adjustments to your tree if needed.
Take a Stance! Construct a Scientific Argument
Using the results of your investigation, make a claim about one of the relationships found in your 5 animals. Support your claim with EVIDENCE and REASONING from your uniprot results.
CLAIM:
EVIDENCE:
REASONING:
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