Module 5: Sequence Alignments

 

Exercise 5

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In this exercise, we will use ClustalW to align multiple sequences and employ an alignment editor tool to present the various, aligned sequences.

ClustalW Alignment

  • Go to the ClustalW site at the European Bioinformatics Institute. Check the "HELP" option for explanation of the different alignment parameters.
  • To perform the multiple alignments, we will use five sequences obtained from the BLAST similarity search for the murine IL-7R sequence (exercise 4). The selected sequences will vary from closely similar to highly divergent when compared to the query sequence. A file has been created for the input of these five sequences into the ClustalW program.
  • For this exercise, use al the default options except to the set the "COLOR ALIGNMENT" to "Yes". Copy and paste the murine IL-7 receptor sequences from the Notepad file into the sequence window. When using Netscape as your browser, you may upload this file either directly or by browsing for it.
  • You may enter a valid e-mail address to perform the alignments offline but we will run it interactively here. Click the "RUN CLUSTALW" button.
  • When you review the results of the alignments, keep in mind the following notations for the consensus line (last line below the five sequences).

    * = identical or conserved residues of all sequences in the alignment

    : = conserved substitutions

    . = semi-conserved substitutions

Answer the following questions regarding the multiple alignments:
  1. What changes do you expect in your output if you change the gap penalty options such as GAPOPEN, ENDGAP, GAPEXT, GAPDIST? Under what circumstances would you change these parameters?
  2. From the output, what can you conclude about conserved residues, regions or any other significant patterns of similarity within the various murine IL-7 receptor and receptor-like sequences?
Boxshade Alignment Editor

We will employ the Boxshade software to edit the aligned sequences from the ClustalW program.
  • Link to the boxshade site at the Institut pasteur in France. Provide your e-mail address. Copy and paste the five sequences from the file used for ClustalW. If you are using Netscape, then you may browse and add the file name.
  • Under "Sequence properties" check the "Display consensus line" box. Leave the foreground and background colors for the 'letters' (amino acids) as the default colors. Under "Comparison to a single sequence" select the "Similarity to a single sequence" option with sequence # 1 as the sequence for comparison.
  • Click "Run Boxshade". From the results window select the 'boxshade.html' file to view the results.
  • Compare the results to the foreground and background color scheme for the different residues in the five sequences. Green residues indicate fairly divergent regions of the polypeptides. Notice that as we go from sequence # 1 to # 5, we find increasing number of green residues. This is to be expected since we chose sequences from the BLAST results in exercise 4 with increasing divergence to the murine IL-7 receptor sequence (#1).


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