Using the Gen Bank Database
Updated 28/11/00

Resources	Objectives	Procedures	Marks
Kuby's Immunology	Immunology Reference Shelf	Carney's Learning Techniques	To Tortora's Microbiology	Immunology Laboratory

This Lab exercise contains a Description of the needed source materials, a list of objectives, a detailed description of each of the objectives and the marking scheme

OBJECTIVES:

1. Obtain Amino Acid Sequences form the GenBank Database
2. Install Clustal X on your computer
3. Align the amino acid sequences from the GenBank Database using Clustal X.
4. Analyse the polymorphism in the aligned sequences.
5. Submit a report

Introduction

The mammalian immune system has innate and adaptive components. The function of the adaptive immune system relies on the ability of immune system to alter the amino acid sequences of specific proteins in order to better recognise and defend against specific pathogens. The sequences of these proteins is altered over several generations in the case of Major Histocompatibility Complex receptors , or over the course of a single infection in the case of Immunoglobulins and T-Cell receptors . The presence of many different forms of a specific protein within a population of animals is call polymorphism. Modern molecular biological and biochemical techniques allow researchers to study these amino acid differences by sequencing genes or proteins from different cells and individuals.and then comparing the sequences. In this laboratory you will obtain several sequences from one of the proteins mentioned above from an online database and analyse their amino acid sequences in order to determine the degree of polymorphism.

OBJECTIVE 1: Obtain amino acid Sequences from the GenBank Database

READ THIS ENTIRE OBJECTIVE BEFORE YOU START !!!

In this objective you will learn to access the National Center for Biotechnology Information website and use their "Entrez" search engine to find and retrieve protein sequences that will be used later in the exercise

To achieve this objective you will

• Access the website
• Become familar with the website and its search engine
• Use a keyword search to obtain a list of files with the appropriate protein sequences
• Open a text file in Notepad or Wordpad to store the retrieved sequences
• Open a file from the NCBI database and find the amino acid sequence
• Copy the amino acid sequence
• Paste the sequence into the Notepad text file
• Repeat the open, copy and paste operation for 10 molecules
• Save the Notepad text file with the 10 different protein sequences
• Close the NCBI database
• Format the text file to be used by the Clustal X program

The National Center for Biotechnology Information (NCBI) is an international repository of data on the sequence of genes and proteins. Researchers from around the world deposit their information here so that it can be used by the scientific community. The data are stored in several different databases; the "proteins" databases contain just the amino acid sequences while the "Nucleotides" databases contain both the nucleotide sequences and the deduced amino acid sequences.You can use either of these databases to retrieve the sequences that you will need for this exercise.

Data on millions of sequences are stored in separate files each with a unique identification code. You can use key-words to find specific sequences deposited in the databases.

The NCBI is constantly updated and modified as they find faster ways to retreive the information. Do not be surprised if the screen layout changes frequently as improvements ae brought online.

As you browse the NCBI website look for answers to the following questions

• What is the function of the NCBI?
• What organizations support the NCBI?
• What kind of information is stored in the NCBI?
• What is GenBank?
• Who are the members of the International Nucleotide sequence Database collaboration?
• How many species or represented in the database?
• How fast is the database expanding?

	http://www.ncbi.nlm.nih.gov/Entrez/

RETRIEVING AMINO ACID SEQUENCES

Before you start your search make certain that you have space on your computer to store the info that you retrieve. While you can store the info on a hard drive, a 3 1/2" floppy disk will be more than enough. As you will have to submit the data on a floppy disk anyway, why not save it directly to floppy disk?

Also, before you start your search make certain that you have opened a text file for future storage. Open a new text file in NotePad or Wordpad

Use the NCBI Entrez search engine to obtain 10 amino acid sequences of a specific protein from a single species and cut and paste these sequences in the opened text file as described below

You want to find specific sequences of proteins that are immunologically important eg, MHC, T cell Receptors, Kappa chain, Lambda chains or any of the heavy chains of immunoglobulins. Use only sequences from a single isotype, for example IgM or IgG, and from only the heavy or the light chain. The Entrez search engine lets you use key words to find these

Examples of Key word searches

• "homo sapiens, MHC II" produces a list of MHCII sequences
• "homo sapiens, MHC I" will produces a list of MHCII sequences
• "homo sapiens, IgM, variable" will produce a list of IgM variable domains.
• "homo sapiens, IgG, variable" will produce a list of IgG variable domains
• "homo sapiens, Kappa, variable" will produce a list of kappa chain variable domains
• "homo sapiens, Lambda, variable" will produce a list of Lambda chain variable domains

After you have completed your search you will have a list of files with the appropriate sequences

SAVING THE SEQUENCE DATA

• Select a file from the list that has been created by the key word search
• Click on the GenPept ot GenBank report link to see the full entry for a specific sequence. The amino acid sequence (in one letter code) will be near the bottom.
• Copy this sequence by highlighting the sequence and using the copy command (CTRL C is faster)
• Go to the text file that you opened in Notepad or Wordpad and use the paste command (CTRL V is faster)
• Move on to another sequence and paste it in the same text file until you have 10
• TIP try an pick sequences at are about the same size
• Save the text file

FORMATTNG THE TEXT FILE TO BE USED BY CLUSTAL X

The text file containing your sequences should be formatted to meet the following:

CLUSTAL FORMAT RULES

• The font must be Courier, 10 point
• The >P1; tells Clustal X that the following is a sequence for alignment
• The words following on the same line after >P1; indicate the sequence name. Use any name that you choose but do not repeat the name. Use the Underscore reather than the space
• Leave a blank line after the title line,
• The sequence must have amino acid letter codes only ( no numbers)
• The sequence must end with a star (*).
• Save the file as "text only"

I usually call the file something that will indicate what I am aligning. The example below would be "stvi.txt" .

EXAMPLE OF A CLUSTAL FORMATTED TEXT FILE

>P1;Stvi-10-8

HSLKFYCTASSGVPNFPEFVAVGLVDDVEISHYDSNTKREEPKQDWMSRVTEDDPQFWQRETEISEGHQQTFKANIEVVRQRLNQT*

>P1;Auha-706

HSLKYFYTASSQVPNFPEFVTVGMVDDVQVDYYDSDTEKAEPKQDWIARNTDQQYWERNTDIYRGSQQSFKANIEIVKQRFNQT*

>P1;Auha-705

HSLKFFCTETPGVQSIPEFVAVAFVDEVQIGDFNNVRGAEPKKDWIKFFADHPEHLEWYSSISKQSHQVFKANIETFRQRLNQT*

>P1;Auha-517

HSLKYFFTGSSQVPNFPEFVVVGMVDDVQVVHYDSDTEKAGPKQDWFARNTDQQYWESQTGNLLGSQQTFKANIETAKQRFNQT*

>P1;Cyca-UAA

HTLQYFYTATSGIENFPEFMTAGVVDGQQIDYYDSIIRKAVQKAEWISGAVDPDYWNRNTQIYAGNEPSFKENINIVKSRFNQT*

>P1;Furu-UAA

HTLKYFYTASSGVPNFPEFVIVGMVDDVQMVRYDSNTRRMQPKQEWMKEFTADDPQYLDTQSQNAFGAQQIYKANIEIAKERFNQT*

>P1;Sasa-UAA

HALKYFYTASSEVPNFPEFVVVGVVDGVQMVHYDSNSQRAVPKQDWVNKAADPQYWERNTGIFKGSQQTFKANIDIAKQRFNQS*

>P1;Plam-sp005

HSLKNFHTASSQVTNIPEFVFVGFIDDVQTEYYDSNTKKSEPKQDWMLKATDAKYWERQTGILRAYQRVFKANMEIAKERFNQT*

>P1;Stvi-10-9

HSLKFYFTAFFGVPNFPEFVSVGLVDDIEISRYDSNTKREEPTQDWMSRVPQEDPPFWPTETEISDGDQQTFQAHIEVVKQRLNQT*

>P1;Stvi-10-10

HSLKIFCTACSGVPNFPEYVSVGLVDGVQMIRYDSNTKRQEPKQDWMSRVTEDDPQYWQRNTEIAEGNQQVYKAGIEILKQRLNQT*

Submit a copy of your file with your report.

OBJECTIVE 2 Install Clustal X on your Computer

http://www-igbmc.u-strasbg.fr/BioInfo/ClustalX/

DownLoad Clustal X

A copy of Clustal X is stored in
Immunology>>Resources >>Software Downloads>> Clustal X under the file name clustalx1_8msw.exe This is a compressed document that has several files. This program will not work until it has been downloaded and installed

To Download this file, goto the appropriate section on the website, Double click on the filename, tell the computer where you want to store the file on your hard drive. Remember where you have stored it!

Install Clustal X

To install Clustal X, find the file clustalx1_8msw.exe that is now stored on your computer. Double click on the file name and follow the install instructions You will have to tell the computer where you want the program ClustalX installed. You could create a subdirectory called Clustal in the Program Files directoryand install CLUSTALX in the subdirectory.An executable file (clustalx.exe) which will run under MS Windows (32 bit). The directory containing the executable (plus the files named *.par,and clustalx.hlp) should be added to your path defined in the autoexec.batfile.

Browse through the menu options and the help files of ClustalX to learn some of its features

	The BioBox website compares both the Clustal W and the Clustal X programs. The Clustal W is a DOS version. The Clustal X is a windows version
http://www.csc.fi/molbio/progs/clustalw/

OBJECTIVE 3 Align the sequences using Clustal X

download the file called Clustal_Example.txt an use it as a test example .

Start the program Clustal X (clustalx.exe).

Select Multiple Alignment Mode

Select file

select load sequences

Find and select the file Clustal_Example.txt If ClustalX recognises you file, it will list the sequences and the number of amino acids in each one. (If it doesn’t do this, check that your file is saved as text only).

The path and the filename should appear at the bottom of the screen

A list of the alignment sequences will appear in the left hand frame

The sequences will appear as multi coloured letter codes on the right hand frame

Select the aligment option from the menu

Selct the Do Complete Alignment option

You will be asked for names for the alignment and dendrogram files. Just use the default names, which will be the same as your text file, with either an .aln (eg stvi.aln) or .dnd (eg stvi.dnd) extension. The alignment will appear on the screen.

Close the clustal window, then using a wordprocessor such as Microsoft Wordpad, Microsoft Word or Wordperfect open up the .aln file (eg stvi.aln). Your alignment should be there and it should look like the example below (note the example below is comparing MHC class I sequences from several different fish species - your sequences should be much more similar!). Deletions within the sequence will be indicated by dashes. The bottom line of the alignment will contain several symbols. Stars (*) indicate amino acids which are identical, colons (:) indicate amino acid changes which are conservative (hydrophobic for hydrophobic or acidic for acidic), while periods (.) indicate changes which may be conservative. If the rows do not line up, change the font to Courier, 10 point.

CLUSTAL X (1.8) multiple sequence alignment

Stvi-10-8 HSLKFYCTASSGVPNFPEFVAVGLVDDVEISHYDSNTKREEPKQDWMSRVTEDDPQFWQR
Stvi-10-9 HSLKFYFTAFFGVPNFPEFVSVGLVDDIEISRYDSNTKREEPTQDWMSRVPQEDPPFWPT
Stvi-10-10 HSLKIFCTACSGVPNFPEYVSVGLVDGVQMIRYDSNTKRQEPKQDWMSRVTEDDPQYWQR
Cyca-UAA HTLQYFYTATSGIENFPEFMTAGVVDGQQIDYYDSIIRKAVQKAEWISG--AVDPDYWNR
Sasa-UAA HALKYFYTASSEVPNFPEFVVVGVVDGVQMVHYDSNSQRAVPKQDWVNK--AADPQYWER
Auha-706 HSLKYFYTASSQVPNFPEFVTVGMVDDVQVDYYDSDTEKAEPKQDWIAR--NTDQQYWER
Auha-517 HSLKYFFTGSSQVPNFPEFVVVGMVDDVQVVHYDSDTEKAGPKQDWFAR--NTDQQYWES
Plam-sp005 HSLKNFHTASSQVTNIPEFVFVGFIDDVQTEYYDSNTKKSEPKQDWMLK--ATDAKYWER
Furu-UAA HTLKYFYTASSGVPNFPEFVIVGMVDDVQMVRYDSNTRRMQPKQEWMKEFTADDPQYLDT
Auha-705 HSLKFFCTETPGVQSIPEFVAVAFVDEVQIGDFN-NVRGAEPKKDWIKFF-ADHPEHLEW

*:*: : * : .:**:: ...:* : :: . . :*. . .

Stvi-10-8 ETEISEGHQQTFKANIEVVRQRLNQT
Stvi-10-9 ETEISDGDQQTFQAHIEVVKQRLNQT
Stvi-10-10 NTEIAEGNQQVYKAGIEILKQRLNQT
Cyca-UAA NTQIYAGNEPSFKENINIVKSRFNQT
Sasa-UAA NTGIFKGSQQTFKANIDIAKQRFNQS
Auha-706 NTDIYRGSQQSFKANIEIVKQRFNQT
Auha-517 QTGNLLGSQQTFKANIETAKQRFNQT
Plam-sp005 QTGILRAYQRVFKANMEIAKERFNQT
Furu-UAA QSQNAFGAQQIYKANIEIAKERFNQT
Auha-705 YSSISKQSHQVFKANIETFRQRLNQT

: . :: :: :.*:**:

OBJECTIVE 4: Analyse the polymorphism in the aligned sequences

Researchers who study polymorphic molecules use a special method to present and interpret their results, called a Wu-Kabat variability plot. This is a graph which indicates the position along the amino acid chain on the X axis and the variability index along the Y axis. The variability index is calculated as follows:

Number of different amino acids at a given position along the amino acid chain

Frequency of the most common amino acid present at a given position along the amino acid chain

For example in the alignment above position 1 is histidine (H) in all ten sequences so the variability index is calculated as follows:

Number of different amino acids = 1

Frequency of the most common =10 out of 10 = 10/10 = 1

Variability index = 1/1 = 1

In the alignment above position 2 is variable and the variability index is calculated as follows:

Number of different amino acids (S, T, A) = 3

Frequency of the most common (S)= 7 out of 10 = 7/10 = 0.7

Variability index = 3/0.7 = 4.29

Position 21, which follows the sequence PEFV, is variable and the variability index is calculated as follows:

Number of different amino acids (A,S,T,V,F,I)= 6

Frequency of the most common (A or S or T or V) =2 out of 10 = 2/10 = 0.2

Variability index = 6/0.2 = 30

In a Wu-Kabat plot these values are plotted against their position in the polypeptide chain. An example of such a plot is given below:

OBJECTIVE 5: Submit a Report

Construct a Wu-Kabat plot for the alignment you have produced. Submit a report indicating the type of sequence you analysed (MHC, immunoglobulin, T- Cell receptor) and the names of the sequences you used. Include the your .pir file, your .aln file and your Wu-Kabat plot. You can hand in all of these items in electronic format on a 3.5 inch computer disk if you wish.

Appendix: The one letter amino acid code

• G - Glycine (Gly)
• P - Proline (Pro)
• A - Alanine (Ala)
• V - Valine (Val)
• L - Leucine (Leu)
• I - Isoleucine (Ile)
• M - Methionine (Met)
• C - Cysteine (Cys)
• F - Phenylalanine (Phe)
• Y - Tyrosine (Tyr)
• W - Tryptophan (Trp)
• H - Histidine (His)
• K - Lysine (Lys)
• R - Arginine (Arg)
• Q - Glutamine (Gln)
• N - Asparagine (Asn)
• E - Glutamic Acid (Glu)
• D - Aspartic Acid (Asp)
• S - Serine (Ser)
• T - Threonine (Thr)

CLUSTAL REFERENCES

Details of algorithms, implementation and useful tips on usage of Clustal
programs can be found in the following publications:

Jeanmougin,F., Thompson,J.D., Gouy,M., Higgins,D.G. and Gibson,T.J. (1998)
Multiple sequence alignment with Clustal X. Trends Biochem Sci, 23, 403-5.

Thompson,J.D., Gibson,T.J., Plewniak,F., Jeanmougin,F. and Higgins,D.G. (1997)
The ClustalX windows interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic Acids Research, 24:4876-4882.

Higgins, D. G., Thompson, J. D. and Gibson, T. J. (1996) Using CLUSTAL for
multiple sequence alignments. Methods Enzymol., 266, 383-402.

Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence
weighting, positions-specific gap penalties and weight matrix choice. Nucleic
Acids Research, 22:4673-4680.

Higgins,D.G., Bleasby,A.J. and Fuchs,R. (1992) CLUSTAL V: improved software for
multiple sequence alignment. CABIOS 8,189-191.

Higgins,D.G. and Sharp,P.M. (1989) Fast and sensitive multiple sequence
alignments on a microcomputer. CABIOS 5,151-153.