Marine Biology
Investigating Evolutionary Questions Using Online Molecular Databases
Lesson Background and Overview
How does an evolutionary biologist decide how closely related two different species are? The simplest
way is to compare the physical features of the species (their morphologies). This method is very similar to
comparing two people to determine how closely related they are. We generally expect that brothers and sisters
will look more similar to each other than two cousins might. If you make a family tree, you find that brothers
and sisters share a common parent, but you must look harder at the tree to find which ancestor the two cousins
share. Cousins do not share the same parents; rather, they share the same grandparents. In other words, the
common ancestor of two brothers is more recent (their parents) than the common ancestor of two cousins (their
grandparents), and in an evolutionary sense, this is why we say that two brothers are more closely related than
two cousins.
Similarly, evolutionary biologists might compare salamanders and frogs and salamanders and fish. More
physical features are shared between frogs and salamanders than between frogs and fish, and an evolutionary
biologist might use this information to infer that frogs and salamanders had a more recent common ancestor
than did frogs and fish.
This methodology certainly has problems. Two very similar looking people are not necessarily related,
and two species that have similar features also may not be closely related. Comparing morphology can also be
difficult if it is hard to find sufficient morphological characteristics to compare. Imagine that you were
responsible for determining which two of three salamander species were most closely related. What physical
features would you compare? When you ran out of physical features, is there anything else you could compare?
Many biologists turn next to comparing genes and proteins. Genes and proteins are not necessarily better than
morphological features except in the sense that differences in morphology can be a result of environmental
conditions rather than genetics, and differences in genes are definitely genetic. Also, there are sometimes more
molecules to compare than physical features.
You will use data in a public protein database of gene products (proteins) to evaluate evolutionary
relationships. You will select a protein to work with. You will obtain your data from a public online database
that contains the amino-acid sequences of proteins coded for by many genes for many different organisms.
Objectives:
1. Find protein sequences of two marine organisms from each of the following Phyla (Porifera, Cnidaria,
Mollusca, Arthropoda, Echinodermata and Chordata.) Try to find the same protein sequence so that
they can be compared. Be careful not to select fragmented sequences. Complete Table 1.
2. Make pair wise comparisons of organisms for which you have found the same protein sequence. Present
results in a data table. In some cases you may need to construct more than one table if you could not
locate sequences for the same protein.
3. Generate of phylogenetic tree for all organisms for which you have the same protein sequence.
4. Write one to two paragraphs discussing the results of your investigation.
* Adapted from Puterbaugh, M.N. & Burleigh, J.G. (2001). “Investigating Evolutionary Questions Using
Online Molecular Databases.” The American Biology Teacher, 63 (6): 422-431, August. © 2001 ENSI
Procedure: (Be sure to download and save the Word document)
1. Search the Protein Knowledgebase (UniProtKB) to find two examples of marine organisms from each of the
following Phyla: (Porifera, Cnidaria, Mollusca, Arthropoda, Echinodermata and Chordata)
a. Try and find the same protein for each organism or at least amongst some of them
Step 1: Begin by going to http://www.uniprot.org/help/uniprotkb.
Step 2: Using the figure above as a guide go towards the top of the page, select Protein Knowledgebase
(UniProtKB), and under “Query” type the phrase Porifera and click “search.” The computer will retrieve many
entries and display them.
Step 3: You are able to scroll through the names of the many entries on this page. Identify two examples of
marine sponges that have the sequence for the same protein. Once you have selected your species, click on the
accession number.
Step 4: The next screen looks confusing, but what we need to pay attention to is the protein sequence under the
"Sequence information" section. You will have to scroll down the page to find it (see figure on next page.) The
amino acids are indicated with their single-letter symbols, and every 10th amino acid is marked with its
position.
Step 5: Click on the “FASTA ” link. See figure above to find the location. This will simply provide you with
the condensed sequence for that species, along with the species identification.
Here is a sample:
>sp|P02140|HBB_CARAU HEMOGLOBIN BETA CHAIN - Carassius auratus (Goldfish).
VEWTDAERSAIIGLWGKLNPDELGPQALARCLIVYPWTQRYFATFGNLSS
PAAIMGNPKVAAHGRTVMGGLERAIKNMDNIKATYAPLSVMHSEKLHVDP
DNFRLLADCITVCAAMKFGPSGFNADVQEAWQKFLSVVVSALCRQYH
Step 6: Copy and paste the sequence into your Word document. Do not reformat the information in any way.
Be sure to record the common name, scientific name and protein being used of the species you chose in the
correct location in Table 1.
Step 7: Repeat steps 3-6 until your word-processing sheet contains the FASTA formatted sequences for twelve
different marine species.
Step 8: Save your Word document, but do not close it.
Step 9: To align the sequences and determine how similar they are, go to the “Align” tab at the top of the page.
Step 10: Select, copy and paste one sequence from your word processing sheet into the sequence box, press
enter, then select, copy and paste a second sequence. Note: the second sequence should be directly under the
first and to obtain reasonable results you must be comparing the same protein.
Step 11: Click on “Align Sequences.” (You may need to wait a short time for this to be done.) When the
alignment is completed your screen should look like the figure below.
Step 12: At this point, we want to figure out the percent similarity between the two protein sequences you
chose. In other words, how close are the two amino acid sequences to being the same? (If all the amino acids
were the same, the percent would be 100). This page will show you the actual alignment of the two sequences.
Identical amino acids are marked with an asterisk symbol (*). If there is one or two dots (. or :), the change in
amino acid is conservative (both amino acids have similar properties and charge). If there are no dots, the two
amino acids have different biochemical properties. See next step to save time!
Step 13: The program will calculate this number for you or you can calculate it yourself. Refer to the following
diagram to identify the % similarity or % identity.
Step 14: Create a table on your Word document showing all of your comparisons. You may need to create
more than one table if you were not able to find sequences for the same protein for all of your organisms.
Example:
Table2: Percent similarity of the 40S ribosomal protein S21 from three different marine organisms in the
Phylum Porifera.
Amphimedon queenslandica
Sycon ciliatum
Suberites domuncula
Amphimedon queenslandica
100
35
41
Sycon ciliatum
XXXXXXXXXXXXXXXX
100
35
Suberites domuncula
XXXXXXXXXXXXXXXX XXXXXXXXXXXXX
100
Step 15: Take all of the sequences that are for the same protein and align them at the same time. This will
allow you to generate a phylogenetic tree to compare evolutionary relationships. See snapshot below. Print
the screen and include this in your data presentation. Be sure to label the species name on the tree!