Assignment #4

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  1. Start clustalx by double-clicking the icon; load globin.pep into the program; calculate an alignment and save the result under different names in different formats possible. Using a text editor (MS Word) and a non-proportional font, inspect the different formats.


    *.MSF is read by GCG; *.PIR output is like FASTA - format, but the seq. all have equal lengths, you have to merge the first and second line in order to be FASTA compatible (you also could use the GDE format and using a text editor replace % with >). Phylip (*.phy) is the "new" phylip - interlaced format.


    You can use the input/output options to reformat an alignment. Some programs require specialized formats. You can use a text editor like MSWord to get your alignment into the desired format, but things are certainly much easier, if you start out with a format close to the desired one.


    Hint: Often you do not want to use the complete alignment, but only those portions which are sufficiently conserved. You can take a file in clustal format (*.aln) and delete columns with a text editor (in MSWord pressing down the Alt key before clicking the mouse switches to column mode -- to see the alignment, you often need to decrease the font size and select a non-proportional font in your editor!). Although the different lines in the resulting alignment have different lengths, clustal reads in the aligned sequences correctly, and you can output the shortened sequences in any desired format you want.



  2. Create an input file using this file to which you should add one additional vacuolar ATPase A-subunits (proteins) of your choice. Load the sequences into clustalx and calculate an alignment using the default parameters. What is the huge indel in the center?

    Create an alignment file with the insertion removed using MSWord, i.e. only the extein portions are included in the file (see hint above - there are other ways but this is by far the easiest) and save the file under a new name. Open this file in clustal and save it again as seq21_1del.txt. (This is necessary to make sure that the blocks in the output all have the same lengths).

  3. Load the file with the intein (seq21_1.txt) and explore the different menu options, in particular explore the effect of different gap penalties on the alignment -- make sure that in alignment options the gaps are reset before you calculate a new alignment. What is the effect of application of different gap penalties?

  4. Explore the dotlet application using the HO Endonuclease and the yeast vacuolar ATPase with intein and intein only. What are your conclusions about the relationshipr between HO Endonuclease and yeast vacuolar ATPase?

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