Downloading the example data

The last base pair, the so-called WIKI:Wobble_base_pair, in a WIKI:Codon changes more frequently during evolution than the first and second base. This will be demonstrated in this tutorial. Please activate "Nucleotides" in the BUTTON:UserProfile#newButton()!.

The following nucleotide sequences need to be downloaded from NCBI:
WIKI:Proteasome alpha 1-subunits:

  NCBI_NT:BC022354         NCBI_NT:M63641          NCBI_NT:NM_017283        NCBI_NT:NM_203782        
  NCBI_NT:XM_001139648     NCBI_NT:XM_001213840    NCBI_NT:XM_001263332     NCBI_NT:XM_749396


WIKI:Proteasome alpha 2-subunits:

  NCBI_NT:BC002900         NCBI_NT:BC026768         NCBI_NT:AB013342         NCBI_NT:NM_001024441     
  NCBI_NT:NM_001090584     NCBI_NT:NM_001046879     NCBI_NT:NM_079604        NCBI_NT:NM_001035980     
  NCBI_NT:XM_628527        NCBI_NT:XM_950392      
Using the dialog BUTTON:DialogFetchSRS! involves three steps:
  1. Copying (CTRL-C and CTRL-V) the IDs into the text field
  2. Highlighting the IDs
  3. Pressing the download LABEL:"ncbi"-button
  4. Loading the downloaded files into STRAP by pressing the LABEL:FilesFetchedFromServer#BUT_LAB_Load-button
You should now see the nucleotide sequences in the alignment pane. Select "nucleotides" in the selector JCOMPONENT:StrapView#choiceShading()! in the tool-bar.
Figure: Nucleotide alignment: Gaps disrupt codons.
 ... TAtAAtGAaGAt-------CTgGAaCTt--------------GAaGAtGCc ... 
 ... TAtAAtGAaGAc-------CTaGAgCTg--------------GAgGAtGCc ... 
 ... TAcAAcGAaGAt-------TTgGAaCTt--------------GAaGAtGCa ... 
 ... TAcAAtGAgGAc-------CTgGAaCTg--------------GAaGAtGCa ... 
 ... TAtAAtGAaGAt-------CTgGAaCTt--------------GAaGAtGCt ... 
 ... TAcACaGAaGAc-------ATgGAaCTt--------------GAcGAtGCc ... 
 ... TAtACaGAaGAc-------TTgGAgTTa--------------GAtGAcGCa ... 
 ... CTgAAgAAcAAg------GAtTAtGCtGAgGGtAGtT---GgGAgGAgGTg ... 
 ... CTgAAaAAcAAg------GAtTAcGCcGAtGGcAAcT---GgGAgGAgGTg ... 
 ... TTcAAaAAgAGtAAaATcGAcCAtATtAAtGAaGAaTCaTGgGAgAAgGTg ... 
 ... GTgAAgAAgAA--------aTTtGAtTG--gACaTTt-----GAaCAgACa ... 
 ... GTaAAgAAgAA--------aTTgGAcTG--gACtTAt-----GAgCAgACt ... 
            

Aligning the nucleotide sequences

Align the nucleotide sequences by pressing the tool-button JCOMPONENT:StrapView#button(BUT_ALIGN)!. Use the tool-bar button W to export the alignment file to your word processor.

Translating the nucleotides sequences:

Now we are going to translate the nucleotide sequences with the dialog BUTTON:DialogGenbank!. After translation you will see amino acid sequences in the alignment pane.
Figure: Amino acid sequence alignment: Wobble base position is less conserved
 ... TAt AAt GAa GAt --- --- --- --- --- --- --- CTg GAa CTt GAa GAt GCc ... 
 ... TAt AAt GAa GAc --- --- --- --- --- --- --- CTa GAg CTg GAg GAt GCc ... 
 ... TAc AAc GAa GAt --- --- --- --- --- --- --- TTg GAa CTt GAa GAt GCa ... 
 ... TAc AAt GAg GAc --- --- --- --- --- --- --- CTg GAa CTg GAa GAt GCa ... 
 ... TAt AAt GAa GAt --- --- --- --- --- --- --- CTg GAa CTt GAa GAt GCt ... 
 ... TAc ACa GAa GAc --- --- --- --- --- --- --- ATg GAa CTt GAc GAt GCc ... 
 ... TAt ACa GAa GAc --- --- --- --- --- --- --- TTg GAg TTa GAt GAc GCa ... 
 ... CTg AAg AAc AAg --- --- --- GAt TAt GCt GAg GGt AGt TGg GAg GAg GTg ... 
 ... CTg AAa AAc AAg --- --- --- GAt TAc GCc GAt GGc AAc TGg GAg GAg GTg ... 
 ... TTc AAa AAg AGt AAa ATc GAc CAt ATt AAt GAa GAa TCa TGg GAg AAg GTg ... 
 ... GTg AAg AAg AAa --- --- --- --- --- TTt GAt TGg ACa TTt GAa CAg ACa ... 
 ... GTa AAg AAg AAa --- --- --- --- --- TTg GAc TGg ACt TAt GAg CAg ACt ... 
            

Aligning the amino acid sequences:

Align the amino acid sequences by pressing JCOMPONENT:StrapView#button(BUT_ALIGN)!. After alignment export the alignment BUTTON:DialogExportAlignment!. In the export dialog select nucleotides instead of amino acids. The wobble base position is written in lower case and the first two base positions are written in upper case. The result should look like the figure below.

Discussion:

Now we compare the result of the alignment computation with nucleotide with the alignment computation with amino acids. There are significant differences. Computation from nucleotides, the three bases that belong to one WIKI:codon are sometimes separated by gaps. In the WIKI:ClustalW output for amino acid sequences it can be seen that the wobble base position (lower case) exhibits high variability compared to the first and second nucleotide position (upper case). Conclusion: Alignments computed with amino acid sequences are more reliable than with nucleotide sequences.