1 Phylogenetic Analysis Homework assignment This assignment will be completed on your own and turned in the week of 11/8-11/10. Introduction Molecular evolution is the study of how proteins and nucleic acids evolve. Included in this field are studies of mutations and chromosomal rearrangements, the evolutionary process, the identification of sequence patterns conferring function in proteins and nucleic acids, and the reconstruction of the evolutionary history of organisms and the molecules that they make. All of these studies rely on comparisons of nucleotide or amino acid sequences. In this tutorial, you will be introduced to some of the fundamental principles of molecular evolution and the types of bioinformatics tools that are used in evolutionary studies. We will begin by carrying out a manual sequence comparison, so that the basic concepts can be introduced, and the remainder of the project will be carried out at The Biology Workbench, a set of bioinformatics analysis programs managed by The San Diego Supercomputing Center at the University of California, San Diego. Objectives • To introduce the principles of molecular evolution • To acquaint you with the tools that are available to compare nucleotide and amino acid sequences • To learn about the use of protein sequences in reconstructions of evolutionary history Project Branching evolution occurs when one ancestral species gives rise to two or more progeny species. However, speciation events don't involve the vast majority of the genes in a genome. That is, for most genes, both of the progeny species inherit identical genes from the ancestor. Following speciation, these genes evolve independently in the separate lineages. Studies of molecular evolution therefore rely heavily on comparisons of related sequences from different organisms. Shown below is an alignment of two homologous sequences that we will use as a starting place. Homologous sequences are sequences that have descended from a common ancestral sequence. You can't meaningfully compare sequences unless they are homologous. This alignment uses the single letter amino acid code, in which G represents glycine, Q represents glutamine, etc. The aligned proteins have been shown to be involved in the metabolism of similar, but different, toxic compounds. As you can see, these amino acid sequences are very similar and it is easy to recognize that they are related by common descent. 2 dntAc: KMGVDDEVIVSRQNDGSVR nahAc: KMGIDDEVIVSRQSDGSIR An expanded version of this alignment is shown below. In this expanded alignment, both the amino acids and the corresponding DNA nucleotides are shown. For ease of analysis, the codons have been broken into separate entries in a table. Alignment of nahAc and dntAc sequences. K M G V D E V I V dntAc AAA ATG GGC GTC GAT GAA GTC ATC GTC nahAc ...

1. 1
Phylogenetic Analysis Homework assignment
This assignment will be completedon your own and
turned in the weekof 11/8-11/10.
Introduction
Molecular evolution is the study of how proteins
and nucleic acidsevolve. Included in this
field are studies of mutations and chromosomal
rearrangements, the evolutionary process,
the identification of sequence patterns conferring
function in proteins and nucleic acids,
and the reconstruction of the evolutionary history of
organisms and the molecules that
they make. All of thesestudies rely on comparisons
of nucleotide or amino acid sequences.
In this tutorial, you will be introduced to someof
the fundamental principles of molecular
evolution and the types of bioinformatics tools that
are used in evolutionary studies. We
will begin by carrying out a manual sequence
comparison, so that the basicconcepts can
be introduced, and the remainder of the project
will be carried out at The Biology
Workbench, a set of bioinformatics analysis
programs managed by The San Diego
Supercomputing Center at the University of

2. California, San Diego.
Objectives
• To introduce the principles of molecular evolution
• To acquaint you with the tools that are available to
compare nucleotide and
amino acid sequences
• To learnabout the use of protein sequences in
reconstructions of evolutionary history
Project
Branchingevolution occurs when one ancestral species
gives rise to two or more progeny
species. However, speciation events don't
involve the vast majority of the genes in a
genome. That is, for most genes, both of the
progeny species inherit identical genes from
the ancestor. Following speciation, these genes
evolve independently in the separate
lineages. Studies of molecular evolution therefore rely
heavily on comparisons of related
sequences from different organisms.
Shown below is an alignment of two homologous
sequences that we will use as a starting
place. Homologous sequences are sequences
that have descended from a common
ancestral sequence. You can't meaningfully
compare sequences unless they are

3. homologous. Thisalignment uses the single letter
amino acid code, in which G represents
glycine, Q represents glutamine, etc. The aligned
proteins have been shown to be involved
in the metabolism of similar, but different, toxic
compounds. As you can see, these amino
acid sequences are very similar and it is easy to
recognize that they are related by common
descent.
2
dntAc: KMGVDDEVIVSRQNDGSVR
nahAc: KMGIDDEVIVSRQSDGSIR
An expanded version of this alignment is shown
below. In this expanded alignment, both
the amino acidsand the corresponding DNAnucleotides
are shown. For ease of analysis,
the codons have been broken into separate entries in
a table.
Alignment of nahAc and dntAc sequences.
K M G V D E V I V
dntAc AAA ATG GGC GTC GAT GAA GTC ATC GTC
nahAc AAA ATG GGT ATT GAC GAG GTC ATC GTC

4. K M G I D E V I V
S R Q N D G S V R
dntAc TCC CGC CAG AAC GAT GGC TCG GTG CGA
nahAc TCT CGG CAG AGC GAC GGT TCG ATT CGT
S R Q S D G S I R
This region was chosen at random to represent
the changes that take place in
nucleotide sequences over time.
Answer the questions below by manually comparing
these sequences (this section is
for your own understanding, you do not need to turn
this in.)
1. Assuming that the dntAc sequence represents the
ancestral sequence, how
many nucleotide changes (mutations) have
occurred in this region to create the
nahAc nucleotide sequence? Remember that in
actuality neither sequence
represents the ancestral sequence.
2. Ofthesenucleotide changes, how many of these
changed the amino acid
encoded by that codon (i.e, how many were
nonsynonymous changes)?

5. 3. How many nucleotide changes were in the
first codon position? How many of
these altered the encoded amino acid?
4. How many nucleotide changes were in the
second codon position? How many
of these altered the encoded amino acid?
5. How many of the nucleotide changes were in
the third codon position? How
many of thesealtered the encoded amino acid?
6. Compare the % identity of thesetwo sequences at
the nucleotide vs protein
3
level. Percent identity is equal to (# of
positions in common / total # positions)
* 100.
Nucleotide % identity
Amino acid % identity

6. 7. Why is therea difference between amino acid %
identity and nucleotide percent
identity?
If needed, a table of the single letter amino
acid code can be found at:
http://umber.sbs.man.ac.uk/dbbrowser/bioactivity/aacodefrm.ht
ml
If needed, a codon table can be found at:
http://www.pangloss.com/seidel/Protocols/codon.html
The manual analysis that you just carried out
introduced you to someof the ways that
molecules evolve. The purpose of that manual
analysis was to get you thinking about
the mechanisms by which genes and proteins
change over time,and the types of forces
that control those changes. For example, when
we do analyses of this type we almost
always see many more changes in the 3rd
position of codons than in the first position.
Why is this? Do you thinkthat thesenucleotides
mutate at a higher rate than
nucleotides in the first position? What else
might be responsible for this
phenomenon?
Several computer tools have been developed that allow

7. you to quickly retrieve, align,
and compare genes and proteins from different
organisms. The remaining portion of
this tutorial will be carried out using The
Biology Workbench, a set of bioinformatics
analysis programs managed by The San Diego
Supercomputing Center at the University
of California, San Diego. The Biology Workbench
integrates a wide variety of different
programs, and this site can be used for many
different kinds of analyses in addition to
molecular evolution studies.
4
Homework Assignment
You are going to retrieve, align, and compare
hemoglobin protein sequences from a variety
of animals. Print out the phylogenetic trees for both b-
hemoglobin & a- and b -hemoglobin.
Don’t forget to writeyour name on the
assignment. Each tree will be worth 10
points. There
are additional questions in the protocol that you should
answer for your lab manuals only.
Bonus points (10):
Do a third separate alignment of a gene of interest
to you from 5 different species, print out
and include the phylogenetic tree.

8. Or
Extend the hemoglobin analysis for 5 additional
species suggested at the end of the
assignment, print out and include the phylogenetic
tree.
Project 1: b-hemoglobin gene alignment
Hemoglobin is a protein in red blood cells
that is involved in oxygen transport. It
belongs to
a family of related globin oxygen-binding
genes that has evolved through a number of
speciation and gene duplication events.
In the instructions below, the actions that you
will need to take on the internet site are
indicated in boldface type.
Computer-Aided Sequence Comparisons
First, you will need to access The Biology Workbench at:
http://workbench.sdsc.edu/
In order to use the site, you need to set up a
free account. To do this, select Register and
follow the instructions.
You will be taken to a new session. You
can carryout several different projects at the
same time using The Biology Workbench, and it
keeps your different projects in different
folders, which are referred to as sessions.
Now, select Protein Tools to begin.
Retrieving protein sequences using The Biology
Workbench.

9. You are going to retrieve, align, and compare
hemoglobin proteins from a variety of
different animals. You will do this using a tool
named Ndjinn. Click on protein tools and
then use the menu to select Ndjinn -
Multiple Database search, then select Run. First,
you need to select the database to use for the
search. In the alphabetical list of
databases select Swissprot database. SWISSPROT is
a protein sequence database that
was begun in 1986 and is maintained
collaboratively by the Swiss Institute for
Bioinformatics and the European Bioinformatics Institute.
This database has recently
been updated, and is now divided into divisions
(human, rodent, mammal, vertebrate,
etc.).Be sure to search the correct division for
each organism.
Secondly, in the search box, enterthe term beta
hemoglobin in order to search for beta
hemoglobin sequences. Change hits per page to 50
Then select Search. A list of
5
sequences will be returned (lots and lots of hemoglobin
sequences are in the databases,
which is one of the reasons why we are using
this protein). You want to select the box
next to the entrylabeled
SWISSPROT:HBB_HUMAN
Hemoglobin subunit beta (Hemoglobin beta chain)

10. (Beta-globin) [Homo sapiens (Human)]
This result won't be at the top of the list.
Make sure that you selected the sequence for
the beta chain, and not the alpha, delta or gamma
sequences.
Now, select Import Sequence from the list of
actions at the bottom of the page.
This will return you to the Protein tools screen,
but now the entryyou just imported will
be underneath the action menu.
For this project, you also want to import 4 more
hemoglobin beta sequences into your
session - from gorilla (Gorilla gorilla), mouse
(Musmusculus), chicken (Gallus gallus),
and bullfrog (Lithobates catesbeiana). The specific
sequences you want are labeled as
follows:
SWISSPROT:HBB1_MOUSE
HEMOGLOBIN BETA-1 CHAIN (B1) (MAJOR) [Mus
musculus (Mouse)]
SWISSPROT:HBB_CHICK
HEMOGLOBIN BETA CHAIN [Gallus gallus
(Chicken)]
SWISSPROT:HBB_LITCT
HEMOGLOBIN BETA CHAIN [Lithobates
catesbeiana (American bullfrog) (Rana
catesbeiana)]
SWISSPROT:HBB_GORGO
(P02024) HEMOGLOBIN BETA CHAIN [Gorilla

11. gorilla]
Note: to get thesesequences quickly, you can use
Ndjinn with the appropriate search
terms. Even faster, you could change the
settings for Ndjinn (so it displayed all hits
instead of 10) and use the 'search in page'
feature of your browser. Select the
sequences, then select Import Sequence from the
list of actions at the bottom of the
page.
This will take you to a page similar to the one
that you were at before, but now there
should be five different hemoglobin sequences listed
on the page. These organisms are
related in a way that should be pretty clear to
you. The human hemoglobin sequence is
identical to that from chimpanzees. Which of the
remaining four amino acid sequences
do you thinkwill be most like the human sequence?
Which sequence will be the next
closest? Which sequence do you thinkwill be the
most different from the human
sequence? Why?
6
Aligning the sequences.
Now we will carryout an alignment of the five
hemoglobin sequences that you have
retrieved. We will use a program called ClustalW to

12. perform the alignment. ClustalW is
a multiple sequence alignment tool that searches out
the best global alignment—that is,
the alignment with the most identity across the entire
range of all of the sequences. The
program is adjustable in that you can give different
weights to different types of
mismatches.
Select the boxes of all five of the hemoglobin
sequences. Then select CLUSTALW
Multiple Sequence Alignment from the action menu,
and select Run. This will take you
to a new page. Change output tree from
'unrooted tree' to 'rooted and unrooted trees',
and Select submit. This will take you to a new
page. Scroll down the page to the section
called ‘Sequencealignment’. Each of the amino
acid sequences that you retrieved is
shown in the alignment.
The alignment is color-coded, so that you can see
different types of changes without
looking at the specific amino acidsin the
sequence. Four different colors are used to
indicate varying levels of sequence conservation.
The bright blue areasare areasthat
are identical across all 5 sequences. The green
and dark blue areasare conserved but
not identical. Thismeans that although different amino
acidsare in this position, the
amino acids are similar to one another--for
example an amino acid with a hydrophobic R
group has been replaced by another amino acid

13. that also has a hydrophobic R group.
The black regions are unconserved--different amino
acidswith different properties are
present in this position in the different proteins.
One thingthat you can see in this alignment is that
thereare someregions in the protein
that are highly conserved, and others that are
only conserved to a lesser extent. This
reflects the fact that someparts of a protein can
and do evolve at different rates than
other parts of a protein. The highly conserved
regions are likely to be directly involved in
the functioning of the protein, which in this
case might be in binding the heme group
or in
interchain interactions. In an enzyme, amino acids
located at the active site are likely to be
highly conserved. The less conserved regions are
unlikely to be directly involved in
the functioning of the protein, for example, they
may have a 'spacer' function to
separate two otherregions.
Mutations happen randomly, however, and occur without
regard to the type of change
in the encoded protein. When changes occur
that produce a nonfunctional protein, the
organisms that have that mutation are likely to be
eliminated via natural selection. The
selection pressure against organisms with nonfunctional or
less-functional hemoglobin is
likely to be high.The highly conserved regions
aren't conserved because mutations never

14. occurred in thesepositions, but rather because natural
selection eliminated those
mutations once they did occur.
7
The Phylogenetic Tree created from the alignment
The ClustalW program also gives you another means
of viewing the information in the
alignment--it also depicts this information in the
form of a phylogenetic tree.
At the bottom of the page is a dendrogram
that depicts the relationships between the
sequences. In this figure, the sequences are
represented at the tips of the lines. A
branchpoint, where two lines diverge, represents a
single ancestor sequence. Two
sequences that have a single branchpoint between
them are more closely related to each
otherthan to the othersequences. The horizontal
length of the branches represents
evolutionary distance (related to amount of
dissimilar sequence), so that two sequences
with a high amount of similarity are connected by a
smaller distance than two sequences
that are less closely related.
This type of diagram can be used to infer the
evolutionary relationships between the
sequences, because you would expect that
sequences which are nearly identical shared a

15. recent common ancestor. Similarly, the evolutionary
relationships between the source
organisms can be inferred from the relationships
between the sequences.
Here, you can see the relationship that you
intuitively expected--the gorilla sequence is
very similar to the human sequence, because these
two organisms are both primates
and therefore shared a common ancestor more
recently than humans and mice did.
Similarly, the mouse, a mammal, shared a
common ancestor with the primates more
recently than it shared a common ancestor with
the chicken or frog.
Different models for evolutionary mechanisms might
produce different evolutionary
distances than those shown here.
• Print out the phylogenetic tree (rooted or
unrooted) and include it in your
homework assignment
The relationship between alpha and beta globin
sequences
Hemoglobin is a tetramer (4 subunits) of two
different polypeptides named alpha and
beta hemoglobin. Alpha and beta hemoglobin are
related to each othervia an ancient
gene duplication event. That is, therewas one gene
(the ancestral globin), and it

16. duplicated to form two genes in the same
organism (alpha and beta globins), and then
thesegenes underwent independent evolution as
the progeny of that organism
replicated. This gene duplication event occurred
before mammals diverged, Therefore,
an alpha globin gene in a mouse is more
closely related to (or, more recently diverged
from) an alpha globin gene from a
chimpanzee than it is to a beta globin
gene from the
same mouse. Gene duplication is a very important
evolutionary process, and it is clear
that it has happened numeroustimes on an
evolutionary timescale.
The alpha and beta sequences arose in an
ancestor common to all of the animals that
we
8
are looking at. That is, this gene duplication
event occurred before the divergence of
the
lineages that led to modern primates, mammals, birds,
and amphibians.
To import the alpha globin sequences into your
session, pressthe gray Return button on
the screen, then deselect the alignment, and then
select Protein Tools. Then use Ndjinn
to import the following sequences. If you
carryout a search for 'hemoglobin alpha' in

17. the
SWISSPROT database using Ndjinn, you will have to
search through several hundred
responses--it may help to use the 'find in page'
feature of your internet browser.
SWISSPROT:HBA_HUMAN
HEMOGLOBIN ALPHA CHAIN [Homo sapiens
(Human), Pan troglodytes
(Chimpanzee), and Pan paniscus (Pygmy chimpanzee)
(Bonobo)]
SWISSPROT:HBA_CHICK
HEMOGLOBIN ALPHA-A CHAIN [Gallus gallus
(Chicken)]
SWISSPROT:HBA_MOUSE
HEMOGLOBIN ALPHA CHAIN [Musmusculus
(Mouse)]
SWISSPROT:HBAB_LITCT
HEMOGLOBIN ALPHA-B CHAIN [Lithobates
catesbeiana (American bull frog)]
SWISSPROT:HBA_GORGO
HEMOGLOBIN ALPHA CHAIN [Gorilla gorilla
gorilla (Lowland gorilla)]
Onceyou have all the sequences imported, select all
ten sequences (your 5 beta
sequences and 5 alpha sequences) and perform
another Clustal W alignment of the
sequences. Make sure that the entire results
page has loaded (this takesa little bit of
time), and then examine the unrooted phylogenetic

18. tree of the sequences.
• Print out the phylogenetic tree (dendrogram) of
alpha and beta globin
sequences from the five organisms. Include it in
your assignment.
Answer thesequestions for your lab manuals:
1 . Do the alpha sequences cluster together
separately from the beta sequences?
2. Is the branching pattern among the alpha
sequences similar to the branching
among the beta sequences?
Independent project: For (10) bonus points you
may do an independent project
where you alignsequences from 5 different species
from a gene you are interested in or
you may extend the beta hemoglobin gene analysis
and complete the project idea below.
Print the phylogenetic tree and include it in your
homework assignment to get the bonus
9
points.

19. Bonus project idea
Estimating relationships between existing mammals.
In the analysis that we carried out, it was fairly
easy to predict the results because we
understand the relationships between humans,
gorillas, mice, chickens, and frogs. The
way in which otheranimals are related can be
difficult to understand. Even among the
mammals, it may be difficult to see how different
organisms are related to each other.
Using beta hemoglobin sequences, investigate
relationships between the organisms
below.
Minke whale Killer
Whale Harbor seal
Indian Elephant
White Rhinoceros
Brazilian Manatee
European River Otter
Polar Bear
Hippopotamus
Before carrying out the analysis, predict the
relationships between the different
organisms.
1. Are whales and seals more closely related to
one another than to any of these
other species?
2. To which organism is the manatee most closely
related?
3. To which organism is the elephant most closely

20. related?
4. To which organism is the otter most closely
related?
After carrying out the analysis:
1. Are whales and seals more closely related to
one another than to any of these
other species?
2. To which organism is the manatee most closely
related?
3. To which organism is the elephant most closely
related?
4. To which organism is the otter most closely
related?
Would you predict that you would see the same
results if you used a different protein for
1
0
the analysis, for example the cytochrome c
protein? Why or why not?
10

21. The following review questions will help to reinforce
what you've learned in this tutorial.
Answer them for your lab manual.
Questions – Review
1. Why was it necessary to carryout an alignment of
the sequences we analyzed?
2. Nucleotide substitutions might lead to changes in
the sequence of amino acidsin a
protein, which can be seen by comparing
homologous positions in a sequence alignment.
How would deletions or insertions appear in a
sequence alignment?
3. Sometimes gene duplications result in the
formation of a pseudogene, a sequence of
nucleotides that is very similar to a gene but

22. isn't expressed. This can occur in a variety
of
ways, for example if the gene is duplicated
but a regulatoryregion or the promoter
region for that gene is not duplicated. Which
would evolve (accumulate nucleotide
substitutions) more rapidly, a pseudogene or a
duplicated gene that was expressed?
Explain.