Download Glutamate Synthase - Blue Valley Schools

PROTEIN BASED PHYLOGENETIC RELATIONS
OF THE FIVE KINGDOMS
Name: ___________________________
Background
Since humans have been cognizant of the life around them, they have been classifying
and trying to determine our relationship to other life forms. In Aristotle’s time, we were
only aware of two categories of organisms: animals and plants. In the 1950’s, after the
advent of the microscope and a more thorough analysis and synthesis of the
information regarding the life on Earth, Whittaker introduced the five Kingdom system of
classification. Twenty years later, Carl Woese used rRNA to reevaluate the relations of
the organisms in the five kingdoms and proposed a higher level of classification: the
Domain. More specifically, he concluded that the Kingdom Monera (bacteria) should be
split into the Eubacteria and Archeabacteria while the remaining organisms should be
classified as the Eukaryota. In the following activity, you will use sequences of the
protein “glutamate synthase” from a number of different species to analyze the
evolutionary relationship between the five kingdoms.
Procedure
A. Acquiring Protein Sequences & Converting to FASTA format
Given the accession numbers associated with the glutamate synthase protein for the
twelve species listed below, proceed to the National Center for Biotechnology
Information website at:
http://www.ncbi.nlm.nih.gov/
Accession Numbers Species
Kingdom
A38596
Zea mays
Plantae
CAC05496
Arabidopsis thaliana
Plantae
AAC08261
Porphyra purpurea
Protista - algae
CAA76602
Plasmodium falciparum
Protista - protozoa
AAF49409
Drosophila melanogasater
Animal
CAB92626
Neurospora crassas
Fungi
CAA61505
Saccharomyces cerevisiae Fungi
BAB05447
Bacillus halodurans
Bacteria
AAA58014
Eschericia coli
Bacteria
AAK94787
Klebsiella areogenes
Bacteria
CAB64595
Nostoc sp.
Bacteria
AAG44102
Staphylococcus aureus
Bacteria
1. In the “Search” window select “protein” from the pull down menu, and then copy and
paste the first species accession number into the “for” window. Then, select “go”
or type the return key. A record for a particular protein should show up in the main
screen area.
2. Select the accession number, which is now a hyperlink, in the record displayed. In
a few seconds a complete page of information about the protein for Zea mays
should be displayed. At the bottom of the page, you should recognize a series of
letters in rows. Each row is separated into 6 segments of ten letters each.
3. Using your mouse, highlight and copy the entire protein sequence including the
numbers used to help one quickly identify particular amino acids. Then, proceed to
the BCM Search Launcher at the Baylor College of Medicine at:
http://searchlauncher.bcm.tmc.edu/seq-util/seq-util.html
4. Paste your copied protein sequence into the window above which it says “cut and
paste formatted sequences here, etc...” (make sure the ReadSeq is selected from
the bullets at the bottom), and select the “submit” button. This will convert the
copied protein sequence into FASTA format, removing all the spaces and row
numbers.
5. Open a word document and type “>” and then immediately following type the
species name for the protein sequence (in this first case, Zea mays). Then, return
to the Baylor website and “copy” just the protein sequence from the converted
data, and “paste” it on the line following the “>Zea mays” identifier.
6. After you have finished this species, complete steps 1 through 5 for each of the
remaining eleven species. You should paste all of the protein sequences into the
same word document, making sure that there are no spaces between each
species entry. For example, the line after the final letters in the Zea mays
sequence should be followed by “>Arabidopsis thaliana” and its sequence.
B. Multiple Sequence Alignment & Tree Diagrams
1. Once you have the word document complete having all twelve species protein
sequences in FASTA format, open the Clustal W Multiple Sequence Aligment
online software at (select the multiple alignment link):
http://align.genome.jp/
2. Open the word document and “highlight” and “copy” the entire file. Then, “paste”
the file into the sequence window below where it says “Enter your sequences,
etc...” Also, make sure to select the “slow/accurate” and “protein” selections above
the window. Then, select the “Execute Multiple Alignment” button.
3. In a few seconds, your Clustal W Results should appear. Print this document for
later analysis (if it doesn’t print accurately then save the html file in your server
folder too).
4. Next, select “N-J Tree” at the bottom of the screen. In a few seconds, a
phylogenetic tree showing the possible evolutionary relations for the species being
compared. Right click and save this picture as a bitmap file in your server folder.
Also, select and save the “Unrooted N-J Tree” as well.
Questions
1. What is the function of “glutamate synthase”? Why would this protein make a good
choice in analyzing the relationship of organisms from each of the five kingdoms (as
opposed to a protein like hemoglobin found in red blood cells)?
2. Using the Clustal W results, what does it appear an “alignment” is? How could such
data be used to determine the possible evolutionary relationship between difference
species?
3. Define the following terms.
Prokaryote
Eukaryote
Unicellular
Multicellular
Autotroph
Heterotroph
4. What morphological (physical) characteristic are known to define the organisms in the
Kingdoms Monera, Protista, Fungi, Plantae, and Animalia? Use the terms
prokaryote, eukaryote, unicellular, multicellular, autotrophic, heterotrophic, and cell
wall in your answer.
K. Monera
K. Protista
K. Fungi
K. Plantae
K. Animalia
5. Based on the characteristics of the five kingdoms,
a. Which kingdom is most likely the oldest? Explain.
b. Which kingdom is most likely the second to have evolved? Explain.
c. How are the remaining three kingdoms similar?
d. Of the three remaining kingdoms, which two are most alike? Explain.
6. Copy the N-J Tree diagram (making sure to label the kingdom for each species
represented).
7. a. Give at least three specific examples of how the tree supports the conclusions that
you have previously made about the relationships between the five kingdoms.
1.
2.
3.
b. Then, give one specific example of how the tree does not support the conclusions
that you have previously made about the relationships between the five kingdoms.
1.