Exercise 3 - Search for Homologues in Sequence Databases

Aim: find homologues of human insulin protein

1- Search for homologues in vertebrates

Go to the BLAST page for search against protein databases at PBIL, and paste the protein sequence of human insulin. Perform the search with BLASTP against the SWISS-PROT database and modify the default options using these parameters: 
Expect:        0.0001
Descriptions:  100
Alignments:    100

On the result page, filter the output to retain only vertebrate sequences that are not partial. For that purpose, use the following options from the Filter your BLAST results by taxon, keyword or date menu: 

Taxon    IS      Vertebrates
Keyword  IS NOT  partial

On the output (***) select, by clicking on the checkboxes left to the sequence names, all the insulin sequences that are longer than 100 amino acids (***). Note that entries annotated as INS1, INS2, PREPROINSULIN or INSULIN PRECURSOR are indeed insulins. On the other hand, do not select insulin-like growth factors or short sequences. After that, click on the Get selected sequences button and save the sequences in text format. These sequences will be useful for the Exercise 4 of the course.

2- Search for homologues in C. elegans with BLAST

Go back to the BLAST page and paste again the protein sequence of human insulin. Perform the search with BLASTP against the SWISS-PROT database using default parameters. 





On the result page, filter the output to retain only C. elegans
sequences. For that purpose, use the following option from
the Filter your BLAST results by taxon, keyword or date
menu:



Taxon  IS  C. elegans






How many nematode insulin homologues did you find?


Now repeat the BLASTP search using these parameters:



Expect:        1000
Descriptions:  10000
Alignments:    0






How many nematode insulin homologues did you find?


3- Search for homologues in C. elegans with PSI-BLAST

Now we will repeat the search with the
PSI-BLAST service
at NCBI. Click on the PSI- and PHI-BLAST link, paste
the protein sequence of human insulin, use the nr database and
modify the Format option as follows:



Descriptions: 1000
Alignments:   1000






In the window that appears, click on the Format button to
start the computation. After the first BLAST, run three other PSI-BLAST
iterations, with the same parameters. For that purpose, first click on
the button Run PSI-Blast iteration, and then on the
Format button in the first window. Now click on the taxonomy
report and found how many nematode sequences have been detected 
(***).






If you have problems or comments...



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