Tricks: The simplest way to edit an html page is to create a text document in MS-Word (*.doc) or in OpenOffice (*.odt) text document  and saving in *.html format. Visualize the page in the browser to see the final presentation.

Practice 1.2: Transfer files to a remote computer.

Connect to a remote computer to the Structural Biocomputing and Modeling Center (SBMC) at IBMC-UMH and transfer a file. For this and many other practices you need a login and a password. Connect to server "proteo.ibmc.umh.es" using your own login and password provided by your supervisor.

Tricks: Use the SSH Client program: File/Quick Connect...   and write the server name and your login (figure left, insert). Then you will be asked for a password. You will see a shell with the confirmation that you have entered in the server (figure left). The command line can be used to run command or programs. Try the following commands: whoami, ls, ls -al, pwd, top (ctrl+c to break).
Now you can transfer a file to an appropriate folder in two steps:
1. At the prompt type "mkdir" to create the folder: $mkdir folder_name. Now change to the new folder: $cd folder_name. Check your new location: $pwd. Tipically you will want to have a folder structure like this: ..../RESULTS/P1/P1.2
2. Go to your SSH shell and select Windows/New File Transfer. A new window appears (figure right). Left panel is your local machine while right panel is the remote one. Select the appropriate folder either in the local or remote computers. As an example download locally the compressed file GetPDBfiles and transfer it to the remote server. For this just select (left+click) the file in the left panel and drag to the desired folder in the right panel.

Practice 1.3: Run programs in a remote computer. Run prochek to check a structure file. Later use the program ps2pdf to transform *.ps to *.pdf.

Connect with ssh to proteo.ibmc.umh.es server (login: name.familyname pass: nick) and run the program prochek to check the structure Model1.pdb at 2.1 Å resolution. Visualize the Ramachandran plot. Could we run prochek from a web server?

Tricks: Connect to the machine and make an appropriate folder (remember: no spaces, no weird characters). Transfer the file to this folder. Run prochek to see the command line format and run the program. Use the following format: %prochek Model1.pdb 2.1. The results are "*.ps". Transform to pdf using the program "ps2pdf"; focus on Model1_01.ps and download the result from the server to your computer. Then store the result correctly in the server.

Practice 1.4: Capture results from the screen.

A useful way to get and store the results obtained is the screen capture. You will use this utility along these practices. Capture any part of your actual screen and paste it in a word processor. Alternatively you can print the page in pdf format.

Tricks: Use the portable software Lightscreen for captures. For printing in pdf use DoPdf.

2.- Scripting & Database Management

Practice 2.1: Get or update structural information from a database: SMART. In this practice we want to know which and how many structures are available for certains  domains in SMART. These domains are WW, SH3, SH2, PDZ. Imagine you want to get a file with all the names of the structure files, but you already have some of these names.

Tricks: This job is quite tedious. It is important to realize that the scriptting programs are invaluable help to automatize work. Use the python script SMART_domain_checker. Edit the file locally in windows with python IDLE, NotePad or EditPlus and follow the instructions inside. Transfer the file and run the script in the SBMC server "proteo.ibmc.umh.es". Run the script four times, one for each domain. Check the text content of the *_XX.txt files 'before' and 'after' the use of the script, as well as the 'new_PDB_files_XX.txt' files that are generated by the script.

Practice 2.2: Get all pdb structure files from PROTEIN DATA BANK. Now we want to get all pdb files found in Pratice 2.1.

Tricks: Again the job is tedious. Use the python script Get_PDBfiles. Copy the script to "proteo" in an appropriate folder (no edition is needed) containing the list of desired *.pdb files obtained in exercise 2.1. Execute script as follows:

$python GetPDBfile.py filename_list.txt (enter)

Practice 2.3: Change all pdb file names to uppercase. This is just for convenience. Most of the times several terminology problems are avoided if names of structures, sequences, etc. are homogeneous.

Tricks: Use python script Change_case_files. Edit the file locally in windows with python IDLE, NotePad or EditPlus and follow the instructions inside. Transfer the file and run the script in the SBMC server "proteo.ibmc.umh.es".

Practice 2.4: Get Information for pdb files. This practice is conceptually very simple: Once you have a list of pdb files from a given domain, you want to extract as many information as possible from each domain, namely: 'Swiss-prot-ENTRY_NAME', 'Swiss-prot-AC_number', 'Sequence domain', 'Sequence ligand', 'Putative partners', 'Cellular localization', 'Drugs', 'Phosphorylated residue type', 'Experimental Technique', 'Protein Source', 'Title', 'Resolution', 'MedLine_ID', 'Reference', 'Protein family', 'Keywords', etc. Visit:

 http://www.uniprot.org/uniprot/Q04439 (Myo5 protein from S. cerevisiae) to check that all these information is easily accesible from UNIPROT. However, the automatation of this data extraction is a bit more complicated.

Tricks: Download python 2.6+modules modules and install them. Download the file Get_Information, modify the necessary lines and run the python script Get_Information.py in Windows (not Linux) using the IDLE editor. Some example files (cleaned and original pdb files) are provided. The domains belong to the PDZ family.

3.- Managing Protein Databases

Practice 3.1: Try to find an alive database (with its corresponding home server address and the date of the latest update) dealing with:                  [solution]

• nucleic acid sequences (in Japan)
• microarray data
• mass spectrometry data repository
• protein protein interaction data
• rat enamel 2D gel electrophoresis
• CFTR mutations

Tricks: You can search databases with Google or with the ExPASy Life Science Directory

Practice 3.2: Human erythropoietin (EPO) in different protein sequence databases. Find, if it exists, one entry corresponding to human erythropoietin protein sequence in the following protein sequence databases:          [solution]

• NCBInr http://www.ncbi.nlm.nih.gov/
• RefSeq http://www.ncbi.nlm.nih.gov/RefSeq/
• UniProtKB http://www.uniprot.org/find sequence(s) in UniProtKB/Swiss-Prot and sequence(s) in UniProtKB/TrEMBL
• PIR-PSD http://pir.georgetown.edu/pirwww/dbinfo/pir_psd.shtml
• UniParc http://www.uniprot.org/
• UniRef50 http://www.uniprot.org/
• Ensembl http://www.ensembl.org/Homo_sapiens/searchview
• IPI IPI query

Find the UniProtKB/Swiss-Prot entry corresponding to:

• RefSeq NP_036231 - GI:584682

Tricks: You can use the query tool provided by each database.
You can use SRS
You can use the crosslinks (if they exist) to go from one database to another...
You can use the ID/AC mapping tool provided by UniProt

Practice 3.3: Searching all the protein sequences for the gene "ken and barbie" with text search tools

• Enter "ken and barbie" in the text search box of UniProt website
• How many sequence do you get? Are there mainly reviewed or unreviewed?
• Do the same search at NCBI

Tricks: [solution]

Practice 3.4: Different looks and tools for the same entry....

• Starting with the UniProt server: (http://www.uniprot.org/):
• Look for the amino acid sequence of human carbonic anhydrase 2.
• Get the corresponding nucleic acid entries in EMBL and GenBank: try to find a nucleic acid sequence derived from genomic DNA sequencing and another one derived from cDNA sequencing.
• From the UniProtKB/Swiss-Prot entry, look at the data available for variant Pro-92 and in particular its position in the 3D structure (Use the “Jmol viewer”).

• Starting with the NCBI server: (http://www.ncbi.nlm.nih.gov/):
• Look for the amino acid sequence of human carbonic anhydrase 2 using ENTREZ Protein.
• Find the UniProtKB/Swiss-Prot entry and as above:
• Get the corresponding nucleic acid entries in EMBL and GenBank.
• Find the data available for the variant Pro-92.

Tricks: [solution]

Practice 3.5: Browsing a database. Search in UNIPROT (http://www.uniprot.org) the sequence of the botulinum toxins (types A to G) from Clostridium botulinum. Optimize the search so that the query produce 10 results or less. Extract and download the sequence in fasta format. [solution]

Tricks: Take advantage of the facilities in the UNIPROT server. Download results in fasta format. This format is as follows:

>bontoxilysin A

Practice 3.6: Search all proteins containing SH3 domains in S. cerevisiae and S. pombe. Use SMART as the source database and download protein architecture, whole protein sequence in fasta, and SH3 protein sequence in fasta, as well.

Tricks: Visit SMART and select Normal Mode. Take advantage of the query capabilities and the taxonomic selection.

Practice 3.7: 3D structure database

• Software: Java Virtual Machine.
• Find the 3D structure of 1IWO at PDB.
• Look at the complete coordinates of the entry (by clicking to Download/Display file) and save it in a text file.
• Edit the former text file and try to find a line starting with "DBREF". Look at the cross-reference to UniProtKB/Swiss-Prot.
• Visualize the structure-sequence relationships with Quick PDB.
• Can you identify the transmembrane helices? How are they annotated in the corresponding UniProtKB/Swiss-Prot entry?
• How many PDB entries are there for the lysozyme T4?

Tricks: [solution]

4.-  Managing Genomic Databases

Practice 4.1: Browsing genes in genome databases

• Look for the UniProtKB/Swiss-Prot entry of the E.coli gene gutQ.
• Follow the link to EcoGene (EcoGene Database of Escherichia coli sequence and function) and find the chromosomal location.
• Get the next E.coli gene on the same strand.
• Follow the link to UniProtKB/Swiss-Prot.
• Find the subcellular localisation of the protein.

Tricks: [solution]

Practice 4.2: Locating genes in chromosomes

• Find the IL-2R alpha gene in the OMIM database? What is its chromosomal location ?
• View the cytogenetic maps of the regions surrounding the gene loci
• Are there known diseases associated with this gene? What are the associated disorders ?
• From OMIM (IL-2R alpha), follow the cross-reference to Entrez Gene. Have a look to the Reference Sequences (RefSeq).
• Find the corresponding UniProtKB/Swiss-Prot entry

Tricks: [solution]

Practice 4.3: Gene ontology database: structure and validation of the data...

• Look at the definition of ontology in wikipedia
• Go to the Gene ontology consortium (AmiGO)
• Compare the GO terms associated with mouse and human erythropoeitin
• Go to the UniProtKB/Swiss-Prot entry for human erythropoietin: look at the associated GO terms How many terms have been 'manually' attributed to the gene (have a look to the GO evidence tag )?

Tricks: [solution]

Practice 4.4: Comparing two putative splice variants of a human gene. The following two protein sequences, from SwissProt and TrEMBL, map to the same locus on human chromosome 1. They are both derived from RNA sequences that were originally isolated from cancerous tissues.

>EKI2_HUMAN
MAVPPSAPQQRASFHLRRHTPCPQCSWGMEEKAAASASCREPPGPPRAAAVAYFGISVD
PDDILPGALRLIQELRPHWKPEQVRTKRFTDGITNKLVACYVEEDMQDCVLVRVYGERT
ELLVDRENEVRNFQLLRAHSCAPKLYCTFQNGLCYEYMQGVALEPEHIREPRLFRLIAL
EMAKIHTIHANGSLPKPILWHKMHNYFTLVKNEINPSLSADVPKVEVLERELAWLKEHL
SQLESPVVFCHNDLLCKNIIYDSIKGHVRFIDYEYAGYNYQAFDIGNHFNEFAGVNEVD
YCLYPARETQLQWLHYYLQAQKGMAVTPREVQRLYVQVNKFALGPSCVSSTMTASLQCC
RVGNRHGEIARLTLSGLFPGVSLLLGSLGPHPEPVLHHRL

>Q96G05
MAVPPSAPQPRASFHLRRHTPCPQCSWGMEEKAAASASCREPPGPPRAAAVAYFGISVD
PDDILPGALRLIQELRPHWKPEQVRTKRFTDGITNKLVACYVEEDMQDCVLVRVYGERT
ELLVDRENEVRNFQLLRAHSCAPKLYCTFQNGLCYEYMQGVALEPEHIREPRLFRLIAL
EMAKIHTIHANGSLPKPILWHKMHNYFTLVKNEINPSLSADVPKVEVLERELAWLKEHL
SQLESPVVFCHNDLLCKNIIYDSIKGHVRFIDYEYAGYNYQAFDIGNHFNEFAGVNEVD
YCLYPARETQLQWLHYYLQAQKGMAVTPREVQRLYVQVNKFALASHFFWALWALIQNQY
STIDFDFLRYAVIRFNQYFKVKPQASALEMPK

Verify that the two proteins can indeed be (nearly) perfectly mapped to the same human genomic region

>HUMAN_TRIMMED_1
GACTTCCCCTGGCCCCTCCTCTACCACTCCCACTCCCTCGCCGGACCCCCCCGCCGGGG
CTAGCGTCTGCCGCGGCTCCGAGGGGGTGGGGCTGCTGGGAATGGCTGTGCCCCCTTCG
GCCCCTCAGCCGCGCGCGTCCTTTCACCTGAGGAGGCACACGCCTTGCCCGCAGTGCTC
ATGGGGCATGGAGGAGAAGGCGGCGGCCAGCGCCAGCTGCCGGGAGCCGCCGGGCCCCC
CGAGGGCCGCCGCCGTCGCGTACTTCGGCATTTCCGTGGACCCGGACGACATCCTTCCC
GGGGCCCTGCGCCTCATCCAGGAGCTGCGGCCGCATTGGAAACCCGAGCAAGTTCGGAC
CAAGGTAGCGGAGTGGGCGCGGGGCCGAGGATGGGGTCCTGCCAGGGCTGCCAGGGGGA
TGCGGGGACCCAGTCCTCGCATCCTTCCGGGTGTCAGTCCCAGGCAAATGAGCACCTCC
TGCTGGAGCTACAGAGCCCAGGAGAGAGACCAGAGAGATCCTGCCCCACCCACCCACCC
ACGCACTGTCCTCTCCCCATGCTAGCGCTTCACGGATGGCATCACCAACAAGCTGGTGG
CCTGCTATGTGGAGGAGGACATGCAGGACTGCGTGCTGGTCCGGGTGTATGGGGAGCGG
ACGGAGCTGCTGGTGGACCGGGAGAATGAGGTCAGAAACTTCCAGCTGCTGCGAGCACA
CAGCTGTGCCCCCAAACTCTACTGCACCTTCCAGAATGGGCTGTGCTATGAGTACATGC
AGGGTGTGGCCCTGGAGCCTGAGCACATCCGTGAGCCCCGGCTTTTCAGGTGAGGAGGG
TGCCAGGGCCTCTGTCTCTACTATTCTTCAGGCCTTGGGCTTGGAAATCCTTGGCATGT
GGGGTCCATGGTGGAGAGTTGTAAGATCTGTTGCCCTGGTGGGCCTGGAGGGTCCCTAT
CTGGCCAGGAGGAAATGAGTGGGGAAGGCAATGTCCCAGCACTGATGCTCTACTCTTCC
TGTTCCCTGCTGTAGGTTAATCGCCTTAGAAATGGCAAAGATTCATACTATCCACGCCA
ACGGCAGCCTGCCCAAGCCCATCCTCTGGCACAAGATGCACAATTATTTCACGCTTGTG
AAGAACGAGATCAACCCCAGGTACAAAGATCTGGGAGGGTCCAAGGCTGCCTTAATATG
ATCTCCCTTCCCCATCCTTTTGGCTCCAGCCCACCCTGCTAGGAGGTTTAAGGGCTTAC
AGTAAGACAGAGGGAGTGATGAGGCTCAGATTCAGGGCTCTTGAGGCCAAGCACCTGTC
TATCAGCCCTCCTTTCACTGGCTACCTTTTTTGCCACTGCCCAGCCTTTCTGCAGATGT
CCCTAAGGTAGAGGTGTTGGAACGGGAGCTGGCCTGGCTGAAGGAGCATCTGTCCCAGC
TGGAGTCCCCTGTGGTGTTTTGTCACAATGACCTGCTCTGCAAGAATATCATCTATGAC
AGCATCAAAGGTATGGCTTCTCTGGCCCTGGGGCAGCAGCAGGGTTTTGGTTGGTTGGT
TAGCTGGCTTCACGGAGCTGTTCTTCCGGCTGGCACTTGGACAAGCAAAAGTGCATCAC
TCAGTGGGTGCATCCCAGAGGTCTGTGGAAGCTTGACCAGGCTCTGTTGCCAGATCTCC
TGGGCCAACTCTCATCGATTCTCCTCTACTTCATAGGTCACGTGCGGTTCATTGACTAT
GAATATGCTGGCTACAACTACCAAGCTTTTGACATTGGCAACCATTTCAATGAGTTTGC
AGGTGAGAGGGGCATTTCTACAGTCATGTGCTTTGTAGCGGTGTTTCAGTCAATGACTG
ACCGCATGTAGATGGTGGTCCTATGAGATTACTGTATATTTACGAAGGAGATGGGAGTT
TGAGGGGAGATGGGAACACCTACTGAGCTTGAGGAGACCCTTGGTGGGCCCATCTCTGA
GCAACTTTGTCTGGCCTGCCTCCCCCTCAGGCGTGAATGAGGTGGATTACTGCCTGTAC
CCGGCGCGGGAGACCCAGCTGCAGTGGCTGCACTACTACCTGCAGGCACAAAAGGGGAT
GGCCGTGACCCCCAGGGAGGTGCAAAGGCTCTACGTGCAAGTCAACAAGTTTGCCCTGG
TGAGTGCCTTATTTCTAGGGCTGGGGCAGGAAAGGAGGAGTTAGCAGGAGGGACTGGTG
ACCCACCTAGCCTCCTACCCAGAGCCCAGGGTCAGGTGAGAGCCTGGGGACACCCATCC
CTGGTTTCCCCAGCTTGTGCTAGGGAGACCCCACCACATCTCTTCCAGGGTCCTAGCTG
TGTGTCTTCCACAATGACTGCATCCCTCCAGTGCTGTAGAGTCGGAAACAGGCATGGGG
AGATTGCCAGGCTGACCCTCTCTGGTCTGTTTCCAGGCGTCTCACTTCTTCTGGGCTCT
CTGGGCCCTCATCCAGAACCAGTACTCCACCATCGACTTTGATTTCCTCAGGTGAGTGT
GGGGGTGATGGTGGGACGTGGAGAAAAGGGAGAGCCTTGGTTGAGCCCAGGGTGTGGGC
AAAGGACAGCCCAGGCAGAATGCCCAGAGTCCGAGGCCTAGAGCCACTGAAAAACTTTT
CTCATGATGTCAGCACCCCCAGCAGGGCCCACCTGGCTTTCTTCCTCACACCCAGCTTC
CTTCTGTCTTCCCCACAGGTACGCAGTGATCCGATTCAACCAGTACTTCAAGGTGAAGC
CTCAAGCGTCAGCCTTGGAGATGCCAAAGTGACCAGCCACCCCATCCCTCCCCTACCCA
TCTGTCTGGCCAGACCTGTTCTCCAGAGCTCAATTCTGCACTCTGGGATCCACACCCTT
GGACAGGGTG

If you do pairwise comparisons between the two protein sequences on one hand, and between the two corresponding cDNAs on the other (you can use lalign), you will notice a discrepancy. Looking at the exon coordinates in the GeneWise output, can you resolve it?

Tricks: Use the GeneWise web server at http://www.ebi.ac.uk/Wise2/. To speed up this task, you are provided with a trimmed version of the genomic human region, which corresponds to all the BLAST HSPs against either protein plus 100 bp on either side of the HSPs. Make sure you select the output option cDNA in the GeneWise form, so that you can retrieve the predicted cDNA for both splice variants! You are advised to copy the full GeneWise output into a text document for further reference.

Practice 4.5: Predicting splice variants in the mouse

The mouse orthologous locus of the human gene studied above, is given here (untrimmed): MOUSE_GENOMIC_1 (about 17 kb). This mouse genomic region region has not been investigated yet.

You are asked to map the two human protein sequences from above to it. Again, select the cDNA and translation output options in GeneWise. Looking at the results, which of the two human protein sequences do you trust more? (you can compare the mouse and human proteins using lalign as in the previous exercise).

Tricks: Each of these analyses will run for about 3 min on the GeneWise server, so maybe you can do these exercises in larger groups together.

5.- Managing Other Databases

Practice 5.1: Metabolic / Enzyme databases

• Go to the Genome Net server (Japan)
• Find a database called KEGG
• In KEGG, find the enzyme number EC 1.2.3.4
• Have a look at BRENDA database from the KEGG entry.
• Get from KEGG the ENZYME entry in ExPASy, then from ENZYME the UniProtKB/Swiss-Prot entry.
• Using Brenda database: find all enzymes using FAD as cofactor.
• At KEGG pathway: look for the Krebs cycle: compare this pathway in E.coliK12 and in human.

Tricks: [solution]

Practice 5.2: Polymorphisms

• Look for information in dbSNP on the human blue eye variant rs12913832
• What is the Craig Venter's 'eye color' (Celera genome assembly) ?
• Follow the link to the Alfred database to look for the population distribution of the 'blue eye allele'.

Tricks: [solution]

Practice 5.3: Publication databases

• How many papers did Nature publish in 1995 ?
• Find the publication dealing with Dolly death and find its DOI number.
• Get the publication thank to this DOI number on the site http://dx.doi.org
• How many articles are dealing with Viagra ? What is the 'generic' name of the molecule ?

Tricks: [solution]

6.- Tools: Pairwise Alignments

Practice 6.1: Compare the sequences OPRM_RAT and SSR1_HUMAN (these are the SWISS-PROT IDs) with lalign using default parameters.

The sequences can be fetched here (choose the "FASTA" format) using the SWISS-PROT IDs.
Don't hesitate to look at the complete SWISS-PROT entries (OPRM_RAT and SSR1_HUMAN), in order to get more information about these two proteins !

Try to answer the following questions:

• Is this a local or global alignment ?
• Switch between local and global alignment . Try to understand the differences.
• Why are there several alignments displayed when performing the local alignment ?
• What does "% identity" mean ? How is it computed ?
• What do the symbols ":" and "." stand for ?
• When two residues are different, there can be either a "." or a blank. Try to understand the difference and what parameters influence this result ?
• Try to modify the gap penalties, examine more closely how these parameters influence the occurrence and the length of gaps ("-").
• Try to modify the scoring matrices used (i.e. BLOSUM35 and BLOSUM80), examine more closely how these parameters influence the scores and the alignments.

Tricks: see the following hints

Practice 6.2: Dotplot using Dotlet

Compare the same sequences (OPRM_RAT and SSR1_HUMAN) using Dotlet.

The sequences can be fetched here (choose the "FASTA" format).

Start with a look onto the Dotlet documentation

• Load the two sequences into Dotlet and compute the dotplot.
• What does the intensity (gray level) of a pixel mean ?
• Try changing the grayscale borders. Where would be an optimal position for the upper and lower limits of the grayscale ?
• What do the diagonal lines represent ?
• Try to identify corresponding aligned regions in the dotplot and the alignments found by LALIGN.
• Try to modify the noise by changing the window size, the threshold, both.
• Try comparing each sequence against itself.

Tricks: Dotlet examples and method comparison. The Dotlet learn by example pages show different typical sequence analysis problems. Take an interested look at the Dotlet examples and try to understand the dotplots.

Practice 6.3: Perform a pairwise sequence alignment for the following analogous proteins at EBI

>539057
MATAGKVIECKAAVAWEAGKPLSIEEVEVAPPHAMEVRVKILYTALCHTDVYFWEAKGQTPVFPRILGHE
AGGIVESVGEGVTELVPGDHVLPVFTGECKDCAHCKSEESNLCDLLRINVDRGVMIGDGQSRFTINGKPI
FHFVGTSTFSEYTVIHVGCLAKINPEAPLDKVCVLSCGISTGLGATLNVAKPKKGSTVAIFGLGAVGLAA
MEGARMAGASRIIGVDLNPAKYEQAKKFGCTDFVNPKDHTKPVQEVLVEMTNGGVDRAVECTGHIDAMIA
AFECVHDGWGVAVLVGVPHKEAVFKTYPMNFLNERTLKGTFFGNYKPRTDLPEVVEMYMRKELELEKFIT
HSVPFSQINTAFDLMLKGEGLRCIMRMDQ

>82347
MATAGKVIKCKAAVAWEAGKPLTMEEVEVAPPQAMEVRVKILFTSLCHTDVYFWEAKGQIPMFPRIFGHE
AGGIVESVGEGVTDVAPGDHVLPVFTGECKECPHCKSAESNMCDLLRINTDRGVMIGDGKSRFSIGGKPI
YHFVGTSTFSEYTVMHVGCVAKINPEAPLDKVCVLSCGISTGLGASINVAKPPKGSTVAIFGLGAVGLAA
AEGARIAGASRIIGVDLNAVRFEEARKFGCTEFVNPKDHTKPVQQVLADMTNGGVDRSVECTGNVNAMIQ
AFECVHDGWGVAVLVGVPHKDAEFKTHPMNFLNERTLKGTFFGNFKPRTDLPNVVEMYMKKELEVEKFIT
HSVPFSEINTAFDLMAKGEGIRCIIRMDN

Practice 6.4: Perform a pairwise global alignment.   [solution]

Download the P57727 protein sequence and compare with the following sequence

>splice variant 
MCSDDWKGHYANVACAQLGFPSYVSSDNLRVSSLEGQFREEFVSIDHLLPDDKVTALHHS
VYVREGCASGHVVTLQCTACGHRRGYSSRIVGGNMSLLSQWPWQASLQFQGYHLCGGSVI
TPLWIITAAHCVYDLYLPKSWTIQVGLVSLLDNPAPSHLVEKIVYHSKYKPKRLGNDIAL
MKLAGPLTFNEMIQPVCLPNSEENFPDGKVCWTSGWGATEDGAGDASPVLNHAAVPLISN
KICNHRDVYGGIISPSMLCAGYLTGGVDSCQGDSGGPLVCQERRLWKLVGATSFGIGCAE
VNKPGVYTRVTSFLDWIHEQMERDLKT

Compare the results you obtained with the results obtained by doing a pairwise local alignment between the same pairs of sequences. How can you explain the differences? Which domain is missing in the splice variant of the protein?

Tricks: Use Align tools at EBI.

7.- Multiple Sequence Alignment

Practice 7.1: Generate a multiple Sequence Alignment of the botulinum toxins types A to G found in former exercises (Managing protein databases, practice 5). Use CLUSTALW in server "proteo.ibmc.umh.es".

Tricks: Use ssh to connect to the server. Make a directory and copy the fasta formated sequences. Invoke the program by typing "clustalw" and following the instructions. If you get "command not found", type "locate clustalw" and follow the absolute path.

Practice 7.2: Orthology and Paralogy. Go to the MyHits web site (myhits.isb-sib.ch), select the PROTEIN Hub from the Hub menu, paste these 3 SwissProt ID or AC:      [solution]

sw:STMN2_HUMAN Q93045

sw:STM2A_XENLA Q09001

sw:STMN1_MOUSE P54227

Send the proteins to either ClustalW or T-Coffee to produce the multiple alignment. Send the resulting alignment to the MSA Hub and start the Jalview applet. From the Calculate menu of the applet, create a Neighbour Joining Tree.

• Discuss the results. What is wrong? Propose a strategy.
  
• Add sequences from SwissProt in your list, make a new multiple sequence alignment and a tree as before (make sure to include the STMN2 sequences from rat, human and mouse)
  
• Discuss the results and propose a new strategy in order to obtain a better tree.

Tricks: Make a multiple sequence alignment with CLUSTALW and set the output to phylip format.  [hints]

Make a MSA with the DNA and restrict the coding regions. To compute the tree, use infobiogene. To plot a tree, you may use phylodendron or TreeView.

Remember: Never use a clustalw guide tree file (.dnd) in place of a phylogenetic tree.

>ens:ENSP00000354426
MAAGPSGCLVPAFGLRLLLATVLQAVSAFGAEFSSEACRELGFSSNLLCSSCDLLGQFNL
LQLDPDCRGCCQEEAQFETKKLYAGAILEVCGUKLGRFPQVQAFVRSDKPKLFRGLQIKY
VRGSDPVLKLLDDNGNIAEELSILKWNTDSVEEFLSEKLERI
>ens:ENSRNOP00000036726
MAAGQGGWLRPALGLRLLLATAFQAVSALGAEFSSEACRELGFSSNLLCSSCDLLGQFNL
LPLDPVCRGCCQEEAQFETKKLYAGAILEVCGUKLGRFPQVQAFVRSDKPKLFRGLQIKY
VRGSDPVLKLLDDNGNIAEELSILKWNTDSVEEFLSEKLERI
>ens:ENSMUSP00000046910
MAAGQGGWLRPALGLRLLLATAFQAASALGAEFASEACRELGFSSNLLCSSCDLLGQFNL
LPLDPVCRGCCQEEAQFETKKLYAGAILEVCGUKLGRFPQVQAFVRSDKPKLFRGLQIKY
VRGSDPVLKLLDDNGNIAEELSILKWNTDSVEEFLSEKLERI
>ens:ENSANGP00000011457
MRLFAITCLLFSIVTVIGAEFSAEDCRELGLIKSQLFCSSCSSLSDYGLIELKEHCLECC
QKDTEADSKLKVYPAAVLEVCTCKFGAYPQIQAFIKSDRPAKFPNLTIKYVRGLDPIVKL
MDEQGNVKETLSINKWNTDTVQEFFETRLAKVEDDDYLKTNRV

Tricks: [hint]

Practice 7.4: Use BioEdit to identify the consensus sequence for the following somatostatins

gi_6678034 ref NM—09215.1 Mouse somatostatin (Smst), mRNA

AGCGGCTGAAGGAGACGCTACCGAAGCCGTCGCTGCTGCCTGAGGACCTGCGACTAGACTGACCCA
CCGCGCTCCAGCTTGGCTGCCTGAGGCAAGGAAGATGCTGTCCTGCCGTCTCCAGTGCGCCCTGGC
TGCGCTCTGCATCGTCCTGGCTTTGGGCGGTGTCACCGGCGCGCCCTCGGACCCCAGACTCCGTCA
GTTTCTGCAGAAGTCTCTGGCGGCTGCCACCGGGAAACAGGAACTGGCCAAGTACTTCTTGGCAGA
GCTGCTGTCCGAGCCCAACCAGACAGAGAATGATGCCCTGGAGCCCGAGGATTTGCCCCAGGCAGC
TGAGCAGGACGAGATGAGGCTGGAGCTGCAGAGGTCTGCCAACTCGAACCCAGCAATGGCACCCCG
GGAACGCAAAGCTGGCTGCAAGAACTTCTTCTGGAAGACATTCACATCCTGTTAGCTTTAATATTG
TTGTCCTAGCCAGACCTCTGATCCCTCTCCCCCAAACCCCATATCTCTTCCTTAACTCCTGGCCCC
CGATGCTCAACTTGACCCTGCATTAGAAATTGAAGACTGTAAATACAAAATAAAATTATGGTGAGA
TTATG

gi_207030_gb_M25890.1_RATSOMX Rat somatostatin mRNA, complete cds

TGCGGACCTGCGTCTAGACTGACCCACCGCGCTCAAGCTCGGCTGTCTGAGGCAGGGGAGATGCTG
TCCTGCCGTCTCCAGTGCGCGCTGGCCGCGCTCTGCATCGTCCTGGCTTTGGGCGGTGTCACCGGG
GCGCCCTCGGACCCCAGACTCCGTCAGTTTCTGCAGAAGTCTCTGGCGGCTGCCACCGGGAAACAG
GAACTGGCCAAGTACTTCTTGGCAGAACTGCTGTCTGAGCCCAACCAGACAGAGAACGATGCCCTG
GAGCCTGAGGATTTGCCCCAGGCAGCTGAGCAGGACGAGATGAGGCTGGAGCTGCAGAGGTCTGCC
AACTCGAACCCAGCCATGGCACCCCGGGAACGCAAAGCTGGCTGCAAGAACTTCTTCTGGAAGACA
TTCACATCCTGTTAGCTTTAATATTGTTGTCTCAGCCAGACCTCTGATCCCTCTCCTCCAAATCCC
ATATCTCTTCCTTAACTCCCAGCCCCCCCCCCAATGCTCAACTAGACCCTGCGTTAGAAATTGAAG
ACTGTAAATACAAAATAAAATTATGGTGAAATTATG

_gi_163636_gb_M31217.1_BOVPSOMA Bovine somatostatin mRNA, complete cds

AAGCTGCTTTAGGAGAGGCAAGGTTCGAGCCGTCGCTGCTGCCTGCGATCAGCTCCTAGAGTTTGA
ACTCTAGCTCGGCTTCGCCGCCGCCGCCGAGATGCTGTCCTGCCGCCTCCAGTGCGCGCTGGCCGC
GCTCTCCATCGTCCTGGCTCTTGGCGGTGTCACCGGCGCGCCCTCGGATCCCCGGCTCCGTCAGTT
TCTGCAGAAATCCCTGGCTGCTGCCGCTGGCAAGCAGGAACTGGCCAAGTACTTCTTGGCAGAGCT
GCTGTCTGAACCCAACCAGACAGAGATTGATGCCCTGGAGCCTGAAGATTTGTCCCAGGCTGCTGA
GCAGGATGAAATGAGGCTGGAGCTGCAGAGATCTGCTAACTCAAACCCGGCCATGGCACCCCGAGA
ACGCAAAGCTGGCTGCAAGAATTTCTTCTGGAAGACTTTCACATCCTGTTAACTTTATTAATGATT
GTTGCCCATATAAGACCTCTGATTCCTCTTCTCCAAACCCCTTCTCACCTCCCTAATCCCTCCAAT
CCTCAATAAGACCCTCGTGTTAGAAATTGAAGACTGTAAATACAAAATAAAATTATGGGAAATTAT
G

Tricks:  Install BioEdit in your computer and run it. Create a new alignment (File/New Alignment). Then add sequences and names in Sequences/New. You will see the sequences in italic and black and white. Save file (File/Save as...) and reload the file. Now you will see the sequences in full colour and most functions activated. Make an MSA in BioEdit (Accessory Applications/CLUSTALW), and  then Alignment/Create consensus sequence. Colour the alignment to highlight the common areas.

Practice 7.5: Make a MSA of the following sequences to align correctly the active center of the catalytic domain of kinases.

>D28     CD28  S. CEREVISIAE CELL CYCLE CONTROL PROTEIN KINASE
ANYKRLEKVGEGTYGVVYKALDLRPGQGQRVVALKKIRLESEDEGVPSTAIREISLLKEL
KDDNIVRLYDIVHSDAHKLYLVFEFLDLDLKRYMEGIPKDQPLGADIVKKFMMQLCKGIA
YCHSHRILHRDLKPQNLLINKDGNLKLGDFGLARAFGVPLRAYTHEIVTLWYRAPEVLLG
GKQYSTGVDTWSIGCIFAEMCNRKPIFSGDSEIDQIFKIFRVLGTPNEAIWPDIVYLPDF
KPSFPQWRRKDLSQVVPSLDPRGIDLLDKLLAYDPINRISARRAAIHPYFQES
>SKH   SKH  HELA MYSTERY PUTATIVE PROTEIN KINASE
AKYDIKALIGRGSFSRVVRVEHRATRQPYAIKMIETKYREGREVCESELRVLRRVRHANI
IQLVEVFETQERVYMVMELATGGELFDRIIAKGSFTERDATRVLQMVLDGVRYLHALGIT
HRDLKPENLLYYHPGTDSKIIITDFGLASARKKGDDCLMKTTCGTPEYIAPEVLVRKPYT
NSVDMWALGVIAYILLSGTMPFEDDNRTRLYRQILRGKYSYSGEPWPSVSNLAKDFIDRL
LTVDPGARMTALQALRHPWVVSM
>APK   CAPK  BOVINE CARDIAC MUSCLE CYCLIC AMP-DEPENDENT (ALPHA)
DQFERIKTLGTGSFGRVMLVKHMETGNHYAMKILDKQKVVKLKQIEHTLNEKRILQAVNF
PFLVKLEFSFKDNSNLYMVMEYVPGGEMFSHLRRIGRFSEPHARFYAAQIVLTFEYLHSL
DLIYRDLKPENLLIDQQGYIQVTDFGFAKRVKGRTWTLCGTPEYLAPEIILSKGYNKAVD
WWALGVLIYEMAAGYPPFFADQPIQIYEKIVSGKVRFPSHFSSDLKDLLRNLLQVDLTKR
FGNLKDGVNDIKNHK
>EE1   WEE1  S. POMBE MITOTIC INHIBITOR
TRFRNVTLLGSGEFSEVFQVEDPVEKTLKYAVKKLKVKFSGPKERNRLLQEVSIQRALKG
HDHIVELMDSWEHGGFLYMQVELCENGSLDRFLEEQGQLSRLDEFRVWKILVEVALGLQF
IHHKNYVHLDLKPANVMITFEGTLKIGDFGMASVWPVPRGMEREGDCEYIAPEVLANHLY
DKPADIFSLGITVFEAAANIVLPDNGQSWQKLRSGDLSDAPRLSSTDNGSSLTSSSRETP
ANSIIGQGGLDRVVEWMLSPEPRNRPTIDQILATDEVCWV
>GFR   EGFR  HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR
TEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASV
DNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLE
DRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHR
IYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVK
CWMIDADSRPKFRELIIEFSKMAR
>DGM  PDGF RECEPTOR, MOUSE KINASE REGION 
DQLVLGRTLGSGAFGQVVEATAHGLSHSQATMKVAVKMLKSTARSSEKQALMSELYGDLV
DYLHRNKHTFLQRHSNKHCPPSAELYSNALPVGFSLPSHLNLTGESDGGYMDMSKDESID
YVPMLDMKGDIKYADIESPSYMAPYDNYVPSAPERTYRATLINDSPVLSYTDLVGFSYQV
ANGMDFLASKNCVHRDLAARNVLICEGKLVKICDFGLARDIMRDSNYISKGSTYLPLKWM
APESIFNSLYTTLSDVWSFGILLWEIFTLGGTPYPELPMNDQFYNAIKRGYRMAQPAHAS
DEIYEIMQKCWEEKFETRPPFSQLVLLLERLLGEGYKKKY
>FES  THIS IS VFES TYROSINE KINASE
VLNRAVPKDKWVLNHEDLVLGEQIGRGNFGEVFSGRLRADNTLVAVKSCRETLPPDIKAK
FLQEAKILKQYSHPNIVRLIGVCTQKQPIYIVMELVQGGDFLTFLRTEGARLRMKTLLQM
VGDAAAGMEYLESKCCIHRDLAARNCLVTEKNVLKISDFGMSREAADGIYAASGGLRQVP
VKWTAPEALNYGRYSSESDVWSFGILLWETFSLGASPYPNLSNQQTREFVEKGGRLPCPE
LCPDAVFRLMEQCWAYEPGQRPSFSAIYQEL
>AF1   RAF1  HUMAN C-RAF-1 ONCOGENE
SEVMLSTRIGSGSFGTVYKGKWHGDVAVKILKVVDPTPEQFQAFRNEVAVLRKTRHVNIL
LFMGYMTKDNLAIVTQWCEGSSLYKHLHVQETKFQMFQLIDIARQTAQGMDYLHAKNIIH
RDMKSNNIFLHEGLTVKIGDFGLATVKSRWSGSQQVEQPTGSVLWMAPEVIRMQDNNPFS
FQSDVYSYGIVLYELMTGELPYSRDQIIFMVGRGYASPDLSKLYKNCPKAMKRLVADCVK
KVKEERPLFPQILSSIELLQH
>MOS   CMOS  HUMAN C-MOS ONCOGENE
EQVCLLQRLGAGGFGSVYKATYRGVPVAIKQVNKCTKNRLASRRSFWAELNVARLRHDNI
VRVVAASTRTPAGSNSLGTIIMEFGGNVTLHQVIYGAAGHPEGDAGEPHCRTGGQLSLGK
CLKYSLDVVNGLLFLHSQSIVHLDLKPANILISEQDVCKISDFGCSEKLEDLLCFQTPSY
PLGGTYTHRAPELLKGEGVTPKADIYSFAITLWQMTTKQAPYSGERQHILYAVVAYDLRP
SLSAAVFEDSLPGQRLGDVIQRCWRPSAAQRPSARLLLVDLTSLKA
>SVK   HSVK  HERPES SIMPLEX VIRUS PUTATIVE PROTEIN KINASE
MGFTIHGALTPGSEGCVFDSSHPDYPQRVIVKAGWYTSTSHEARLLRRLDHPAILPLLDL
HVVSGVTCLVLPKYQADLYTYLSRRLNPLGRPQIAAVSRQLLSAVDYIHRQGIIHRDIKT
ENIFINTPEDICLGDFGAACFVQGSRSSPFPYGIAGTIDTNAPEVLAGDPYTTTVDIWSA
GLVIFETAVHNASLFSAPRGPKRGPCDSQITRIIRQAQVHVDEFSPHPESRLTSRYRSRA
AGNNRPPYTRPAWTRYYKMDIDV

Tricks: Evaluate the performance of the four types of alignments: CLUSTALW, MAP, PIMA and BlockMaker in the BCM server. To simplificate the evaluation, classificate the alignment type as "all or none", whether all sequences have been correctly aligned in the server, or not.

8.- Similarity Searches: BLAST and PSI-BLAST

Practice 8.1: Make a similarity search with the protein insulin from D. rerio

>gi|12053668|emb|CAC20109.1|insulin[Danio rerio]
MAVWIQAGALLVLLVVSSVSTNPGTPQHLCGSHLVDALYLVC
GPTGFFYNPKRDVEPLLGFLPPKSAQETEVADFAFKDHAELIR
KRGIVEQCCHKPCSIFELQNYCN

Practice 8.9: Try to characterize the gene Xrcc1 for DNA reparing in humans. Answer the following questions: How this protein acts? Which domains? What it looks like?

Tricks: Use BLAST to find homologues. Compare the sequences using DOTTLET or DOTTER. Make a MSA of the homologue fragments. Use PSI-BLAST to find remote homologues with one of the fragments obtained before.

9.- Patterns, Profiles, and HMMs

Practice 9.1: PROSITE database

The aim of the exercise is to explore and understand the PROSITE database. First have a look at the following PROSITE entry: PS50235.

PS50235 is a Pattern or a Profile? How do you distinguish a Pattern from a Profile?

If this descriptor match your sequences (using standard parameters), will you believe the result? And if it doesn't match?

What is the function of PS50235? Is the PS50235 related to other PROSITE entries? If yes, are they Patterns or Profiles?

Tricks: Look at the Numerical results section. Check also the PDOC documentation

Practice 9.2: PROSITE vs. InterPro

Analyze the following sequence using InterProScan and ScanProsite:

>seq1
MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEV
CDVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFYYESDA
DTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLRLGPLSKAGFYLAFQD
QGACMALLSLHLFYKKCAQLTVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCR
EDGQWAEQPVTGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSA
VCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALR
CRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSLATGPV
PFEPVNVTTDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAVLDYEVKYHEKGAEGPS
SVRFLKTSENRAELRGLKRGASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQLAL
IAGTAVVGVVLVLVVIVVAVLCLRKQSNGREAEYSDKHGQYLIGHGTKVYIDPFTYEDPN
EAVREFAKEIDVSYVKIEEVIGAGEFGEVCRGRLKAPGKKESCVAIKTLKGGYTERQRRE
FLSEASIMGQFEHPNIIRLEGVVTNSMPVMILTEFMENGALDSFLRLNDGQFTVIQLVGM
LRGIASGMRYLAEMSYVHRDLAARNILVNSNLVCKVSDFGLSRFLEENSSDPTYTSSLGG
KIPIRWTAPEAIAFRKFTSASDAWSYGIVMWEVMSFGERPYWDMSNQDVINAIEQDYRLP
PPPDCPTSLHQLMLDCWQKDRNARPRFPQVVSALDKMIRNPASLKIVARENGGASHPLLD
QRQPHYSAFGSVGEWLRAIKMGRYEESFAAAGFGSFELVSQISAEDLLRIGVTLAGHQKK
ILASVQHMKSQAKPGTPGGTGGPAPQY

What is the domain composition of the protein? What is its function? Can you predict binding sites with InterPro? With PROSITE?

Tricks: Move the mouse on the sequences and images in the result page to highlight information.

Practice 9.3. Protein function discovery.    [solution]

• Use different methods to find protein domains in the following sequence:

>mysterious sequence
NNRNQDNYVSWSDSEDDDEDEEIEEKEKPETNFPSPFTNILCGIIFVERRYTAVVLNRLI
KEAGKQDPELAYISSNFITGHGIGKNQPRNKQMEAEFRKQEEVLRKFRAHETNLLIATSI
VEEGVDIPKCNLVVRFDLPTEYRSYVQSKGRARAPISNYIMLADTDKIKSFEEDLKTYKA
IEKILRNKCSKSVDTGETDIDPVMDDDDVFPPYVLRPDDGGPRVTINTAIGHINRYCARL
PSDPFTHLAPKCRTRELPDGTFYSTLYLPINSPLRASIVGPPMSCVRLAERVVALICCEK
LHKIGELDDHLMPVGKETVKYEEELDLHDEEETSVPGRPGSTKRRQCYPKAIPECLRDSY
PRPDQPCYLYVIGMVLTTPLPDELNFRRRKLYPPEDTTRCFGILTAKPIPQIPHFPVYTR
SGEVTISIELKKSGFMLSLQMLELITRLHQYIFSHILRLEKPALEFKPTDADSAYCVLPL
NVVNDSSTLDIDFKFMEDIEKSEARIGIPSTKYTKETPFVFKLEDYQDAVIIPRYRNFDQ
PHRFYVADVYTDLTPLSKFPSPEYETFAEYYKTKYNLDLTNLNQPLLDVDHTSSRLNLLT
PRHLNQKGKALPLSSAEKRKAKWESLQNKQILVPELCAIHPIPASLWRKAVCLPSILYRL
HCLLTAEELRAQTASDAGVGVRSLPADFRYPNLDFGWKKSIDSKSFISISNSSSAENDNY
CKHSTIVPENAAHQGANRTSSLENHDQMSVNCRTLLSESPGKLHVEVSADLTAINGLSYN
QNLANGSYDLANRDFCQGNQLNYYKQEIPVQPTTSYSIQNLYSYENQPQPSDECTLLSNK
YLDGNANKSTSDGSPVMAVMPGTTDTIQVLKGRMDSEQSPSIGYSSRTLGPNPGLILQAL
TLSNASDGFNLERLEMLGDSFLKHAITTYLFCTYPDAHEGRLSYMRSKKVSNCNLYRLGK
KKGLPSRMVVSIFDPPVNWLPPGYVVNQDKSNTDKWEKDEMTKDCMLANGKLDEDYEEED
EEEESLMWRAPKEEADYEDDFLEYDQEHIRFIDNMLMGSGAFVKKISLSPFSTTDSAYEW
KMPKKSSLGSMPFSSDFEDFDYSSWDAMCYLDPSKAVEEDDFVVGFWNPSEENCGVDTGK
QSISYDLHTEQCIADKSIADCVEALLGCYLTSCGERAAQLFLCSLGLKVLPVIKRTDREK
ALCPTRENFNSQQKNLSVSCAAASVASSRSSVLKDSEYGCLKIPPRCMFDHPDADKTLNH
LISGFENFEKKINYRFKNKAYLLQAFTHASYHYNTITDCYQRLEFLGDAILDYLITKHLY
EDPRQHSPGVLTDLRSALVNNTIFASLAVKYDYHKYFKAVSPELFHVIDDFVQFQLEKNE
MQGMDSELRRSEEDEEKEEDIEVPKAMGDIFESLAGAIYMDSGMSLETVWQVYYPMMRPL
IEKFSANVPRSPVRELLEMEPETAKFSPAERTYDGKVRVTVEVVGKGKFKGVGRSYRIAK
SAAARRALRSLKANQPQVPNS

• Use InterPro to assign to family membership, identify functional domains, etc.
• What domains/sites does this protein contain?
• Can you predict the possible function of the protein by its domain composition?
• Find other proteins containing the same domains in the same order. To do this use the Pfam Stockholm server and the Metamotif server.
• How many proteins contain the same domain structure in SWISS-PROT/TrEMBL?

Tricks: InterPro is an integrated documentation resource for protein families, domains and sites. InterPro combines a number of databases (referred to as member databases) that use different methodologies and a varying degree of biological information on well-characterized proteins to derive protein signatures. By uniting the member databases, InterPro capitalizes on their individual strengths, producing a powerful integrated diagnostic tool. InterPro unifies:

• PROSITE regular expressions and profiles
• Pfam, SMART, TIGRFAMs, PIRSF, PANTHER, Gene3D and SUPERFAMILY hidden Markov models (HMMs)
• PRINTS, provider of fingerprints (groups of aligned, un-weighted motifs)
• PRODOM who use Clustr analysis to group sequences

Signatures describing the same protein family, domain repeat or site are grouped into unique InterPro entries. Each combined InterPro entry has a unique accession number, an abstract describing the features of proteins associated with the entry and literature references and has links to the relevant member database(s). All UniProtKB protein sequences that have matches to a particular InterPro entry are listed in the Match Table associated with that entry. There are also links to the InterPro graphical views. The graphical views, which can be sorted by UniProtKB accession number, structure or taxonomy, show the position of the signatures on the protein, mousing over the signature brings up a pop-box, giving the accession, name and position.

InterPro graphically represents the location of a protein domain and information pertaining to the origin of that domain and the proteins that contain it. Families are also defined and may contain several InterPro domains which are often, but not always, in the same order. Through the InterPro Domain Architecture view, the composition and order of the different domains within a family are clearly displayed for easy comparison, as well as for simple navigation between the entries for individual domains.

InterPro and InterProScan are accessible for interactive use over the EBI web server (www/ebi.ac.uk/interpro), they are distributed as stand-alone copies by anonymous ftp.

InterPro entries are linked to one another through PARENT/CHILD and CONTAINS/FOUND IN relationships. PARENT/CHILD relationships indicate superfamily/family/subfamily relationships, as well as domain hierarchies, where sequences can be subdivided into more specific sub-sets. CONTAINS/FOUND IN relationships apply to domains, repeats and sites within families, and are used to describe the composition of protein sequences.

Practice 9.4. PSI-BLAST. Given the following sequence:    [solution]

>myseq
MRTSSEDVFISDEVPTISPNTMLTSALMYMKDYITAVVVVDENRINVLVGMISTEDILDV
KVGGFFKPAIVCNGDDTLDECRSAIPEGNAQHIPVLEGFDKFHGIIYDKDVLHFGPPPRR
KPVAGGRGQAGAPGVLPKKRPGGPGSAGDDGAPPPPPSPKGVVGSKAAGGRGGTRGTGYA
GEPAEHAGIAGGMRGPQPGPPTGPSGGPAGSGERGAKGDPGPPGQPSMAKKGTAGRESVT
GILGSNGSGKSTFLNIMYGQDVFNEPTNGSNGWHNQDGQVIPMSVIGQKGVSPSLDVGDN
LMEFNHVIERSGQLSKADYLGADSVLVMTEVKELYEELPNCLSGGWKQWVNLCLEMITKS

Questions:

• Can you give a possible function to the protein based on the homologies found?
• Can you distinguish some protein domains?
• What is particular to our protein in respect to its homologous?
• What does it append when you run the PSI-BLAST search without the low complexity filter?

Tricks: Go to the NCBI BLAST service and run a PSI-BLAST search using the myseq sequence against the SWISS-PROT database. Select the low complexity filter and keep the rest of the options as they are. Run a few rounds (at least 5) of PSI-BLAST.

Practice 9.5. Build a pattern.       [solution]

• Given the following MSA:

  	Seq1  WFFKGIADKDAERHLLA
  	Seq2  WFFKNLEQKDAEARLLA
  	Seq3  WFFKR---KDAERQLLA
  	Seq4  WFFGTI---DAERQLLA
  	Seq5  WFFKDIPTKDAERQLLA
  	Seq6  WYFG----RESERLLLA
  	Seq7  WYFGKIPLKDAERQLLA
  	Seq8  WYFGKLRAKDTERLLLL
  

  build a pattern using a text editor.
  
  
• Submit the patten to the ScanProsite server and search against SWISS-PROT. Could you say something about the proteins matching your pattern?
• A possible way to validate the output of the pattern search is to search against a randomized database. To validate you pattern, use again ScanProsite, but this time against a reversed SWISS-PROT. Do you find any false positive sequence?
• Repeat the exercise with the following sequences:

  	seq1 ERGLR
  	seq2 DRASR
  	seq3 DRLGR
  	seq4 ERAAR
  	seq5 ERGVR
  

• What's append with a random database

Tricks: Use the Prosite syntax (you can find the syntax rules here).

Practice 9.6. Search the Prosite pattern database.     [solution]

• Scan the human protein VAV_HUMAN against Prosite patterns using ScanProsite. Have a look to the results.
• Repeat the same scan but including patterns with a high probability of occurrence. What's the difference between this and the previous result? Which are the characteristics of the masked patterns?

Tricks:

Practice 9.7. Build PSSMs with MEME.      [solution]

• Go to the MEME motif discovery service.
• Paste the following sequences (FASTA format) in the 'sequences box' and select the option Any number of repetitions.

  >seq1
  MGFSSALQSRAAHEALIVRQDAELRLMETMKRSIQMKAKCDKEYAISLTAVAQQGLKIDRADEMQGSLISKSWRSYMDEL
  DHQAKQFKFNAEQLEVVCDKLTHLSQDKRKARKAYQEEHAKIAARLNHLTDEVVRKKSEYQKHLEGYKALRTRFEENYIK
  APSRSGRKLDDVRDKYQKACRKLHLTHNEYVLSITEAIEVEKDFRNVLLPGLLEHQQSVQESFILLWRNILQEAAQYGDL
  TADKYKEIQKRIDTVIGSINPTEEYGEFTEKYKTSPTTPLLFQFDETLIQDIPGKLQSSTLTVDNLTVDWLRNRLQELEG
  AVRDCQEKQMKMIEHVNGGSPVANGSIISNGSNTSNGIQSNKDSLCRQSKDLNALRCQEKQKQKLVDMIKCALNEVGCEE
  LPSGCDDDLTLEQNFIENGYNNEQQISLSTNRPLYEEEWFHGVLPREEVVRLLNNDGDFLVRETIRNEESQIVLSVCWNG
  HKHFIVQTTGEGNFRFEGPPFASIQELIMHQYHSELPVTVKSGAILRRPVCRERWELSNDDVVLLERIGRGNFGDVYKAK
  LKSTKLDVAVKTCRMTLPDEQKRKFLQEGRILKQYDHPNIVKLIGICVQKQPIMIVMELVLGGSLLTYLRKNSNGLTTRQ
  QMGMCRDAAAGMRYLESKNCIHRDLAARNCLVDLEHSVKISDFGMSREEEEYIVSDGMKQIPVKWTAPEALNFGKYTSLC
  DVWSYGILMWEIFSKGDTPYSGMTNSRARERIDTGYRMPTPKSTPEEMYRLMLQCWAADAESRPHFDEIYNVVDALILRL
  DNSH
  >seq2
  MEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKGGFIPKNYIEMKPHPWFFGKIPRAKAEEMLGKQRHD
  GAFLIRESESAPGDFSLSVKFGNDVQQFKVLRDGAGKYLLWVVKFNSLNELVDYHRSTSVSRNQQIFLRDIEQVPQQPTY
  VQALFDFDPQEEGELGFRRGDFIQVLDNSDPNWWKGACHGQTGMFPRNYVTPVNRNI
  >seq3
  MSAEGYQYRALYDYKKEREEDIDLHLGDILTVNKGSLVALGFSDGQEARPEEIGWLNGYNETTGERGDFPGTYVEYIGRK
  KISPPTPKPRPPRPLPVAPGSSKTEADVEQQALTLPDLAEQFAPPDIAPPLLIKLVEAIEKKGLECSTLYRTQSSSNLAE
  LRQLLDCDTPSVDLEMIDVHVLADAFKRYLLDLPNPVIPAAVYSEMISLAPEVQSSEEYIQLLKKLIRSPSIPHQYWLTL
  QYLLKHFFKLSQTSSKNLLNARVLSEIFSPMLFRFSAASSDNTENLIKVIEILISTEWNERQPAPALPPKPPKPTTVANN
  GMNNNMSLQNAEWYWGDISREEVNEKLRDTADGTFLVRDASTKMHGDYTLTLRKGGNNKLIKIFHRDGKYGFSDPLTFSS
  VVELINHYRNESLAQYNPKLDVKLLYPVSKYQQDQVVKEDNIEAVGKKLHEYNTQFQEKSREYDRLYEEYTRTSQEIQMK
  RTAIEAFNETIKIFEEQCQTQERYSKEYIEKFKREGNEKEIQRIMHNYDKLKSRISEIIDSRRRLEEDLKKQAAEYREID
  KRMNSIKPDLIQLRKTRDQYLMWLTQKGVRQKKLNEWLGNENTEDQYSLVEDDEDLPHHDEKTWNVGSSNRNKAENLLRG
  KRDGTFLVRESSKQGCYACSVVVDGEVKHCVINKTATGYGFAEPYNLYSSLKELVLHYQHTSLVQHNDSLNVTLAYPVYA
  QQRR
  >seq4
  MDLLPPKPKYNPLRNESLSSLEEGASGSTPPEELPSPSASSLGPILPPLPGDDSPTTLCSFFPRMSNLRLANPAGGRPGS
  KGEPGRAADDGEGIDGAAMPESGPLPLLQDMNKLSGGGGRRTRVEGGQLGGEEWTRHGSFVNKPTRGWLHPNDKVMGPGV
  SYLVRYMGCVEVLQSMRALDFNTRTQVTREAISLVCEAVPGAKGATRRRKPCSRPLSSILGRSNLKFAGMPITLTVSTSS
  LNLMAADCKQIIANHHMQSISFASGGDPDTAEYVAYVAKDPVNQRACHILECPEGLAQDVISTIGQAFELRFKQYLRNPP
  KLVTPHDRMAGFDGSAWDEEEEEPPDHQYYNDFPGKEPPLGGVVDMRLREGAAPGAARPTAPNAQTPSHLGATLPVGQPV
  GGDPEVRKQMPPPPPCPGRELFDDPSYVNVQNLDKARQAVGGAGPPNPAINGSAPRDLFDMKPFEDALRVPPPPQSVSMA
  EQLRGEPWFHGKLSRREAEALLQLNGDFLVRESTTTPGQYVLTGLQSGQPKHLLLVDPEGVVRTKDHRFESVSHLISYHM
  DNHLPIISAGSELCLQQPVERKL
  >seq5
  MAQWNQLQQLDTRYLEQLHQLYSDSFPMELRQFLAPWIESQDWAYAASKESHATLVFHNLLGEIDQQYSRFLQESNVLYQ
  HNLRRIKQFLQSRYLEKPMEIARIVARCLWEESRLLQTAATAAQQGGQANHPTAAVVTEKQQMLEQHLQDVRKRVQDLEQ
  KMKVVENLQDDFDFNYKTLKSQGDMQDLNGNNQSVTRQKMQQLEQMLTALDQMRRSIVSELAGLLSAMEYVQKTLTDEEL
  ADWKRRQQIACIGGPPNICLDRLENWITSLAESQLQTRQQIKKLEELQQKVSYKGDPIVQHRPMLEERIVELFRNLMKSA
  FVVERQPCMPMHPDRPLVIKTGVQFTTKVRLLVKFPELNYQLKIKVCIDKDSGDVAALRGSRKFNILGTNTKVMNMEESN
  NGSLSAEFKHLTLREQRCGNGGRANCDASLIVTEELHLITFETEVYHQGLKIDLETHSLPVVVISNICQMPNAWASILWY
  NMLTNNPKNVNFFTKPPIGTWDQVAEVLSWQFSSTTKRGLSIEQLTTLAEKLLGPGVNYSGCQITWAKFCKENMAGKGFS
  FWVWLDNIIDLVKKYILALWNEGYIMGFISKERERAILSTKPPGTFLLRFSESSKEGGVTFTWVEKDISGKTQIQSVEPY
  TKQQLNNMSFAEIIMGYKIMDATNILVSPLVYLYPDIPKEEAFGKYCRPESQEHPEADPGSAAPYLKTKFICVTPTTCSN
  TIDLPMSPRTLDSLMQFGNNGEGAEPSAGGQFESLTFDMDLTSECATSPM
  >seq6
  MELWRQCTHWLIQCRVLPPSHRVTWEGAQVCELAQALRDGVLLCQLLNNLLPQAINLREVNLRPQMSQFLCLKNIRTFLS
  TCCEKFGLKRSELFEAFDLFDVQDFGKVIYTLSALSWTPIAQNKGIMPFPTEDSALNDEDIYSGLSDQIDDTAEEDEDLY
  DCVENEEAEGDEIYEDLMRLESVPTPPKMTEYDKRCCCLREIQQTEEKYTDTLGSIQQHFMKPLQRFLKPQDMETIFVNI
  EELFSVHTHFLKELKDALAGPGATTLYQVFIKYKERFLVYGRYCSQVESASKHLDQVATAREDVQMKLEECSQRANNGRF
  TLRDLLMVPMQRVLKYHLLLQELVKHTQDATEKENLRLALDAMRDLAQCVNEVKRDNETLRQITNFQLSIENLDQSLANY
  GRPKIDGELKITSVERRSKTDRYAFLLDKALLICKRRGDSYDLKASVNLHSFQVRDDSSGERDNKKWSHMFLLIEDQGAQ
  GYELFFKTRELKKKWMEQFEMAISNIYPENATANGHDFQMFSFEETTSCKACQMLLRGTFYQGYRCYRCRAPAHKECLGR
  VPPCGRHGQDFAGTMKKDKLHRRAQDKKRNELGLPKMEVFQEYYGIPPPPGAFGPFLRLNPGDIVELTKAEAEHNWWEGR
  NTATNEVGWFPCNRVHPYVHGPPQDLSVHLWYAGPMERAGAEGILTNRSDGTYLVRQRVKDTAEFAISIKYNVEVKHIKI
  MTSEGLYRITEKKAFRGLLELVEFYQQNSLKDCFKSLDTTLQFPYKEPERRAISKPPAGSTKYFGTAKARYDFCARDRSE
  LSLKEGDIIKILNKKGQQGWWRGEIYGRIGWFPSNYVEEDYSEYC
  

• Start the search ... and check the mails with a web browser. How many motifs are found? Which one of the motifs is common to all the 6 sequences?
• Can you see any link between the motifs found by MEME and domains in Pfam (paste a sequence in the Pfam Protein search service and look at the results). Can you describe the motifs found by MEME using the Pfam annotation?

Tricks: The result will be sent by e-mail, and it could take a bit long, so just move to the next exercise until you get the result.

10.- Genomics

Practice 10.1: Mitochondria of various organisms use different genetic code. Search the Internet site to obtain information for the mitochondrial codons from either vertebrates or invertebrates. Discuss usage differences between the standard codons and mitochondrial codons.

Practice 10.2: Retrieve one each of the cytosolic tRNA specific for the following amino acids from Saccharomyces cerevisiae:(a) Aspartic acid, (b) Phenylalanine, and identify their anticodons.

Practice 10.3: Retrieve nucleotide sequences (in fasta format) and restriction maps for one each of bacterial plasmid, cosmid and shuttle vector.

Practice 10.4: Retrieve DNA sequence encoding for human glucagon mRNA. Subject the sequence to Webcutter and construct the restriction map using REBASE restriction enzymes.

Practice 10.5: Search the candidate primers of PCR for the human pro-optiomelanocortin gene with the following sequence:

_gi _4505948 _ ref _ NM—000939.1 _ Homo sapiens proopiomelanocortin (POMC)
AGCGGCGGCGAAGGAGGGGAAGAAGAGCCGCGACCGAGAGAGGCCGCCGAGCGTCCCCGCCCTCAG
AGAGCAGCCTCCCGAGACAGAGCCTCAGCCTGCCTGGAAGATGCCGAGATCGTGCTGCAGCCGCTC
GGGGGCCCTGTTGCTGGCCTTGCTGCTTCAGGCCTCCATGGAAGTGCGTGGCTGGTGCCTGGAGAG
CAGCCAGTGTCAGGACCTCACCACGGAAAGCAACCTGCTGGAGTGCATCCGGGCCTGCAAGCCCGA
CCTCTCGGCCGAGACTCCCATGTTCCCGGGAAATGGCGACGAGCAGCCTCTGACCGAGAACCCCCG
GAAGTACGTCATGGGCCACTTCCGCTGGGACCGATTCGGCCGCCGCAACAGCAGCAGCAGCGGCAG
CAGCGGCGCAGGGCAGAAGCGCGAGGACGTCTCAGCGGGCGAAGACTGCGGCCCGCTGCCTGAGGG
CGGCCCCGAGCCCCGCAGCGATGGTGCCAAGCCGGGCCCGCGCGAGGGCAAGCGCTCCTACTCCAT
GGAGCACTTCCGCTGGGGCAAGCCGGTGGGCAAGAAGCGGCGCCCAGTGAAGGTGTACCCTAACGG
CGCCGAGGACGAGTCGGCCGAGGCCTTCCCCCTGGAGTTCAAGAGGGAGCTGACTGGCCAGCGACT
CCGGGAGGGAGATGGCCCCGACGGCCCTGCCGATGACGGCGCAGGGGCCCAGGCCGACCTGGAGCA
CAGCCTGCTGGTGGCGGCCGAGAAGAAGGACGAGGGCCCCTACAGGATGGAGCACTTCCGCTGGGG
CAGCCCGCCCAAGGACAAGCGCTACGGCGGTTTCATGACCTCCGAGAAGAGCCAGACGCCCCTGGT
GACGCTGTTCAAAAACGCCATCATCAAGAACGCCTACAAGAAGGGCGAGTGAGGGCACAGCGGGCC
CCAGGGCTACCCTCCCCCAGGAGGTCGACCCCAAAGCCCCTTGCTCTCCCCTGCCCTGCTGCCGCC
TCCCAGCCTGGGGGGTCGTGGCAGATAATCAGCCTCTTAAAGCTGCCTGTAGTTAGGAAATAAAAC
CTTTCAAATTTCACA

Suggest which of the candidate pairs lead to the synthesis of the constituent hormone(s) (adrenocorticotropin, _-lipotropin, _-melanocyte stimulating hormone, _-melanocyte stimulating hormone, and/or _-endorphin).

Practice 10.6: Use BioEdit to translate the human pro-optomelanocortin. Deduce the frame from which the constituent hormones are likely to be translated.

Practice 10.7: Retrieve complete CDS of human alcohol dehydrogenase (ADH) isozymes from UniGene and record the comparison with other organisms (UniGene listings).

Practice 10.8: The file F352SS4 has a sequence of a nucleic acid from a comercial automatic sequencer. Discarding the beggining and the end, get the DNA sequence. Check if there are any problem with nucleotide identification. If so, correct the error/s according to the peaks in the electropherogram. Use the program Chromas.

Practice 10.9: Producing a restriction map.

Use the DNA sequence of the gene that codes for protein P57727 and search for enzymes that cut a minimum of once and a maximum of twice, and have a recognition site length of at least six bases.