Developmental Proteins in Drosophila
Primary Author: Nick Exner

Introduction

You are one of the worlds leading scientists, and you have been doing genetic research with the species Drosophila Melanogaster (a.k.a. fruit flies).   One day you stumble into the lab and notice that one of your specimens has a pair of legs growing out of its head!  You scratch your head and take a second look.  Unable to explain, you pull out your handy "Answers to Strange Questions" encyclopedia and learn that this phenomenon is caused by a genetic mutation in a gene that encodes for the Antennapedia protein.  This protein is responsible for suppressing the formation of legs on the head during development. 

Being the world reknown scientist that you are, you decide to pursue the matter even further.  Since you were taught that a protein's function determines its function, a question repeats in your mind "What´s the difference in the structure between the mutated protein and the normal protein?"  Unable to find the solution in your book, you decide to use the world-wide web to find an answer.  After doing a search on the net you find a nifty tool called the Biology Workbench.  The Biology Workbench is a state -of-the-art research tool which will allow even the common person to search through thousands of protein sequences and perform complex comparisons with the greatest of ease.

 

Developmental Proteins in Drosophila:
NDJinn Multiple Database Search

If possible, open a second web browser window and open the Biology Workbench V.3.2 in it. The best  way to proceed through the tutorial is to set up the two  browser windows next to each other. Alternatively, we recommend printing the tutorial.

In the Biology Workbench window, log in (setting up a new account, if necessary) and click on the "Protein Tools" button.

Highlight the NDjinn Multiple Database Search from the scrollbar menu. The Ndjinn Search  is a feature of the Biology Workbench that allows the user to find information on a topic of interest, using specific databases that may be useful.  For example, if one is looking for amino acid compositions for different proteins, the  Protein Infomation Resources (PIR 1,2,3, and 4) databases are useful. After highlighting the Search, click on the "Run" button.

Scroll down the page, and click the checkbox near thePDBFINDER database. The PDBFINDER (Protein Databank) database contains the three dimensional structures of numerous proteins. All of these proteins are in a downloadable ".pdb" format which allows  anyone to view this with a pdb protein molecular viewer described later.

Type the name of the protein sequence that we are interested in:

 antennapedia

in the query space at the top of the page.

Submit your query (by clicking on the "Search" button). For further ease, you may also select "Show All Hits" in the box to the right of the query search.

The next step is to align the sequences.


 

 

Developmental Proteins in Drosophila:
CLUSTAL-W Sequence Alignments

In the scroll menu, you will see that the database found four or more protein structures from your query.
Scroll through the list and highlight the following records:

pdbfinder:2hoa - heard: dna-binding protein
pdbfinder:1hom - header: dna-binding protein
pdbfinder:1san - header: dna-binding protein

This can be done by deselecting the other two sequences.

Import them to the workbench (by  clicking the button "Import Sequence ").

Select each sequence by clicking in the small boxes next to the sequences  and align them using CLUSTAL-W - Multiple Sequence Alignment. This is accomplished  by highlighting the CLUSTAL-W program in the scroll menu and clicking the  "Run " button. The next screen presents an  assortment of settings for the CLUSTAL-W program. Since these a re best left at the default, just click the "Submit" button.   CLUSTAL-W is a tool on the Biology Workbench that is used to align a group of protein sequences by their common elements so that they can be compared. When the results are returned, import them to the workbench (by clicking the "Import Alignments" button).

Continue on to compare the sequences.


 

Developmental Proteins in Drosophila Tutorial: Comparing the Sequences

Select the aligned sequences by clicking in the small boxes next to them  and  determine  their similarities using BOXSHADE.  Highlight BOXSHADE in the scroll menu and click on the "Run " button. As with CLUSTAL-W, screen, click the "Submit" button to use the default settings.

BOXSHADE can be  used on groups of proteins that have been aligned (as by CLUSTAL-W) to  determine their similarities. It produces a color-coded output of the protein sequences.
Green is for amino acids that a re the same (conserved) in all the proteins examined. Yellow is for amino acids that are the same in nearly all protiens and cyan means that the amino acid has the similar structure and charge bu t is a different amino acid.

Look at your aligned sequences. Notice that a majority of the amino acids are highly conserved. The 1hom protein sequence is the wild type. The other two proteins are mutations. If a closer look is taken, it is evident that the 2hoa and the 1hom sequen ces differ by only one amino acid. This single amino acid substitution, all by itself, from cys to ser at the 39th amino acid is responsible for causing the fruit fly to express the development of legs on its head.

Now let's prepare to take a look at the structure of the antennapedia protein to help visualize the effect of this slight mutation.


 

Developmental Proteins in Drosophila Tutorial: One Mutation Causes the Condition

Now that you have seen the mutation in the antennapedia protein sequence that causes the pronounced change in the fruit flies phenotype, let's look at what this mutation does to the structure of the protein itself.

For proteins, there are 4 levels of structure. The first, primary structure, is composed of the nucleotides (A,G,C,T) that make up the DNA sequence that contains the code to make the protein. The secondary structure is the amino acid sequence that make s up the protein. The tertiary structure is the 3-D structure of the protein that allows it to perform its functions. The quaternary structure is the total protein structure that is made when all the subunits of the protein are in place.

Click on images below to manipulate proteins with CHIME:

From the visualization above, we notice that the molecules, marked in red, are arranged slightly different between the mutant protein and the normal protein.  The very subtle difference in this arrangement and content gives rise to the inability o f the mutant protein to function as an inhibitor to prevent the formation of legs on the head during early developmental stages.

Developmental Proteins in Drosophila Tutorial: Links

General Information

http://copan.bioz.unibas.ch/homeo.html
Information relating to the definition and classification of homeobox genes.

http://zygote.swarthmore.edu/droso4.html
Contains recent information and diagrams of the effects of the  Antennapedia protein.

http://zygote.swarthmore.edu/cyto6.html
Description of HOX genes with a mention of the Antennapedia gene.

 http://iubio.bio.indiana.edu/IUBio-Software%2BData/flybase/allied-data/interacti ve-fly/segment/antenap1.htm
Biological o verview of the history of research dealing with the  Antennapedia protein.

http://www.csa.ru:81/Inst/gorb_dep/inbios/genet/s1antp.htm
Offers insight to the location of the gene which is responsible for the  Antennapedia protein.


This page created and maintained by Kristian N. Engelsen.
E-mail any questions or comments.