Bioinformatics: Investigating Molecular/Biochemical Evidence for Evolution
Background
How does an evolutionary biologist decide how closely related two different species are? The simplest way is to
compare physical features of the species (as we did with the wild and domestic canines.) 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 most look harder at the tree to find which ancestor the two
cousins shore. Cousins do not share the same parents; rather, they share some of 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 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 biologists might
use this information to infer that frogs and salamanders had a more recent common ancestor than did frogs and fish.
But, no process is without 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. Remember the problems with the canids! 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.
There are three exercises that follow in which you will use protein databases that are in the public domain. You will
be able to investigate gene products (which are proteins) and evaluate evolutionary relationships. The protein you
will work with is the hemoglobin beta chain. You will obtain your data from public online databases that contains
the amino-acid sequences of proteins coded for by many different organisms. Hemoglobin, the molecule that carries
oxygen in our bloodstream, is composed of four subunits. In adult hemoglobin, two of these subunits are identical
and coded for by the alpha hemoglobin gene located on 16 chromosome. The other two are identical and coded for
by the beta-hemoglobin gene found on the 11 chromosome. We will use the protein sequences of the beta
hemoglobin as a set of traits to compare among species.
Part I “Are Bats Birds?”
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In Parts I and II, there are no hypotheses. Hence, you are not collecting data to determine the validity of your
hypothesis. You are learning a new skill – the mining of molecular information available to all on the internet. In
Part III, you will use your skills to test a hypothesis. Then, you will need to determine the Experimental Design
Questions. We’ll discuss these in class. However, since Part III does have a hypothesis, you will collect data to
determine the validity of your hypothesis.
Procedure Part I To be completed as a team.
Answer questions in spaces provided on Data Tables
Table 1: Morphological comparison of birds, bats, and other mammals
Feature
Birds
Bats
Presence of hair
Presence of feathers
Presence of mammary
glands
Presence of wings
Homeothermy
Four-chambered hearts
1a. What morphological features do bats share with mammals?
Other mammals
1b. Based on morphology, are bats more similar to birds or mammals?
A distance matrix is a table that shows all the pairwise comparisons between species. Continue to make all pairwise
comparisons until Table 3 is filled. For each comparison, use the percent identity for the overlap of all the 146
amino acids.
Table 2 The distance matrix for Part I
Bat 1
Bat 2
Bat 1
100%
Bat 2
100%
Bird 1
Bird 2
Mammal 1
Mammal 2
Shaded boxes are simply repeat data.
Bird 1
Bird 2
Mammal 1
Mammal 2
100%
100%
100%
100%
Use your internet access to complete Part I
2. Generate a distance matrix for the beta-hemoglobin chain for two birds species, two bat species\, and two non-bat
mammal species into a word processing worksheet. Follow the steps below to do this.
Step 1: Begin by going to www.uniprot.org
Step 2. In the Search In dialogue box, use down arrow to select Protein Knowledgebase(UniProt). In the Query box,
type “ hemoglobin beta”. Click “Search”.
Step 3. This will take you to the beginning of the database.
http://www.uniprot.org/uniprot/?query=hemoglobin&sort=score
Step 4. Use the right-hand scroll bar to scroll through the names of the many entries. Find one for either a bird, a
bat, or some other mammal. (I selected chicken) When you find one, check to make sure that it is the hemoglobin
beta chain (preferably one without a number after it) and not the alpha or gamma or other hemoglobin subunit. If
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the sequence is for the beta chain and it is for an appropriate species, click on it and the computer will retrieve the
sequence.
Step 5. Once you have selected your organisms, then hit Retreive at the bottom of the page.
Step6. Once you are at the next page, you will see the UniProt Identifiers
Step 7. Hit Align at the bottom of the page.
Step 8. Go to the FASTA choice and click Open
FASTA
Sequence data in FASTA format.
[ Download ( 2 KB* ) | Open ]
9. You will then see the FASTA data using the single letter identifiers for amino acids.
>sp|P68871|HBB_HUMAN Hemoglobin subunit beta OS=Homo sapiens GN=HBB PE=1 SV=2
MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPK
VKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFG
KEFTPPVQAAYQKVVAGVANALAHKYH
>sp|P02112|HBB_CHICK Hemoglobin subunit beta OS=Gallus gallus GN=HBB PE=1
SV=2
MVHWTAEEKQLITGLWGKVNVAECGAEALARLLIVYPWTQRFFASFGNLSSPTAILGNPM
VRAHGKKVLTSFGDAVKNLDNIKNTFSQLSELHCDKLHVDPENFRLLGDILIIVLAAHFS
KDFTPECQAAWQKLVRVVAHALARKYH
10. However, you do not need to actually use these FASTA format, but you now know what they look like.
11. What you need to do is click Align which is found at the bottom of the page next to Retrieve.
Scroll down to see what was found in the Uniprot database.
Alignment
1 --MLTAEEKAAVTAFWGKVKVDEVGGEALGRLLVVYPWTQRFFESFGDLSTADAVMNNPK 58 P02070 HBB_BOVIN1
MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPK 60 P68871 HBB_HUMAN
*********.:**:*: ***:******:****************:******* ***::**
59 VKAHGKKVLDSFSNGMKHLDDLKGTFAALSELHCDKLHVDPENFKLLGNVLVVVLARNFG 118 P02070 HBB_BOVIN
61 VKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFG 120 P68871 HBB_HUMAN
*********.:**:*: ***:******:****************:******* ***::**
119 KEFTPVLQADFQKVVAGVANALAHRYH 145 P02070 HBB_BOVIN
121 KEFTPPVQAAYQKVVAGVANALAHKYH 147 P68871 HBB_HUMAN
***** :** :*************:**
Here are some items to note in this “alignment”
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Query
>sp|P02112|HBB_CHICK Hemoglobin subunit beta OS=Gallus gallus GN=HBB
PE=1 SV=2
MVHWTAEEKQLITGLWGKVNVAECGAEALARLLIVYPWTQRFFASFGNLSSPTAILGNPM
VRAHGKKVLTSFGDAVKNLDNIKNTFSQLSELHCDKLHVDPENFRLLGDILIIVLAAHFS
KDFTPECQAAWQKLVRVVAHALARKYH
>sp|P68871|HBB_HUMAN Hemoglobin subunit beta OS=Homo sapiens GN=HBB PE=1
SV=2
MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPK
VKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFG
KEFTPPVQAAYQKVVAGVANALAHKYH
Date run
Sept 2, 2011
Running time
9.2 seconds
Identical
positions
102
Identity
69.388%
Similar
positions
29
Algorithm
clustalw




147 amino acids in each HBB
* indicated identical amino acids
: and . indicate similarities between two amino acids (conserved properties)
Blank space indicates no similarity at all between two amino acids.
12. Continue scrolling down until you see Job Information
Some analysis of this information:
 102/147 amino acids are identical = 69.388%
 29/147 are similar
 16/147 are not identical
13. Now go back and do a search, retrieval and alignment for Human HBB and Bovine HBB. What would you
expect to see to the % identity? What do your results tell you?
14. Of course, you could compare all three or any number of organisms at once.
At this point, you should have the tools to complete Parts I, II and III.
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Table 3 . Amino Acid Symbols
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Procedure Part II “Reptiles with Feathers?” You may complete as a team.
Some phylogenetic systematists (scientists who work to make the classification of organisms match their
evolutionary history) complain that the vertebrate class Reptilia is improper because it should include birds. In
technical terms, the vertebrate class Reptilia is paraphyletic because it contains some but not all of the species that
arose from the most recent common ancestor to this group. Just how similar are reptiles and birds in terms of
the beta-hemoglobin chain? Should birds be considered a type of reptile? Evaluate this question using a
BLAST (Best Local Alignment Search Tool) search.
A BLAST (Best Local Alignment Search Tool) search takes a particular sequence and then locates the most similar
sequences in the entire database. A BLAST search will result in a list of sequences with the first sequence being
most close to the one entered and the last sequence being least similar
Repeat steps in Procedure Part I to gather data to answer the questions posed above.
Table 4: Results of a BLAST search on the crocodile beta-hemoglobin sequence
Similarity
Species name & name of protein
First most similar
(do not use crocodile)
Second most familiar
Third most familiar
Fourth most familiar
Fifth most familiar
Sixth most familiar
Seventh most familiar
Eighth most familiar
Ninth most familiar
Tenth most familiar
1.
2.
3.
4.
5.
Were any of those species birds?
One unusual reptile is the tuatara, whose name is Sphenodon punctatus. How similar is the tuatara to the
crocodile?
Does the tuatara appear in your list of ten? If not, how far down on the BLAST search list does it occur,
fifteenth, twentieth?
Most importantly, which species are more similar to the crocodile? (birds, or other reptiles?)
Do the molecular data suggest that Reptilia is paraphyletic, or monophyletic? Explain.
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Part III
Problem: The first two parts of this exercise gave you background and tools to consider phylogenetic relationships
among a variety of organisms. Do you ever wonder how closely related certain organisms were to each other?
Well, now you have the background and tools to develop a working hypothesis. Select a group of 5-7 organisms
(plants, animals, fungus, protists, bacteria, viruses) and develop a cladogram that depicts the phylogenetic
relationship among the organisms. You should have data to back-up your cladogram arrangement.
Hypothesis:
Materials:
Procedure:
See Parts I and II above.
Data:
Analysis
Conclusion
Do your data support or not support your hypothesis? Cite specific reference to data.
Error Analysis:
Further Question/s to Investigate
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