NAME: ____________________________________ DATE: ______________ BLOCK: ____
Are polar bears closely related to American brown bears? Do you think the giant panda is
more closely related to the American brown bear or the polar bear? Is the giant panda a bear
at all? What about the red panda – is it a giant panda relative? What does the evolutionary
history of bears look like? In this activity you will use real gene sequences to figure out which
animals are more closely related. You will create a family tree for bears and help to solve the
mystery of bear history!
TABLE 1: Accession numbers and scientific names different species
Organism
1.
2.
3.
4.
5.
6.
Scientific Name
Accession Number
American black bear
Y08520.1
American brown bear
L21889.1
Spectacled bear
L21883.1
Asiatic bear
L21890.1
Polar bear
L22164.1
Giant panda
Y08521.1
Red Panda
S80939.1
Northern raccoon
U78345.1
Leopard seal
AY377288.1
Go to www.ncbi.nlm.nih.gov. Click the arrow beside “all databases” and select
Nucleotide. Enter the accession number for the American Black bear (see Table 1 for
number) in the search space.
You will see lots of information. Near the top you will see the definition listed as:
U. americanus mitochondrial 12S rRNA gene. U. americanus is the scientific name for
the American black bear. The gene is part of the mitochondrial DNA and makes up part of
each ribosome. Write the scientific name for the American black bear in Table 1.
Click FASTA at the top of the page. You will then see the sequence of DNA that makes
up this 12S rRNA gene.
On the right, towards the top, is a drop-down arrow next to “Send”. Click the arrow and
choose Clipboard as your destination. Then click “Add to Clipboard.”
Continue by searching each animal’s accession number, clicking on the FASTA, and
sending the sequences to the clipboard.
After you have added all 9 FASTA sequences to the clipboard, click on the “9 items” next
to clipboard to open your clipboard.
1
7.
8.
9.
10.
11.
12.
Select each animal by checking the box.
After all nine have been selected, click the “Send to” in the upper right hand corner.
Chose “File” and under Format select “FASTA”.
Chose “Organism Name” under “Sort By”.
Click “Create File”
Your file will be created with the following name: sequence (1).fasta, which will be
automatically downloaded. You will be able to find this file in your download files.
You will now take these sequences for the 12S rRNA gene and align them using an online
website. The website uses algorisms (math) to find areas of similarity across the 9 organisms.
This requires significant computation so this is always done via computers.
13.
14.
15.
16.
17.
Now you will go to http://www.genome.jp/tools/clustalw.
Chose “CLUSTAL” for the output format.
Check “SLOW/ACCURATE” for the pairwise alignment.
Check “DNA” for Enter your sequences.
You will upload the sequence file you just made by choosing “Choose File”. Navigate to
find the file you downloaded called sequence (1).fasta.
18. Click “Execute Multiple Alignment”.
19. The program will align the sequences to see the results.
Interpreting your results
20. At the top you will see the 9 sequences with their accession numbers and the number of
base pairs (bp) for the sequence. The program numbered your organisms 1 – 9. In the
table below, write the common name beside each number. You must look back at the
accession numbers to match the right name to the correct number.
Organism
Number
Organism Name
1
2
3
4
5
6
7
8
9
2
21. Now scroll down slightly and you will see alignment scores like below:
Sequences (1:2) Aligned. Score: 75.
22. This is just an example and will not match your data! This is the score between organism
1 and organism 2. The higher the alignment score between two organisms, the more
similar their DNA is for this gene.
23. Scroll down farther and look at the sequence alignments; you will see nucleotide
sequences (the A’s, G’s, T’s, and C’s). The symbol “–” indicates that there is no
information for this location in the sequence. In other words the base (letter) is not
known. Because some sequences are longer than others there may be many sections
where information is missing. The symbol “*” indicates a location where all nine
organisms have the exact same nucleotide, meaning the base is conserved. If a
nucleotide is conserved then that same nucleotide should be in the common ancestor.
QUESTIONS ABOUT THE ALIGNMENT
a. Which two species have the highest alignment scores? You will need to check the
accession numbers so you know which organism is which.
b. Which two species have the lowest alignment scores?
c. Which group has the highest score with the red panda?
d. Which group has the second highest score with the red panda?
e. Which group has the lowest score with the red panda?
f. What does it mean if two groups have a high alignment score?
24. Now you will use this website to build a phylogenic tree. This is like a family tree based
on genetics and evolution. The website takes the data that you have looked at and draws
a tree, which is more visually appealing.
25. At the top of the page (above the alignment data), select “Rooted phylogenetic tree with
branch length (UPGMA)” from the drop-down menu.
26. Click on “Exec”.
27. You will see the phylogenic tree.
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28. Draw the tree in the space provided below. Write the name of each animal instead of
the accession number and other information.
Phylogenic Tree
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QUESTIONS ABOUT THE PHYLOGENIC TREE
g. Is the red panda more similar to the giant panda, the raccoon, leopard seal, or the
American black bear? Explain your reasoning.
h. Can you make the assumption that the red panda and raccoon definitely share a
common ancestor? Are there any problems with making that assumption from this
data? Again, explain your reasoning.
i.
Which two bears could possibly be mated together to create a hybrid. Explain your
answer. Remember that animals that create hybrids must be very similar.
j.
If an Asiatic bear needed a kidney transplant, which species of bear do you think would
be the best match as an organ donor? Explain your answer.
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TAXONOMY
29. Go to http://animaldiversity.ummz.umich.edu. Answer the following
questions for each animal. DO NOT copy directly from this website. PUT
YOUR ANSWERS IN YOUR OWN WORDS!
30. Click on classification (right side of page). List the Suborder, Family, and
Genus Species (same as scientific name) for each animal. You should put
this information in the table below.
Table 2: Classification Information
Organism
American
black bear
American
brown bear
Spectacled
bear
Accession
Number
Suborder
Y08520.1
L21889.1
L21883.1
Asiatic bear
L21890.1
Polar bear
L22164.1
Giant panda
Y08521.1
Red Panda
S80939.1
Northern
raccoon
U78345.1
Leopard seal
AY377288.1
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Family
Genus Species
31. Now read about each animal and complete the chart below. Briefly describe the following
for each animal and type your answers into the table below:
 located geographically
 habitat
 diet
 conservation status
Table 3: General Information
Organism
Geographic location
Habitat
Eats
American
black bear
American
brown
bear
Spectacled
bear
Asiatic
bear
Polar bear
Giant
panda
Red
Panda
Northern
raccoon
Leopard
seal
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Conservation Status
TAXONOMY v. PHYLOGENY QUESTION
k. Does this taxonomic information match the phylogeny data? Look at their classification
in Table 2.Figure out the lowest level that the animals are similar. For example, if they
shared only to subphylum vertebrata, the big trait they shared would be that they both
have backbones (vertebrate). You can click on the taxonomic levels to see descriptions.
Explain your answer.
ANOTHER WAY TO ANALYZE THE DATA
32. Go back to NCBI (http://blast.ncbi.nlm.nih.gov/) and chose nucleotide blast.
Enter the leopard seal accession number (AY377288.1) in the query sequence.
33. Click the small box labeled Align Two or More Sequences. This process will take these
sequences for the 12S rRNA gene and align them. The website uses algorisms (math) to
find areas of similarity across the nine organisms. This requires significant computation
so this is always done via computers.
34. Enter the accession numbers for the rest of the organisms in the subject box. See the
screen shot and the list of accession numbers below.
Accession Numbers
Y08520.1
L21889.1
L21883.1
L21890.1
L22164.1
Y08521.1
U78345.1
Y08521.1
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35. The results of the BLAST search are displayed in three ways as you scroll down the
page:
a. First, a graphical overview illustrates how significant matches, or hits, align with
the query sequence. Matches of differing lengths are coded by color.
b. This is followed by a list of “Sequences Producing Significant Alignments", or
hits, with links to Accession information. An E value close to zero indicates a
more significant match (closer) match.
c. Next, there is a detailed view of the query aligned to the nucleotide sequence of
the search hit (subjects).
36. When you look at the alignments, check out the top information for each alignment. The
identities refer to the number of bases that are identical. These bases are connected by
lines. The gaps are listed and are symbolized by a dash (-). Bases that do not match do
not have a line connecting them. See this example below.
Query
718
Sbjct
252
GAAATGGGCTACATTTTCTATTCAAGAACAACTTCTACGAAAACTTTTATGAAACTAAAA
|||||||||||||||||||||||||||||||||| |||||| ||||||||||||||||
GAAATGGGCTACATTTTCTATTCAAGAACAACTT--ACGAAAGTTTTTATGAAACTAAAA
777
309
37. Scroll down through the alignments and then answer the questions below.
QUESTIONS ABOUT THE NCBI ALIGNMENT
l.
Which species have the lowest E value with the leopard seal? You will need to check
the accession numbers so you know which organism is which.
m. Which species have the highest E value with the seal?
n. What does it mean if two groups have a high E value?
Now you will use this website to build a phylogenic tree. This is like a family tree based on
genetics and evolution. The website takes the data that you have looked at and draws a tree,
which is more visually appealing.
38. At the top of the page (above the alignment data), select “Rooted phylogenetic tree with
branch length (UPGMA)” from the drop-down menu.
39. Click on “Exec”.
40. You will see the phylogenic tree.
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41. Draw the tree in the space provided below. Write the name of each animal beside the
branch.
Phylogenic Tree
QUESTIONS ABOUT THE TWO ALIGNMENTS AND TREES
o. Do the two trees look the same? Why or why not?
p. If you have time, try putting the red panda in as the query and the other organisms as
the subjects. This will change the look of the tree. Why?
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