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• (20 minutes) Using Entrez download the protein sequences with the following gi numbers: 2506213, 2493127, 4323566, 2983405, 1303679 [Hint: you can paste multiple space/comma separated gi numbers to retrieve all 5 proteins simultaneously]. Use PRSS determine if there is significant similarity between these proteins. Choose one of these sequences and compare it to all the other ones. (The 1st row (closest to the door) will use the 1st sequence, the 2nd row the 2nd sequence, etc..) We will combine the findings from the different rows into a single table. Please enter the pseudo E-value for 10000 comparisons into the table on the whiteboard and below..

  You sequence 1:

  E-values for comparison to
  2506213:
  2493127:
  4323566:
  2983405:
  1303679:
  

• (15 minutes) Repeat a few of the pairwise comparisons using Pairwise BLAST (go here). You can paste the GI numbers directly. You have to choose blastp as the program to use. (You can force the program to report insignificant alignments by increasing the expect value).

  What do the E-values mean in case of pairwise blast? (Hint: check the output after clicking align.)
  
  
  How do these E-values compare to the ones obtained using PRSS?
  
  
• (5 minutes) You find that sequence A (gi|1303679) has a significant similarity to sequence B (gi|2506213) and sequence B has significant similarity to C (gi|2983405), but C and A are not significantly similar. Can you nevertheless conclude that A is homologous to C?
  
  
  
  
• (10 minutes) Does the same reasoning hold for gi|6320016, gi|1303679, gi|2507047?
  Why might this case be different from the previous one?
  How does the output from the pairwise blast comparison help you to draw a conclusion (compare 6320016 with 1303679 and 6320016 with 2507047)?
  

 

• (10 minutes) An archaeal gene encoding PylS (gi|33466218) has been horizontally transferred into the genome of Desulfitobacterium hafniense Y51. Using genomic blast, search for the transferred gene in D.hafniense. Make sure to select blastp (protein-protein blast), and to check the graphical overview box on the format page. How many proteins show significant sequence similarity to PylS? What parts of the PylS protein do they match? Repeat using tblastn (searching against the translated nucleotide sequence of D.hafniense). Are the same regions of sequence similarity found? Observe the genomic location of the aligned translated regions. Do they provide any clues as to why PylS aligns to more than one protein sequence in D.hafniense?
  

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