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PB 150 Bioinformatics lab
Objective:
1. An overview of genomes and plant biological resources databases.
2. Obtaining relevant scientific information starting from a keyword
3. Analyzing information and creating a database: Sequence alignment & Analysis
at protein and nucleic acid level.
Methodology
NOTE: All the needed links for this lab, homework and more are listed below.
* Access NCBI home page.
http://www.ncbi.nlm.nih.gov/
From the main page, or ENTREZ, select [dropdown menu]: “Protein”
* Use a keyword “tyrosine phosphatase” to search and retrieve sequences.
Note be careful about spelling.
* Look for mammalian and plant sequences [species name is in the title], leave the rest.
Tip: Arabidopsis does not seem to have receptor-like tyrosine phosphatases, these
sequence tend to result BLAST hits in MAPKs of Arabidopsis genome, you may want to
gather as few of these as possible for your subsequent search.
* Click on different sequences that fulfill these criteria: protein tyrosine phosphatase,
from plant or mammal origin.
* Select FASTA [dropdown menu, next to Display button] and click “Display”.
 Save the output in FASTA format to a text file [Copy and Paste is fine].
Note: FASTA format contains on the first line, behind a “>” information identifying
each sequence, like accession number, species name, brief description of the
sequence. Keep this information through the subsequent steps together with the actual
protein sequence.
* Access TAIR homepage, BLAST,
http://arabidopsis.org/Blast/
select BLASTP [AA query, AA database; AGI Proteins (Protein)] from the dropdown
menus, BLAST program and Datasets, respectively. Query sequence must be checked [it
is default].
* Run a BLAST search with your candidate sequences.
* Collect Arabidopsis sequences that share some homology.
Look for hits that contain “phosphatase”, “myotubularin”, “PTEN”, “unknown
protein”, “expressed protein” or “hypothetical protein”, in the title, save in FASTA
format.
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* Run each one individually at the Conserved Domain Database (integrates PFAM,
SMART, etc)
http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml
Evaluate for relevance:
- does it contain a DSPc or PTPc domain?
A. Yes: it is a “good” sequence.
B. No: that is the nature of BLAST: some of the hits may have only a distant
homology, and/or homology in a domain that is different from the PTP/DSP
catalytic domain.
Collect all “good” sequences and save to a new text file.
This is your small database (DB) that fulfills the following criteria:
Contains
- protein sequences of the
- plant Arabidopsis, that are
- protein tyrosine phosphatases.
Question:
Do you expect your database containing all Arabidopsis protein tyrosine phosphatases?
Why?
Homework:
I. At protein level:
i. Collect the gene accession numbers. Gene accession numbers are in the following
format: Atxgyyyyy , where At is identifier for the organism: Arabidopsis thaliana,
x is a chromosome number, which is from 1 to 5 for At,
yyyyy is a gene specific number.
Gene accession numbers may be available from the identifier string (1st line) of your
sequences in FASTA format, so look first there. Unfortunately, it’s not necessarily the
case.
If not present, the Gene accession numbers can easily be retrieved as follow:
Copy your sequence, one at the time, paste into TAIR BLAST search window.
http://arabidopsis.org/Blast/
Select BLASTP and AGI Proteins (Protein) from the dropdown menus when working
with protein sequence (the default is nucleotide, BLASTN). Run a BLAST search. The
first hit(s) with E Value of 0.0 reveal(s) database entries with 100% identity over a
certain stretch of sequence. This (these) must be the self-score(s) of your protein.
The gene accession # is right after the hyperlinks.
Save to your DB. [it can be saved in the FASTA > line]
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ii. Start organizing your DB in a hierarchical manner:
1. numbers to each entry for your own reference, followed by gene accession
numbers.
2. sequences in FASTA format corresponding to the #s you gave and the gene
accession numbers.
iii. Analyze your protein sequences for presence of:
A. Chloroplast, Mitochondrion, Nucleus targeting signal peptides.
B. Transmembrane domains.
[choose appropriate links from the listed below in Specialty Tasks, Protein Specific,
Subcellular Localization, or Transmembrane domains]
Save your findings to your database in an organized, hierarchical manner.
iv. Perform a ClustalW multiple sequence alignment with your sequence.
http://www.ebi.ac.uk/clustalw/
( or http://www.ch.embnet.org/software/ClustalW.html)
Note: use FASTA format as in all your previous work.
[scoring matrix BLOSUM is good, you may leave all the rest on default too, or try
changing settings as “color”, etc]
v. Build a phylogenetic tree with this program.
It is automatically generated with the multiple sequence alignment. You can view the
image of it at the bottom of you results page when using http://www.ebi.ac.uk/clustalw/
[this function may not work with all browsers!]. Alternatively, you will have to download
/install software for viewing the tree (also called “dendrogram”).
Note: Clustalw will display on the phylogenetic tree only the first string with limited
number of characters as identifiers for your sequence(s). When running Clustalw, you
may want to use your own reference numbers at the beginning of the FASTA “>” for
easier identification/orientation in the tree’s homology relations.
For example:
>DSP1At3g1000gi……
ILOVETHISSEQENCEWORK……
Will display DSP1At3g1000gi to the branch with your
ILOVETHISSEQENCEWORK…… sequence.
II. At nucleic acid level:
1. Look for entries for “tyrosine phosphatase” in nucleic acid database. Following
the same steps as during the demonstration, collect gene sequences for
Arabidopsis protein tyrosine phosphatases. To verify if they are “real” tyrosine
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phosphatases perform a BLAST search in the NT database [BLASTN, and AGI
Genes (+ Introns, + UTRs) from dropdown menus].
2. Crossreference with the gene accession numbers you already have to validate the
entries.
A. If you get new entries, not already present in your database, run their
respective AA seq. in the CDD, you might just have found a new member
of the PTP family.
[AA sequence will be easy to find if you click on TAIR or TIGR hyperlinks,
they are on the summary page of each sequence.]
Email your results.
A summary of your results will be posted temporarily on the unofficial web page of the
course.
In case of any questions, do not hesitate to ask Prof Luan, or Lubo.
General useful links
where and how to find (plant) science related information
Literature:
PubMed http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed
ISI
http://isi3.newisiknowledge.com/portal.cgi?DestApp=WOS&Func=Frame
(address keeps changing, check it from here if link doesn’t work:
http://isi3.newisiknowledge.com/portal.cgi )
Search in particular journal db and crossreferences therein i.e.
http://www.plantcell.org/
http://www.plantphysiol.org/
Biochemical Pathways
http://us.expasy.org/tools/pathways/
Enzyme nomenclature DB
http://us.expasy.org/enzyme/
Protocols and procedures:
Current Protocols
http://www.mrw2.interscience.wiley.com/cponline/tserver.dll?command=doGetD
oc&sUI=&database=CP&useScheme=WIS_Framed.Scheme&getDoc=cp_toc_fs.
html
Specialty tasks
Sequences Retrieval
ENTREZ @ NCBI (National Center for Biotechnology Information)
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http://www3.ncbi.nlm.nih.gov/Entrez/
Sequences Analysis: Both Nucleic Acids & Protein
Similarity and homology search:
BLAST & FASTA concept
HMM http://www.soe.ucsc.edu/research/compbio/HMMapps/T02-query.html
Seq alignment
2 sequences
http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html
Multiple sequence analysis
ClustalW
http://www.ch.embnet.org/software/ClustalW.html
T-Cofee
http://www.es.embnet.org/Services/MolBio/t-coffee/
WebLogo
http://weblogo.berkeley.edu/logo.cgi
GCG
registration required, if someone really interested in using it,
contact the webmaster for arrangements.
http://socrates.berkeley.edu:7029/gcg-bin/seqweb.cgi
Phylogenetic trees
ClustalW
http://www.ch.embnet.org/software/ClustalW.html
GCG
http://socrates.berkeley.edu:7029/gcg-bin/seqweb.cgi
DNA specific
cis-acting regulatory DNA elements db-s (Promoter analysis)
PLACE - Plant cis-acting regulatory DNA elements db
PlantCARE - Plant cis-acting regulatory DNA elements db
gene prediction:
GENSCAN http://genes.mit.edu/GENSCAN.html
GRAIL: http://compbio.ornl.gov/Grail-1.3/
Primer design:
Primer3 http://www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi
T-DNA mapping tool: http://signal.salk.edu/cgi-bin/tdnaexpress
RNA specific:
dbEST (Expressed Sequence Tags) db (NCBI)
http://www.ncbi.nlm.nih.gov/dbEST/
or select EST from dropdown menu in BLAST search
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Structure
Global Gene expression: microarray (genechip) data
http://www.arabidopsis.org/tools/bulk/microarray/index.html
Protein Specific
Domain analysis:
CDD (Conserved Domain Database) @ NCBI
http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml
PFAM
http://pfam.wustl.edu/
SMART
http://smart.embl-heidelberg.de/
Patterns & Profile
Post-translational modification, O-glycosylation sites prediction
YinOYang
http://www.cbs.dtu.dk/services/YinOYang/
NetOGlyc
http://www.cbs.dtu.dk/services/NetOGlyc/
Subcellular localization prediction
SignalP: Prediction of signal peptide cleavage sites
http://www.cbs.dtu.dk/services/SignalP/
ChloroP: Prediction of chloroplast transit peptides
http://www.cbs.dtu.dk/services/ChloroP/
MITOPROT: Prediction of mitochondrial targeting sequences
http://www.mips.biochem.mpg.de/cgi-bin/proj/medgen/mitofilter
TargetP: Prediction of subcellular location
http://www.cbs.dtu.dk/services/TargetP/
Transmembrane domains
SOSUI
http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html
TMpred
http://www.ch.embnet.org/software/TMPRED_form.html
Alignment & sequence features of 2 (or more) seq
http://us.expasy.org/tools/sim-prot.html (general info: LANVIEW:
http://us.expasy.org/tools/lalnview.html)
NetPhos - Prediction of Ser, Thr and Tyr phosphorylation sites in
eukaryotic proteins
Structure: primary, secondary, tertiary (not covered in this lab)
Best Plant Biology DBs: (some of these reference other organisms too)
TAIR http://www.arabidopsis.org/home.html
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TIGR http://www.tigr.org/
MIPS http://mips.gsf.de/
Best general DBs:
Sequences analysis and related tools
Expasy http://www.ncbi.nlm.nih.gov
NCBI http://us.expasy.org/
Odd (but still useful) DBs

Allergen sequence db

Allergome - A db of allergenic molecules
Botanical catalogs (from Expasy)

NAL - AGIS - Access to many plant genome databases at the National
Agricultural Library

Mendel - Plant gene nomenclature database from CPGN

Arabidopsis Swiss-Prot list - Links to A.thaliana WWW sites and to Swiss-Prot
entries

TAIR - The Arabidopsis Information Resource

MATDB - MIPS Arabidopsis thaliana db

TIGR At - TIGR Arabidopsis thaliana db

Arabidopsis ABC transporters

AMPL - Arabidopsis Membrane Protein Library

Arabidopsis at PlaCe - Site with info on P450, glucosyltransferases, etc.

Gramene - A comparative mapping resource for grains

RGP - Rice Genome Research Program

Oryzabase - Japanese rice genome db

Rice genome project at Wisconsin

Rice-research.org - Monsanto rice genome site

TIGR OsGI - TIGR Rice Genome project

BeanGenes - Beans genome db

BeanRef - Beans genome db and other resources
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
Chlamydomonas resource center

ChlamyDB - Chlamydomonas reinhardtii genome db

CottonDB - Cotton genome db

MaizeDb - Maize genome db

INRA Maize - INRA Maize genome db

Pisum sativum (pea) web site

TIGR Potato - TIGR Potato Functional Genomics project

TIGR GmGI - TIGR Soybean Gene Index

Snapdragon (A.majus) web site

SorghumDB - Sorghum genome db

SoyBase - Soybean genome db

SoyBase metabolic db - Metabolic subset of the soybean genome db

TIGR LeGI - TIGR Tomato Gene Index

Dendrome - Forest trees genome db
Further reading:
Pedestrian guide to analysing sequence databases (oldish, but good general info)
http://cubic.bioc.columbia.edu/papers/1999_pedestrian/paper.html
Current Protocols in Bioinformatics (subscription required, capmus computers/IP
#s should e fine):
http://www.mrw2.interscience.wiley.com/cponline/tserver.dll?command=doGetD
oc&sUI=&database=CP&useScheme=WIS_Framed.Scheme&getDoc=cp_toc_fs.html