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Bioinformatics
Adapted from a paper (http://www.lifescied.org/cgi/content/full/4/3/207;
http://www.nslc.wustl.edu/elgin/genomics/Bio3055/manual.pdf) by April Bednarski and
Himadri Pakrasi that was funded by a grant from the Howard Hughes Medical Institute of
Washington University.
Glossary
Genome – The entire amount of genetic information for an organism. The human genome
is the set of 46 chromosomes.
Homologous – With regard to amino acids, homologous amino acids have similar chemical
properties and sizes. For example, glutamate can be considered homologous to aspartate
because both residues have similar sizes and both residues contain a carboxylic acid side
chain.
Sequence alignment – a sequence alignment is a way of arranging the sequences present
in DNA, RNA, or proteins so as to identify regions that are similar.
Multiple sequence alignment – a sequence alignment of three or more biological
sequences.
Conserved – the amino acid residues at a position in a multiple sequence alignment are
identical throughout the alignment.
Conservative residue change – the amino acid residues at a position in a multiple
sequence alignment are homologous.
ClustalW – A program for making multiple sequence alignments.
www.ebi.ac.uk/clustalw/index.html
EC number - Enzyme Commission number - Assigned by the IUBMB (International Union
of Biochemistry and Molecular Biology); classifies enzymes according to the reaction
catalyzed. An EC Number is composed of four numbers separated by dots. For example the
alcohol dehydrogenase has the EC Number 1.1.1.1.
BLOSUM – BLOcks of Amino Acid SUbstitution Matrix – A type of substitution matrix that is
used by programs like BLAST to give sequences a score based on similarity to another
sequence. The scoring matrix gives a score to conservative substitutions of amino acids. A
conservative substitution is a substitution of an amino acid similar in size and chemical
properties to the amino acid in the query sequence.
BLAST – Basic Local Alignment Search Tool – can be accessed from the NCBI website,
blast.ncbi.nlm.nih.gov/Blast.cgi. A program that compares a given input sequence to all the
sequences in a specified database. This program aligns the most similar segments between
sequences. BLAST aligns sequences using a scoring matrix similar to BLOSUM (see entry).
This scoring method gives penalties for gaps and gives the highest score for identical
residues. Substitutions are scored based on how conservative the changes are. The output
is a list of sequences, with the highest scoring sequence at the top. The scoring output is
given as an E-value. The lower the E-value, the higher scoring the sequence is. E-values in
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the range of 10-100 to 10-50 are very similar (or even identical) sequences. Sequences with
E-values 10-10 and higher need to be examined based on other methods to determine
homology. An E-value of 10-10 for a sequence can be interpreted as, “a 1 in 1010 chance that
the sequence was pulled from the database by chance alone (has no homology to the query
sequence).”
ExPASy – Expert Protein Analysis System - us.expasy.org/ A server maintained by the
Swiss Institute of Bioinformatics. Home of SWISS-PROT, the most extensive and annotated
protein database. The Swiss-Pdb Viewer protein-viewing program is also available at this
site for free download.
FASTA – Fast Alignment Search Tool-All (since it works on both nucleotide and amino acid
sequences). Associated with this software is a way of formatting a nucleic acid or protein
sequence. It is important because many bioinformatics programs require that the
sequence be in FASTA format. The FASTA format has a title line for each sequence that
begins with a “>” followed by any needed text to name the sequence. The end of the
title line is signified by a paragraph mark (hit the return key). Bioinformatics
programs will know that the title line isn’t part of the sequence if you have it formatted
correctly. The sequence itself does NOT have any returns, spaces, or formatting of any
kind. The sequence is given in one-letter code. An example of a protein in correct FASTA
format is shown below:
>K-Ras protein Homo sapiens
MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDI
LDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVP
MVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRK
HKEKMSKDGKKKKKKSKTKCVIM
GenBank - a database of nucleotide sequences from over 260,000 organisms.
http://www.ncbi.nlm.nih.gov/genbank/ This is the main database for nucleotide
sequences. It is a historical database, meaning it is redundant. When new or updated
information is entered into GenBank, it is given a new entry, but the older sequence
information is also kept in the database. GenBank belongs to an international collaboration
of sequence databases, which also includes EMBL (European Molecular Biology
Laboratory) and DDBJ (DNA Data Bank of Japan). In contrast, the RefSeq database (see
entry) is non-redundant and contains only the most current sequence information for
genetic loci.
Gene – an NCBI database of genetic loci. It may be accessed through the NCBI homepage by
selecting “Gene” from the Search drop-down menu. This database used to be called
LocusLink. Entries provide links to RefSeqs, articles in PubMed, and other descriptive
information about genetic loci. The database also provides information on official
nomenclature, aliases, sequence accession numbers, phenotypes, EC numbers, OMIM
numbers, UniGene clusters, map information, and relevant web sites.
KEGG – Kyoto Encyclopedia of Genes and Genomes – http://www.genome.ad.jp/kegg/
This website is used for accessing metabolic pathways. At this website, you can search a
process, gene, protein, or metabolite and obtain diagrams of all the metabolic pathways
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associated with your query. You will see a link to the KEGG entry at the end of the Gene
entry for a gene.
NCBI – National Center for Biotechnology Information – www.ncbi.nlm.nih.gov This center
was formed in 1988 as a division of the NLM (National Library of Medicine) at the NIH
(National Institute of Health). As part of the NIH, NCBI is funded by the US government.
The main goal of the center is to provide resources for biomedical researchers as well as
the general public. The center is continually developing new materials and updating
databases. The entire human genome is freely available on this website and is updated
daily as new and better data become available. NCBI also maintains an extensive education
site, which offers online tutorials of its databases and programs:
www.ncbi.nlm.nih.gov/About/outreach/courses.html
OMIM - Online Mendelian Inheritance in Man –
www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM a continuously updated catalog of
human genes and genetic disorders, with links to associated literature references, sequence
records, maps, and related databases.
PubMed – http://www.ncbi.nlm.nih.gov/pubmed/ When writing a paper on a particular
science/medical topic, you should always check PubMed. It is a retrieval system
containing citations, abstracts, and indexing terms for journal articles in the biomedical
sciences. PubMed contains the complete contents of the MEDLINE and PREMEDLINE
databases. It also contains some articles and journals considered out of scope for
MEDLINE, based on either content or on a period of time when the journal was not indexed,
and therefore is a superset of MEDLINE.
RefSeq - NCBI database of Reference Sequences. Curated, non-redundant set including
genomic DNA contigs, mRNAs, proteins, and entire chromosomes. Accession numbers have
the format of two letters, an underscore bar, and six digits. Example: NT_123456. Code:
NT, NC, NG = genomic; NM = mRNA; NP = protein (for more of the two letter codes, see the
NCBI site map).
Sequence Manipulation Suite – bioinformatics.org/sms/ a website that contains a
collection of web-based programs for analyzing and formatting DNA and protein
sequences.
Bioinformatics is a field of study that merges math, biology, and computer science.
Researchers in this field have developed a wide range of tools to help biomedical
researchers work with genomic, biochemical, and medical information. Some types of
bioinformatics tools include data base storage and search programs as well as software
programs for analyzing genomic and proteomic data.
We will be working through a tutorial on web-based bioinformatics programs. The
tutorial is based on the enzymes phospholipase C-gamma (believed to be the major enzyme
of fertilization), and cyclooxygenase-2 (COX-2), which also has the name prostaglandin
synthase-2 (PTGS2). In this tutorial, the bioinformatics tools from the NCBI (National
Center for Biotechnology Information) website will be introduced. NCBI is a division of the
National Institute of Health (NIH).
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These tools include Gene, GenBank, RefSeq, and PubMed. Gene is a database of
genes in which each entry contains a brief summary, the common gene symbol, information
about the gene function, and links to websites, articles, and sequence information for that
gene.
GenBank is a historical database of gene sequences, which means it contains every
sequence that was published, even if the same sequence was published more than once.
Therefore, GenBank is considered a redundant database.
RefSeq is a database of sequences that is edited by NCBI and is NON-redundant,
meaning that it contains what NCBI determines is the most reliable sequence data for each
gene.
Finally, we will be learning to use ClustalW, which is a multiple sequence alignment
program. It allows you to enter a series of gene or protein sequences that you believe are
similar and may be evolutionarily related. These sequences are usually obtained by
performing a BLAST search. ClustalW then aligns the sequences, so that the fewest gaps
are introduced and the largest number of similar residues is aligned with each other.
ClustalW uses a scoring matrix similar to BLOSUM-62, which will be presented in a lecture.
Introduction to Phospholipase C-gamma and COX-2 (PTGS2)
Phospholipase C-gamma is believed to be a major enzyme of fertilization. The
pathway of fertilization in Xenopus laevis is thought to be the following:
1) Sperm binds to the egg.
2) This binding somehow activates the 1b form of phospholipase D (PLD1b)
3) PLD1b breaks the lipid phosphatidylcholine down into phosphatidic acid (PA) and
choline.
4) PA stimulates a tyrosine kinase called Src. Tyrosine kinases are enzymes that
transfer a phosphate from ATP to other proteins. This “phosphorylation” can turn
another protein on or off.
5) The activated Src phosphorylates the gamma form of Phospholipase C (PLC-γ).
6) PLC-γ breaks the lipid “PIP2” down to “IP3” and “DAG”. IP3 diffuses from the cell
membrane to release calcium stored in the endoplasmic reticulum.
7) The calcium floods into the cytoplasm to cause the events of fertilization.
The calcium travels across the zygote from the sperm binding site, causing a
wave of cortical granule exocytosis, a wave of elevation of the fertilization
envelop, a wave surface contraction (that we visualized); and initiation of
other developmental events leading to first cleavage (or cytokinesis.
COX-2 (PTGS2) is called prostaglandin H2 synthase-2 and cyclooxygenase-2 (COX-2).
COX-2 has been thoroughly studied because of its role in prostaglandin synthesis.
Prostaglandins have a wide range of roles in our body from aiding in digestion to
propagating pain and inflammation. Aspirin is a general inhibitor of prostaglandin
synthesis and, therefore, helps reduce pain. However, aspirin also inhibits the synthesis of
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prostaglandins that aid in digestion. Therefore, aspirin is a poor choice for pain and
inflammation management for those with ulcers or other digestion problems. Recent
advances in targeting specific prostaglandin-synthesizing enzymes have led to the
development of Celebrex, which is marketed as an arthritis therapy. Celebrex is a potent
and specific inhibitor of COX-2. Celebrex is considered specific because it doesn’t inhibit
COX-1, which is involved in synthesizing prostaglandins that aid in digestion. This is a
remarkable accomplishment given the great similarity between COX-1 and COX-2. This
achievement has paved the way for developing new therapies that bind more specifically to
their target and therefore have fewer side effects.
Understanding the enzyme structures of COX-1 and COX-2 helped researchers develop
a drug that would only bind and inhibit COX-2. Many of the types of information and tools
used by researchers for these types of studies are freely available on the web. In this
tutorial, and throughout this lab course, you will be introduced to the databases and freely
available software programs that are commonly used by professionals in research and
medicine to study genes, proteins, protein structure and function, and genetic disease.
Gene Database:
Follow these directions to access the entries for PTGS1 and PTGS2 in the “Gene” database
at the NCBI Website:
1) Go to the NCBI homepage: http://www.ncbi.nlm.nih.gov
2) Just after the word “Search,” select “Gene” from the database drop-down menu.
Enter “PTGS” in the “for” textbox, and click the Search button.
3) Find the results for the “Homo sapiens” entries called “PTGS1” and one called
“PTGS2.” (In Firefox, try Ctrl-F, and enter Homo sapiens.)
4) Select each entry by clicking on its name, then read the paragraph under the
Summary section for each entry.
Answer the following questions.
1. PTGS1 and PTGS2 are isozymes: Isozymes catalyze the same reaction, but are coded by
separate genes. Based on the summary, what types of reactions do PTGS enzymes
catalyze?
2. Which gene forms multiple transcript variants?
3. Which isozyme would you want to inhibit to stop inflammation?
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4. According to the Pathways section, what KEGG pathways are listed for these enzymes
(other than “Metabolic pathways”)?
The next two questions are not discussed in the summaries- just read the questions and
think about the answers.
5.
The drug Celebrex selectively inhibits PTGS2 while aspirin and other NSAID’s
inhibit both PTGS1 and PTGS2 in the same way. Why do you think researchers wanted
to discover a selective inhibitor to PTGS2?
6.
Describe how studying 3-D structures of PTGS1 and PTGS2 could help researchers
design a drug that binds to PTGS1, but not to PTGS2.
7. Now start over and search for the gene for “Phospholipase C-gamma” in Homo sapiens.
Find the PLCG1 and PLCG2 entries (case matters). On what chromosome are these
found?
8. Now, go to the PLCG1 entry. From the summary, what do IP3 and PIP2 stand for (spell
out the complete chemical name):
9. What is the official symbol of phospholipase C, gamma 1?
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HUGO is the acronym for the Human Genome Organization. The HUGO Gene Nomenclature
Committee’s acronym is HGNC. Click on the HGNC:9065 link next to “Primary Source.”
This brings up the “Symbol Report” page. Find the section, “OMIM ID”, and click on the link
associated with the entry 172420. OMIM stands for the Online Mendelian Inheritance in
Man database. The OMIM database was started at John Hopkins University and is now
maintained by NCBI. The OMIM database contains entries for both diseases with known
genetic links and entries for the genes that have been linked to a disease. Each OMIM entry
is a summary of the research that has been performed on the disease or gene and contains
links to the research articles that it summarizes. You will be able to read about the clinical
and biochemical research that has been performed related to the mutation you are
studying. Is any information available related to mutations or mutants for PLC gamma?
YES NO
Each link in the OMIM entry will open an abstract from the PubMed database. PubMed is a
literature database, and is also maintained by NCBI. PubMed is a searchable database of
medical and life science journal articles. Most of the abstracts for these articles can be
accessed through PubMed, but in order to access the entire article, you need to go to each
individual journal website and have a subscription to the journal. The Troy University
library has subscriptions to electronic versions of many of these journals that you can
access through the E-journal link on the library home page. Most journals have their
articles available online as .pdf files for articles published between 1995 to present.
However, the older articles must still be accessed through the paper versions stored in
libraries.
Go back to the “Symbol Report” page. In the section, “Accession Numbers”, click on the
GenBank link. An example of a GenBank entry is shown below.
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For PLC-gamma 1, fill in the following info:
Number of base pairs:
Gene sequence was obtained from “Molecule Type”:
Date of latest modification:
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Accession number (Very important number):
Both the AMINO ACID (beginning with “/translation”) and then the GENE sequences
(in ATGC) are listed. Amino acids have both a 3-letter and 1-letter abbreviation—
databases use the 1-letter abbreviations.
Table 1. 1- and 3-Letter
Go back to the original page on PLCG1 (the page
with “Primary Source” and the HGNC:9065” link that you Abbreviations of Amino Acids.
followed). In your browser use Ctrl-F to find “SH3” on in Amino Acid 3-Letter 1-Letter
the Bibliography section of that page. Which journal
Alanine Ala
A
published this entry?
Arginine Arg
R
Asparagine Asn
N
Aspartic acid Asp
D
Then, search for “RET9” in the Interactions section.
Cysteine Cys
C
Which journal published an article listed in PubMed
Glutamic acid Glu
E
about this entry?
Glutamine Gln
Q
Glycine Gly
G
Histidine His
H
You have explored human forms of the enzyme and
Isoleucine Ile
I
its gene. Next, in the Entrez Gene database, search for a
Leucine Leu
L
reference to the presence of the PLC-gamma enzyme in
Lysine Lys
K
Xenopus laevis. You have to go back to the original page
Methionine Met
M
that had “Gene” for the database and “Phospholipase CPhenylalanine Phe
F
gamma Xenopus laevis” for the search string. How many
Proline Pro
P
references for Xenopus PLC-gamma did you find?
Serine Ser
S
Threonine Thr
T
Tryptophan Trp
W
What is the preferred name (the name before the “Other
Tyrosine
Tyr
Y
Aliases” line) of the enzyme in each reference (how do
Valine Val
V
they differ?)?
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For the first reference that you find, under “Related Sequences,” note that there are three
listed:
Nucleotide
Protein
mRNA
AB287408.1 BAF64273.1
mRNA
AF090111.1 AAD03594.1
mRNA
BC070837.1 AAH70837.1
The second column is a sequence of nucleotide bases; the third is the amino acid list for the
base sequence.
Go back and select the second Xenopus PLC gamma reference. Under General gene
information, you see “Pathways.” KEGG stands for the Kyoto Encyclopedia of Genes and
Genomes. It is a database of metabolic pathways that is maintained by a research institute
in Japan. It contains all the known metabolic and signaling pathways. Each protein in the
pathway and each small molecule metabolite (e.g., ATP) has its own entry in the database
that can be accessed by clicking on the protein or metabolite in the pathway figure. By
using this website, you can make predictions about what would happen to downstream
events in the pathway if the protein you are studying is either less active or more active.
There are several links to click on to show how PLC-gamma1b is involved in metabolism.
Click on the link related to inositol metabolism.
In the first link/path, the red arrow below shows where PLC gamma 1b is located- it
has an enzyme number of 3.1.4.11.
PIP2, the reactant, is to the right (1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate).
What is the full name of the product IP3 according to this metabolic pathway?
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Click on the next to last KEGG link, about a signaling system; what is the name of this
pathway?
Essentially, you now have two names for equivalent pathways involving PLC. Note that
they show PLC in red lettering and in a green box.
Locate PIP2 (top center; a substrate for PLCγ) and write how they abbreviate it here in this
second path:
Write down how they prefer to abbreviate IP3 (look for IP3 with some numbers in
parentheses):
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