EXPLORING MOLECULAR EVOLUTION
STUDENT INSTRUCTIONS
You will be using Biology Workbench to run nuclear gene sequence alignments.
These alignments will then be used to construct phylogenetic trees to examine the
relatedness of various species based on their beta globin gene.
The Biology WorkBench is a web-based tool for biologists. It allows biologists to
search for protein and nucleic acid sequences from a variety of databases. This is
integrated with access to a wide variety of analysis and modelling tools, all within a
point and click interface.
Let’s Begin:
Either type the URL address for Biology WorkBench: http://workbench.sdsc.edu/ into
your internet browser or click the link from PowerPoint slide 19.
1. If you have never used the Biology Workbench, you must first register a free
account. Click on Set up free account, enter your details in the boxes provided and
then click on Register.
2. You will be sent back to the main page where you should click on Enter the
Biology Workbench 3.2
3. Click on the Session Tools button
4. We will begin by creating a new session, so click on Start New Session in the
white box so it highlights blue, and click Run
5. Type in a Session Description titled ‘Beta globin’ and click on Start New Session
6. We will be importing nucleotide sequences. To do this, select Nucleic Tools and
import the sequences by clicking on Add New Nucleic Sequence and then clicking
on Run
7. Hit the Browse button to find the beta globin sequence files that you saved to the
desktop. Double click on the file name so it appears in the box. Then click on
Upload File. NB. If you have not saved the sequences to desktop, get them from the
PowerPoint link on slide 18.
8. The sequences should now be imported into Workbench. Now click on Save to
save these sequences
9. First we will run a pairwise alignment for the human and chimp sequence data.
Select the box next to human and the one next to chimp by clicking on it. A
green arrow should appear in each box (see diagram on page 2).
10. Now highlight CLUSTALW – Multiple Sequence Alignment found in the white
box and click on Run (see diagram on page 2).
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11. The next page will show you the sequences you have selected. If they are
correct, click on Submit. If they are not correct go back to the previous screen
and repeat step 10.
12. Scroll down until you find the Sequence alignment. The nucleotides in blue are
completely conserved (*). The black nucleotides indicate that the nucleotides are
not totally conserved.
ANSWER QUESTIONS 1 - 3 IN STUDENT WORKSHEET
13. Click on Return to take you back to your sequences. Follow steps 9 – 12 to run a
pairwise alignment for the human and chicken sequence data. You should select
the boxes next to ‘chicken’ and ‘human’ in step 9 and follow step 10 – 12 to look
at your results.
ANSWER QUESTIONS 4 – 6 IN STUDENT WORKSHEET
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14. Now you will run a multiple alignment for the beta globin sequences from all six
organisms. Click on Return to take you back to your sequences. Click on Select
all sequences and then click on Run.
15. Follow steps 10 – 12 shown above to get your sequence alignment. Look at the
alignment and then scroll down until you find the Unrooted tree. This tree uses
similarities and differences in beta globin genes to confer relationships between
the six organisms.
ANSWER QUESTIONS 7 - 9 IN STUDENT WORKSHEET
16. When you have finished answering the questions, click on the Import
Alignment(s) button.
17. Select the box to the left of your imported sequences which reads CLUSTALW Nucleic by clicking on it. Select the DRAWGRAM – Draw Rooted Phylogenetic
Tree From Alignment option by scrolling down to find it in the white box. It
should now be highlighted in blue. Click on Run.
18. When the next screen comes up, click on the Submit button. Scroll down the
page to find your rooted phylogenetic tree.
ANSWER REMAINING QUESTIONS IN STUDENT WORKSHEET
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Name: _______________________
EXPLORING MOLECULAR EVOLUTION
STUDENT WORKSHEET
Results of your pairwise alignment comparing the beta globin gene in humans and in
chimps:
1. Each line in this alignment contains 60 nucleotides. How many nucleotides are there in the
beta globin gene for:
a) The chimp?
b) The human?
2. A blue asterix indicates that the nucleotides in both sequences are the same, we say they are
conserved. What percentage of the beta globin sequence is conserved in chimps and humans?
(Don’t include the insertion at the beginning of the human gene when making your
calculations). Show your working.
3. Would you expect the protein structure to be highly similar or markedly different in the chimp
and the human? Explain.
RETURN TO BIOLOGY WORKBENCH INSTRUCTIONS
Results of your pairwise alignment comparing the beta globin gene in humans and in
chickens:
4. What is the percentage of sequence conservation between the beta globin gene in chickens
and humans? Work this out using the second line of nucleotides only.
5. Looking at the two pairwise alignments you have performed, would you expect the beta
globin protein found in humans to be more similar to that found in chickens or that found in
chimps? Explain.
6. Do the results achieved by running these alignments support the results on evolutionary
relationships determined by scientists using anatomical homology? Explain.
RETURN TO BIOLOGY WORKBENCH INSTRUCTIONS
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Results of your multiple sequence alignment comparing the beta globin gene in a variety
of animal species:
7. Examine the Unrooted Tree produced.
Record the species at the end of each branch on the unrooted tree shown below.
8. Based on the information in the unrooted tree:
a) Which two species appear to be most closely related to each other? Explain your
choice.
b) Which two species seem to be the least closely related to each other? Explain your
choice.
9. Comparative evolutionary distance between species is indicated by the length of the clades
they are on. Give the comparative evolutionary distance (in mm) between:
a) The mouse and human
b) The wallaby and the human
c) The chimp and the human
Comment on the significance of these results given your knowledge of mammalian groups.
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RETURN TO BIOLOGY WORKBENCH INSTRUCTIONS
Results of your Rooted Phylogenetic Tree:
10. Examine your Rooted Phylogenetic Tree and record the species at the end of each branch.
11. Based on this tree diagram, which species is/are most closely related to:
a) The goldfish:
b) The mouse:
12. Homology is a term used to refer to a feature in two or more species that is similar because of
descent; it evolved from the same feature in the last common ancestor of the species. Hence,
similarity in DNA or protein sequences between individuals of the same species or among
different species is referred to as sequence homology. Which two species in the tree above
share greatest homology with respect to the beta globin gene?
13. A node is a branch point representing a divergence event from a common ancestor. Which
two species have the most ancestral nodes (divergence events) in the tree above? Explain
your answer giving the number of nodes leading to these species.
14. Looking at the phylogentic tree above, which two organisms:
a) Diverged from their common ancestor most recently?
b) Diverged from their common ancestor least recently?
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15. Draw a modified phylogenetic tree to show how the tree above might change if the beta
globin gene for a kangaroo was added to the multiple sequence alignment.
16. It is important to understand that the phylogenetic trees you generated using bioinformatics
tools are based on sequence data alone. While sequence relatedness can be very powerful as
a predictor of the relatedness of species, other methods must be used in addition to sequence
homology, to determine evolutionary relationships. Briefly describe 3 other methods used to
determine evolutionary relationships.
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