ANTENNAPEDIA LAB
Name:___________ Section:____
Part I: Drosophila melanogaster is a species of fruit flies and have been greatly studied
in many subfields of biology. To begin, please observe the two fruit flies found in the
laboratory. Make a quick sketch of what the fruit fly looks like under a microscope.
Then, describe in detail the phenotypic differences between the wild type Drosophila and
the mutant Drosophila provided for you by answering the questions below.
Wild type Drosophila:
Total Magnification:_________
Mutant Drosophila:
Total Magnification:________
1. What are the similarities between the wild type and mutant fruit flies provided?
Describe in detail.
2. What is the main phenotypic difference between the wild type and mutant fruit
flies provided? Describe in detail.
3. How important do you think this mutation is to the fruit fly?
Part II: Registration on “The Next Generation Biology Workbench” (NGBW)
In today’s laboratory, we will be using a bioinformatics research tool called NGBW to
search through thousands of DNA and protein sequences to help us explore the difference
between the similarities between Drosophila and humans.
To Register on NGBW:
A) Open up a web browser window and enter http://jayne.ibest.uidaho.edu/
B) Click on “Use the Workbench Now”
C) If you do not have an account yet, click “Create an Account”
D) Complete all the required parts of the registration form and click “Register”
E) Remember your username and password so you can retrieve information in
the future.
To Create a Folder of NGBW:
A) For this lab, you will need to create one working folder. This folder will allow
you to store data collected during this laboratory and complete tasks to analyze
the stored data. Click on “Create New Folder”.
B) Type “Antennapedia” for the label and “Bio 115 Lab” under the description.
Then, click “Save”.
C) After you click save, a page will show up welcoming you to the Biology
Workbench, notice that the folder in the left top corner has a “data” function and
a “task” function. We will be using these two functions for the next exercises.
D) Take a look at the buttons across the top of the page and answer the questions
below.
1. Click on “my profile”. What is found under “my profile”?
2. What five information pages can you reach by clicking on the “Toolkit”
button?
Part III: Find Antennapedia Protein Sequences
The Antennapedia protein is a protein sequence that helps suppress the formation of legs
on the head region of fruit flies. This protein is important for the proper development of
the head and works correctly in the wild-type fruit flies observed in part one. However, a
mutation occurs allowing legs to grow where antenna should be placed in the mutant.
This part of the laboratory will allow you to search for this sequence so you can store it
for further analyses.
Review:
1. If you have a protein sequence that reads A C T, what do those letters stand
for?
Find Sequences:
A) Under the folder labeled “Antennapedia” on the left top corner of your screen,
click on the “Data” button.
B) Click “Search for Data”
C) In the “Basic Search” window, completely fill in these items:
Query: “Antennapedia”
Entity Type: “Protein”
Data Type: “Sequence”
Data Sets: “SwissProt”
D) Click “Submit Search”
E) At the top of the page, a number of resulting matches are shown, how many
results do you have today?______________________
F) Locate and check the box by the following sequence:
P02833
ANTP_DROME
G) Click “Save Results” at the bottom of the list. This process places the sequence in
the top left “Data” folder. You should notice that you now have one item in this
folder.
H) Go to the Data folder and click on the new entry.
1) What different kinds of information do you see?
2) Where is the actual protein sequence in this file?
I) This is in the EMBL format, notice all of the interesting information provided.
J) We must now convert this file to the Fasta format. Click on the “Tasks” folder
then click “Create New Task”
K) Type “Change file” by description then click “Set Description”
L) Click “Select Input Data”
M) Check the box by P02833, and hit “Select Data” at the bottom of the page.
N) Click on “Select Tool” and click on the Protein Sequence Tools” at the top of the
page.
O) Click on “Readseq”
P) Click on “5 Parameters Set”
Q) Change the first drop down box to EMBL and the second to FASTA, then “Save
Paramters”
R) Save and Run Task
S) Click “Refresh Tasks” until you see the “View Output” signal.
T) Click “View Output” and “View File” button next to “outfile.fasta” file.
U) Click on “Save to Current Folder” button.
V) Change the label to P02833.fasta (WHY?)
W) Change the dropdown menus to: Entity Type: Protein, Data Type: Sequence, Data
Format: Fasta
X) Click the “Save” button. You now have two files in the Data folder.
Part IV: Hox Genes
The antennapedia protein has a region called the homeodomain. This region of the
protein binds to DNA and helps regulate the transcription of some developmental genes.
Homeobox genes or Hox genes are DNA sequences that code for these homeodomain
proteins and are highly conserved in all animal phyla. This means that Hox genes and
their resulting protein sequences are found in most groups of animals and help regulate
early developmental pathways.
Review: Using the textbook chapter 21, glossary, and the information above, answer the
following questions.
3) What are Hox Genes or Homeobox Genes?
4) What groups of living things include these sets of genes?
To Find Sequences Related to Antennapedia Protein in Other Animals:
In this part of the laboratory, we are going to use the program BLASTP to help us search
for other organisms that have sequences of proteins related to the Antennapedia protein
we have been discussing.
A) Type “Blastp” by description then click “Set Description”
B) Click “Select Input Data”
C) Check the boxes by P02388.fasta and hit “Select Data” at the bottom of the page.
D) Click “Select Tool” and click on the “Protein Sequence Tools” at the top of the
page.
E) Click on “BlastP”
F) Click “Set Parameters Tab”
G) From the protein database select “SWISSPROT” from menu.
H) Click “Save Parameters” and then “Save and Run Task”
I) Click “Refresh Tasks” until you see the “View Output” signal.
J) Click “View Output” and “View File” button next to “blast2.txt” file.
K) To view your results, click on the “View” tab at the top of the page.
L) Select the 8 HXB7 sequences, and click on the “Save Results” button. You now
have 9 (10) sequences in your Data folder.
3) What are the other animals that have sequences related to Antennapedia?
Part V: Align the Sequences
Today, you will be using a program called CLUSTALW. CLUSTALW matches up or
aligns protein sequences based on amino acids that they hold in common. To use this
program, students will input the sequences located in Part V and select the CLUSTALW
program to align them. The CLUSTALW program will “read” the sequences, predict
based on evolutionary rules which amino acids are “homologous” to each other and align
these homologous amino acids.
Example:
Sequence 1) GGGGMATCH
Sequence 2) MATCH
Aligned As: GGGGMATCH
MATCH
1. You can do by hand what CLUSTALW does as a computer program (but we
generally use the computer because it is much faster). Review the following
sequences and determine where you think they match up. Write the two
sequences as you would predict them to be aligned.
Sequence 1: AVMTCSRGQCNPGP Aligned As:
Sequence 2: MTCSRGQCN
To Align the HOX Protein Sequences:
A) Click the “Tasks” folder
B) Select “Create New Task”
C) Enter the name “Align 1” by the description and click “Set Description”
D) Click “Select Input Data”. This will open a window that allows you to select the
three sequences you want to align.
E) Check the boxes in front of the Antennapedia and HXB7 proteins DO NOT
INCLUDE THE FILE NAMED P02833.fasta and click on the “Select Data”
button at the bottom of the page. You should now see that you have 9 input sets
by the input button.
F) Click “Select Tool”. A window will open with 5 tools tabs listed toward the top
of the page. Select the Protein Sequence Tools (since you want to align protein
sequences)
G) Click CLUSTALW P (This should set around 42 parameters for you)
H) Hit “Save and Run Tasks” to align the sequences.
I) Click the “Refresh Tasks” button (once per minute is plenty) until it reads “View
Output” under the action column.
J) Click “View Output”
K) Select either View File button labeled “outfile.aln”. Do NOT close this window,
you will need it for the next section.
Questions:
1) Looking at the first few blocks of aligned sequence, which sequence is from
Drosophila, and why do you know this is true? The other sequences are the
vertebrate HXB7 proteins.
2) Starting with the first aligned amino acid designated as “M” for methionine,
and going down each column within a block, how well do the vertebrate
HXB7 proteins align to each other? Use words like identical, mostly the
same, very different.
3) The human HXB7 protein sequence is labeled P09629. How is the human
sequence different from the Drosophila sequence? How are they the same?
Part VII: Observing the Antennapedia Protein Structure
The part of the proteins aligned above that are nearly identical are in the homeodomain of
each protein. This part of the protein is important because it binds to DNA, thus
regulating gene transcription in both humans and Drosophila. Protein Structure Models
allow us to look at a protein’s general structure. We will use a program called Cn3D to
access protein structures and even overlay two proteins so we can compare how one is
different from the other. In this exercise, we will see how the homeodomain interacts
with the DNA.
A) Open another browser window and go to http://www.ncbi.nlm.nih.gov/.
B) Use the “All Databases” dropdown menu to select “Structure”.
C) In the box type in 1AHD. This is the structure of the homeo-box bound to DNA.
D) Click on “Go”.
E) A thumbnail picture of the structure will come up, along with information about
the structure. Click on the link for the description.
F) On the right hand side of the page is a button “View structure”. The drop down
menus above it should read “Cn3D”, “3D structure”, and “Single 3D structure.”
G) Two windows will pop up – one with the amino acid and DNA sequences and
one with the structure of the homeodomain protein bound to the DNA.
H) You should see two molecules in this view, although it may be hard to recognize
them at first. The blue and pink strands wound around each other are the two
strands of the DNA double helix. The rest of the other molecule (brown) is the
homeodomain protein. Can you see them?
I) You can move the protein around to get different views again by clicking on it
and dragging.
J) Finally, go to “Style” and “rendering shortcuts” again from the dropdown menu.
Select “ball and stick” to get a final view of all the elements making up both the
DNA molecule and the homeodomain protein. Pretty cool, huh?
K) Look back at your alignment of the Drosophila sequence with the vertebrate
sequences. Find the two differences in the homeobox domain between human and
Drosophila. Go back to the Cn3D program, and find these amino acids in the
sequence window. Click on these amino acids, and they will turn yellow in the
structure.
4) Where are these amino acids relative to the DNA?
5) Do you think they will affect the binding of the protein to the DNA?
L) Change the style to wire. Move the structure around until you are looking down
the center of the DNA double helix.
6) Can you see how complimentary base-pairing is working in the DNA
molecule?