@*~RSC_HELP_TUTORIAL_WOBBLEBASE

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.

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

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


  WIKI:Proteasome alpha 2-subunits:

  GB:BC002900         GB:BC026768         GB:AB013342         GB:NM_001024441
  GB:NM_001090584     GB:NM_001046879     GB:NM_079604        GB:NM_001035980
  GB:XM_628527        GB:XM_950392
Using the dialog HTMLDOC_BUTTON:BUT_C1(DialogFetchSRS)! requires three steps:
  1. Copying (Ctrl+C anld Ctrl+V) the IDs into the text field
  2. Highlighting the IDs
  3. Pressing the download NCBI-button
  4. Loading the downloaded files into STRAP by pressing HTMLDOC_BUTTON:SBUTS_TAB_FETCH_PDB_Load
You should now see the nucleotide sequences in the alignment pane. Select "nucleotides" in the selector HTMLDOC_JCOMPONENT:HTMLDOC_JC_Strap_comboColorScheme! 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 HTMLDOC_BUTTON:SBUT_ALIGN!. Keep a copy of the current alignment by exporting the current alignment.

Translating the nucleotides sequences:

Now we are going to translate the nucleotide sequences with the dialog HTMLDOC_BUTTON:BUT_C1(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 HTMLDOC_BUTTON:SBUT_ALIGN!. After alignment export the alignment HTMLDOC_BUTTON:BUT_C1(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. WIKI:codon are sometimes disrupted 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. *@