Practical: Alignments, Motifs and Profiles

(An example: a DOF ZINC FINGER PROTEIN from ARABIDOPSIS THALIANA)

Step 1 Extract the Sequence of any Arabidopsis DOF gene using SRS.

Open SRS (EMBL v. 5) & click START.
Select SPTREMBL databank and Continue.
Write DOF in any AllText box and select Organism and write Arabidopsis in another one. Click Do Query.
Click on any entry (e.g. SPTREMBL:O82155)
Save the result as text:

ID   O82155      PRELIMINARY;      PRT;   194 AA.
AC   O82155;
DT   01-NOV-1998 (TrEMBLrel. 08, Created)
DT   01-NOV-1998 (TrEMBLrel. 08, Last sequence update)
DT   01-NOV-1998 (TrEMBLrel. 08, Last annotation update)
DE   DOF ZINC FINGER PROTEIN.
GN   ADOF1.
OS   Arabidopsis thaliana (Mouse-ear cress).
OC   Eukaryota; Viridiplantae; Embryophyta; Tracheophyta; Spermatophyta;
OC   Magnoliophyta; eudicotyledons; core eudicots; Rosidae; eurosids II;
OC   Brassicales; Brassicaceae; Arabidopsis.
OX   NCBI_TaxID=3702;
RN   [1]
RP   SEQUENCE FROM N.A.
RC   STRAIN=COLUMBIA; TISSUE=SEEDLING;
RA   Itagaki T., Kisu Y., Esaka M.;
RT   "cDNA cloning and gene expression of Dof zinc finger protein in
RT   Arabidopsis thaliana.";
RL   Submitted (SEP-1998) to the EMBL/GenBank/DDBJ databases.
DR   EMBL; AB017564; BAA33196.1; -.
SQ   SEQUENCE   194 AA; 21682 MW; 960CB9CF86C63BEA CRC64;
     MQDLTSAAAY YHQSMMMTTA KQNQPELPEQ EQLKCPRCDS PNTKFCYYNN YNLSQPRHFC
     KNCRRYWTKG GALRNIPVGG GTRKSNKRSG SSPSSNLKNQ TVAEKPDHHG SGSEEKEERV
     SGQEMNPTRM LYGLPVGDPN GASFSSLLAS NMQMGGLVYE SGSRWLPGMD LGLGSVRRSD
     DTWTDLAMNR MEKN
//

Step 2 Blast searching using Dof sequence.

Open the Avanced WU-BLAST2 Search Web page (EMBL)
Enter the Dof sequence (Copy & Paste)
Select the nrdb database.
Change the Descriptions and Alignments values to 100. [Look at this FIGURE]
Now, you can launch the search (click Submit Query), but the results page, to save time, is also HERE.
Results analysis.

Take a look to the sequences (they are linked to SRS) and to the different blast results fields.

The "lines" graphic is very informative. What it means?

The query sequence has been filtered (some residues have been substituted by X) Why?

Click on Get the selected sequences to obtain the ones homologous to Dof.

Save this sequences as text. (Again, the results are HERE.)

Step 3 Alignment of the Dof sequences using ClustalW.

Open the ClustalW Server (at EBI)

Look at the different Parameters and Help links.

Enter the sequences previously obtained. [Look at this FIGURE]
Same as before, you can launch the program (click RUN CLUSTALW), but the results are HERE.
Alignment analysis.

What can we say about the Dof Proteins?

Is this result compatible with the "lines" graphic from the Blast search?

To visualize the alignments using good programs makes the analysis easier. As an example, take a look to:

Then, we have found the clear homologous to the Dof protein and we have defined a conserved region into the family sequences alignment.
Another question to be resolved could be: There are some other protein families far related to Dof?

Step 4 Analysis of Dof Sequence using Prosite (Profiles database)

Open the Prosite server (at ISREC)
Select Prosite profiles (NScore) and Prosite patterns (no score)
Enter a Dof sequence into the text field. [Look at this FIGURE]
Click on Run ProfileScan (the results are HERE.)

Results analysis: There is any informative pattern hit?

Step 5 Pfam analysis of the Dof sequence (hidden Markov models)

Open the Pfam server (at Sanger Center)

Look at the different search possibilities in this Alignments Database.

Click on PROTEIN SEARCH.
Enter a family conserved Dof sequence into the text field.

An example:

LKCPRCDSPNTKFCYYNNYNLSQPRHFCKSCRRY
WTKGGALRNVPVGGG SRKNATKRSTSSSSSAS

Change the E-value cutoff level to 100 (more permisive search) [Look at this FIGURE]
Click on Search Pfam (again, the results are HERE.)
Pfam results analysis

What can we say about the Pfam families that appear as results?

Click on the most interesting domains (both are Transcription factors):

TFIIS
GATA

Pfam fields

Links to PROSITE
Alignments (select Full)
Domain organization (select seed) (Look at the GATA domain containing proteins)

Que podemos decir de la organizacion de Dominios de la familia GATA?

Look at the Species Distribution, too.

Joining this results to the predicted Secondary Structure of Dof (simmilar to GATA's one):

AA         LKCPRCDSPNTKFCYYNNYNLSQPRHFCKSCRRYWTKGGALRNVPVGGGSRKN..ATKRSTSS
PHD_sec                EEEE        HHHHHHHHHEE    EEEE                    
Rel_sec    999999999995476449999997134423443114799465637999997699999998999
SUB_sec    LLLLLLLLLLLL.EE..LLLLLLL............LLL.EEE.LLLLLLLLLLLLLLLLLLL

then, some interesting conclusions could be obtained (as in this ARTICLE).

THIS IS THE STRUCTURE OF GATA:

Look at the Cys residues coordinating the Zn atom.

Other figures are:

All Dof genes table.

Dof proteins phylogenetic tree.

The tree alignment.

PGP-17-10-00