Mol Biotechnol (2008) 38:165–177
DOI 10.1007/s12033-007-9003-x
REVIEW
In Silico Characterization of Proteins: UniProt, InterPro and
Integr8
Nicola Jane Mulder Æ Paul Kersey Æ
Manuela Pruess Æ Rolf Apweiler
Published online: 4 October 2007

Humana Press Inc. 2007
Abstract Nucleic acid sequences from genome
sequencing projects are submitted as raw data, from which
biologists attempt to elucidate the function of the predicted
gene products. The protein sequences are stored in public
databases, such as the UniProt Knowledgebase (Uni-
ProtKB), where curators try to add predicted and
experimental functional information. Protein function pre-
diction can be done using sequence similarity searches, but
an alternative approach is to use protein signatures, which
classify proteins into families and domains. The major
protein signature databases are available through the inte-
grated InterPro database, which provides a classification of
UniProtKB sequences. As well as characterization of pro-
teins through protein families, many researchers are
interested in analyzing the complete set of proteins from a
genome (i.e. the proteome), and there are databases and
resources that provide non-redundant proteome sets and
analyses of proteins from organisms with completely
sequenced genomes. This article reviews the tools and
resources available on the web for single and large-scale
protein characterization and whole proteome analysis.
Keywords Bioinformatics
Databases

Protein sequences
Protein signatures
Annotation

Proteomics
N. J. Mulder (&)
P. Kersey
M. Pruess
R. Apweiler
EMBL Outstation - European Bioinformatics Institute,
Wellcome Trust Genome Campus, Hinxton, Cambridge CB10
1SD, UK
e-mail: mulder@ebi.ac.uk
Introduction
Advances in DNA sequencing technologies have made the
sequencing of whole genomes commonplace. However, the
major problem associated with the influx of sequence data
is the quick and reliable elucidation of protein function and
large-scale analysis of whole proteomes (protein compo-
nent of genomes). The protein sequences are stored in
public databases, such as the UniProt Knowledgebase
(UniProtKB) [1], where curators try to add value to the raw
sequences through biological data curation. The curators
retrieve experimental information from the literature, but
also make use of automated function prediction methods to
supplement the information, and where there is no exper-
imental data. These curated protein sequence records enter
the highly curated UniProtKB/Swiss-Prot portion of the
knowledgebase, and the rest of the sequences, derived from
EMBL nucleotide sequence [2] entries and direct protein
sequence submissions, remain in UniProtKB/TrEMBL,
where they are either unannotated or have preliminary
automatically derived annotation. In this article we will
provide a detailed description of the main public protein
sequence repositories and specialized sequence databases.
Traditionally, scientists use sequence similarity searches
to compare a query sequence to those of known function, but
this method has its limitations and relies on the quality of
existing data. Alternative methods for protein sequence
classification use protein signatures. A number of different
databases developing protein signatures diagnostic for
known protein families or domains have arisen. Each has its
own focus, criteria and method for creating the signatures,
and as a result also its own strengths and limitations. In
addition, the shear number of these databases is daunting for
a bench scientist who is not necessarily qualified to under-
stand the similarities, differences and idiosyncrasies of each.
166
Here we will explain these differences, and describe InterPro
[3], an integrated resource that aims to solve the above-
mentioned problem. InterPro unifies the major protein sig-
nature databases into a single, comprehensive resource with
manual intervention by trained biologists to turn a database
and software application into an understandable, usable tool
for scientists world wide. InterPro has been used by bench
biologists analyzing a single gene product and by genome
sequencing centers for the annotation of entire genomes.
The analysis and comparison of whole genomes provides
a powerful tool for identifying unique or target genes in the
reference organism. For example, a comparison between
pathogenic and non-pathogenic organisms may shed light on
which genes or proteins are responsible for pathogenesis if
they are only found in the former organisms. The Integr8
resource [4], aims to present an analysis of completely
sequenced genomes and proteomes for this purpose. This
resource will also be described in more detail below.
Protein Sequence Databases
A range of new technologies in protein science make it
possible to quickly identify large numbers of proteins in a
complex, to map their interactions in a cellular context, to
determine their location within the cell and to analyze their
biological activities. Protein sequence databases play a
vital role as a central resource for storing the data gener-
ated by these efforts and making them freely available to
the scientific community. Data from large-scale experi-
ments are often no longer published in a conventional sense
but are deposited in a database. This means that protein
sequence databases are the most comprehensive resource
of information on proteins available to scientists.
In order to exploit the various resources fully, it is
essential to distinguish between them and to identify the
types of data they contain. Universal protein databases cover
proteins from all species, while specialized data collections
contain information about a particular protein family or
group of proteins, or related to a specific organism. Universal
protein sequence databases can be further subdivided into
two categories: simple archives of sequence data, or
sequence repositories, where the data are stored with little or
no manual intervention in the creation of the records; and
expertly curated databases, in which the original data are
enhanced by the addition of further information derived from
sources such as published scientific literature. One family of
protein sequence databases, the Universal Protein Resource
(UniProt), will be discussed in detail.
Sequence Repositories
A number of protein sequence databases act as repositories
of protein sequences. These databases add little or no
Mol Biotechnol (2008) 38:165–177
additional information to the sequence records they contain
and generally make no effort to provide a non-redundant
collection of sequences to users. One example for this is
the GenBank Gene Products Data Bank, or GenPept, which
is produced by the National Center of Biotechnology
Information [5]. The entries in the database are derived
from translations of the sequences contained in the nucle-
otide database maintained collaboratively by DDBJ [6],
EMBL Nucleotide Sequence Database [2] and GenBank
[7], and contain minimal annotation which has been
extracted primarily from the corresponding nucleotide
entry. The entries lack any additional annotation and the
database does not contain proteins derived from amino acid
sequencing. It presents a redundant view of the protein
world, which means that each protein may be represented
by multiple records and no attempt is made to group these
records into a single database entry. NCBI’s Entrez Protein
[5] is another example of a sequence repository. The
database contains sequence data translated from the
nucleotide sequences of the DDBJ/EMBL/GenBank data-
base as well as sequences from UniProt, RefSeq and the
Protein Data Bank (PDB) (see all below). The database
differs from GenPept in that many of the entries contain
additional information but much of the annotated data has
been extracted from curated databases so there is little
novel information added to the entries, which cannot be
found in other data collections. As with GenPept, the
sequence collection is redundant. A more ambitious
approach is taken by the Reference Sequence (RefSeq)
collection produced by the NCBI [8]. The aim of the
project is to provide a comprehensive, integrated, non-
redundant set of sequences, including genomic DNA,
transcript (RNA), and protein products, for major research
organisms. NCBI staff and collaborators provide ongoing
curation, with review status indicated on each record.
However, the majority of the records are automatically
generated with minimal manual intervention, so the data-
base is closer to a sequence repository than to any of the
curated databases discussed below.
Universal Curated Databases
Although repositories are an essential means of providing
the user with sequences as quickly as possible, it is clear
that, when additional information is added to a sequence,
this greatly increases the value of the resource for users.
The curated databases take basic sequence information and
enrich it by adding additional information from a range of
sources such as scientific literature. This information is
extracted and validated by expert biologists before being
added to the databases and this means that the data in these
collections can be considered to be highly reliable. There is
Mol Biotechnol (2008) 38:165–177 167

also a large effort invested in maintaining non-redundant • The UniProt Archive (UniParc) provides a non-redun-
datasets by compiling all reports for a given protein dant sequence collection of all publicly available
sequence into a single record. protein sequence data.
• The UniProt Knowledgebase (UniProtKB) combines
the work originally done with the expertly curated
PIR, Swiss-Prot and TrEMBL: A Short History Swiss-Prot, TrEMBL and PIR-PSD databases.
• The UniProt Reference Clusters (UniRef) offer non-
The oldest universal curated protein sequence database has redundant views of the data contained in the UniProt
been the Protein Information Resource Protein Sequence Knowledgebase and UniParc.
Database (PIR-PSD), established in 1984 as a successor to • The UniProt Metagenomic and Environmental
the original NBRF Protein Sequence Database. It has been Sequences (UniMES) section stores metagenomic data
developed over a 20-year-period by the late Margaret O. as it becomes available.
Dayhoff and published as the ‘Atlas of Protein Sequence
and Structure’ from 1965 to 1978 [9]. Later it became a
joint effort by Georgetown University Medical Centre and
UniParc
the National Biomedical Research Foundation in Wash-
ington D.C. Another universal curated protein sequence
The UniProt Archive (UniParc) is the main sequence store-
database, Swiss-Prot, has been established in 1986 as a
house and is a comprehensive repository that reflects the
collaboration between the European Bioinformatics Insti-
history of all protein sequences [10]. UniParc houses all new
tute (EBI) and the Swiss Institute of Bioinformatics (SIB).
and revised protein sequences from various sources to ensure
In 1996, the TrEMBL (Translation from EMBL) database
that complete coverage is available at a single site. It includes
was introduced as a supplement to Swiss-Prot to make new
not only UniProtKB but also translations from the EMBL-
sequences available as quickly as possible while preventing
Bank/DDBJ/GenBank Nucleotide Sequence Databases, the
the dilution of the high-quality annotation in Swiss-Prot. It
Ensembl database of animal genomes [11], the International
consisted of computer-annotated entries derived from the
Protein Index (IPI) (see IPI section), the Protein Data Bank
translation of all coding sequences in the DDBJ/EMBL/
(PDB) [12], NCBI’s Reference Sequence Collection (Ref-
GenBank nucleotide sequence database which were not yet
Seq), some model organism databases and protein sequences
included in Swiss-Prot. To ensure completeness, it also
from the European, American and Japanese Patent Offices.
contained a number of protein sequences extracted from
To avoid redundancy, sequences are handled as strings—all
the literature or submitted directly by the user community.
sequences 100% identical over the entire length are merged,
The increasing volume and complexity of protein data
regardless of source organism. New and updated sequences
has meant that the protein databases have had to find ways
are loaded on a daily basis, cross-referenced to the source
to adapt to this data influx so that they can continue to play
database accession number and provided with a sequence
a central role in the proteomics era. Therefore, in 2002 the
version that increases upon changes to the underlying
Swiss-Prot + TrEMBL groups at SIB and EBI and the PIR-
sequence. The basic information stored within each UniParc
PSD group at the Georgetown University Medical Center
entry is the identifier, the sequence, cyclic redundancy check
and National Biomedical Research Foundation joined for-
number, source database(s) with accession and version
ces as the UniProt Consortium, substantially funded by the
numbers and a time stamp. In addition, each source database
National Institutes of Health (NIH, USA). The Universal
accession number is tagged with its status in that database,
Protein Resource (UniProt) resulted, which contains the
indicating if the sequence still exists or has been deleted in
now-leading universal curated protein sequence database,
the source database. UniParc records are designed to be
the UniProt Knowledgebase or UniProtKB [1].
without annotation since the annotation will be only true in
the real biological context of the sequence: proteins with the
same sequence may have different functions depending on
The Universal Protein Resource species, tissue, developmental stage, etc.

The Universal Protein Resource (UniProt) (http://www.

uniprot.org) is the central resource for storing and inter-
connecting information from large and disparate sources and
the most comprehensive catalogue of protein sequence and
functional annotation. It has four components optimized for
different uses:
UniProtKB
The UniProt Knowledgebase (UniProtKB) consists of two
sections, UniProtKB/Swiss-Prot and UniProtKB/TrEMBL.
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The former contains manually annotated records with
information extracted from literature and curator-evaluated
computational analysis. To achieve accuracy, annotations
are performed by biologists with specific expertise. Infor-
mation including function, catalytic activity, subcellular
location, disease, structure and post-translational modifi-
cations is annotated. An important part of the annotation
process involves the merging of different reports for a
single protein. After a careful inspection of the sequences,
the annotator selects the reference sequence, does the
corresponding merging and lists the splice and genetic
variants along with disease information when available.
Any discrepancies between the different sequence sources
are also annotated. Cross-references are provided to the
underlying nucleotide sequence sources as well as to many
other useful databases including organism-specific,
domain, family and disease databases. UniProtKB/TrEM-
BL contains high-quality computationally analyzed records
enriched with automatic annotation and classification. The
computer-assisted annotation is created using automati-
cally generated rules such as those in Spearmint [13] or
manually curated rules based on protein families, including
HAMAP family rules [14], RuleBase rules [15] and PIRSF
classification-based name rules and site rules [16, 17].
UniProtKB/TrEMBL contains the translations of all coding
sequences present in the EMBL/GenBank/DDBJ Nucleo-
tide Sequence Databases, the sequences of PDB structures
and data derived from amino acid sequences that are
directly submitted to the UniProtKB or scanned from the
literature. Some types of data are excluded, such as DDBJ/
EMBL/DDBJ entries that encode small fragments, syn-
thetic sequences, most non-germline immunoglobulins and
T-cell receptors, most patent sequences and some highly
over-represented data. Records are selected for full manual
annotation and integration into UniProtKB/Swiss-Prot
according to defined annotation priorities.
UniRef
The UniProt Reference Clusters (UniRef) databases pro-
vide three clustered sets (UniRef100, 90 and 50) of
sequences from UniProtKB and selected UniParc records
in order to obtain complete coverage of sequence space at
several resolutions while hiding redundant sequences from
view. The UniRef100 database combines identical
sequences and sub-fragments with 11 or more residues into
a single UniRef entry, which displays the sequence of a
representative protein, with the accession numbers of all
the UniProtKB entries within the cluster and links to the
corresponding UniProtKB and UniParc records. UniRef90
and UniRef50 are built by further clustering UniRef100
sequences with 11 or more residues using the CD-HIT
Mol Biotechnol (2008) 38:165–177
algorithm [18] such that each cluster is composed of
sequences that have at least 90% or 50% sequence identity,
respectively, to the representative sequence. Selection of
the representative sequence in each UniRef cluster is based
on the ranking of all the sequences in the cluster using as
criteria quality of the entry, meaningful name, organism,
and length of the sequence. The UniRef databases provide
up-to-date collections of sequences. UniRef100 is the most
comprehensive and non-redundant protein sequence data-
set. UniRef90 and UniRef50 yield a database size reduction
of 40% and 65%, respectively, providing for significantly
faster sequence similarity searches. In addition, UniRef
databases reduce the bias in sequence searches by provid-
ing a more even sampling of sequence space.
UniMES
The UniProt Metagenomic and Environmental Sequences
(UniMES) section has been set up to deal with the new
field of metagenomics, the large-scale genomic analysis of
microbes recovered from environmental samples (as
opposed to laboratory-grown organisms which represent
only a small proportion of the microbial world). UniMES
currently contains the data from the Global Ocean Sam-
pling Expedition (GOS), which was originally submitted to
the International Nucleotide Sequence Databases (INSDC).
The initial GOS dataset is composed of 28 million DNA
sequences from oceanic microbes and predicts nearly
6 million proteins. By combining the predicted protein
sequences with automatic classification by InterPro, a
resource for protein families, domains and functional sites,
which will be described in detail in the ‘‘Protein sequence
classification’’ section, UniMES uniquely provides free
access to the array of genomic information gathered from
the sampling expeditions, enhanced by links to further
analytical resources.
Specialized Databases
In addition to the universal protein sequence databases,
there are numerous specialized databases available to the
life science community. Some are devoted to one particular
aspect of proteins or protein groups or families, or on a
specific organism, while others seek to consolidate and
exploit already existing resources to their full potential.
These vary in size and in the scope of the data they contain.
One example of the former type of database is the Protein
Data Bank (PDB) archiving three-dimensional structural
data. The Gene Ontology (GO) [19] is a dynamic-controlled
vocabulary that can be applied to all organisms, even while
knowledge of gene and protein roles in cells is still
Mol Biotechnol (2008) 38:165–177
accumulating and changing. GOA, the Gene Ontology
Annotation project [20], annotates proteins to GO terms.
IntAct [21] stores protein interaction data. Other examples
are protein family databases and model organism databases.
On the other hand, InterPro is an example of an integrated
protein resource combining a number of databases that use
different methodologies and a varying degree of biological
information on well-characterized proteins to derive protein
signatures for protein families, domains and sites. This
database and its member databases are described in ‘‘Protein
sequence classification’’ section below.
Protein Family Databases
Protein family databases contain information related to a
specific family or group of proteins. These databases are
generally maintained by experts in the field, and due to the
restricted nature of the data they contain, they are able to
offer a finer level of granularity than may be possible in the
universal databases. An example of such a database is
MEROPS, an information resource for peptidases and their
inhibitors [22]. A summary page is provided for each
peptidase and this describes the classification and nomen-
clature of the protein and offers links to supplementary
pages showing sequence identifiers, any known structures,
and literature references. The proteins are classified using a
hierarchical, structure-based approach in which each pep-
tidase is assigned to a family on the basis of statistically
significant similarities in amino acid sequence, and fami-
lies that are thought to be homologous are grouped together
in a clan. Information is also provided about naturally
occurring peptidase inhibitors.
Organism-specific Databases
Model organisms are an invaluable tool for understanding
the basis of human diseases and biological processes.
Increasing amounts of genetic, phenotypic and protein-
related information are being generated in a variety of
model organism systems, and a number of databases
dealing specifically with the biology of these organisms
have been established to capture this information and
provide it to the scientific community. Examples include
FlyBase [23], which covers Drosophila species, the Mouse
Genome Database [24], which contains information related
to the laboratory mouse, WormBase [25], which covers
Caenorhabdidtis elegans, the Saccharomyces Genome
Database (SGD) [26], for Saccharomyces proteins and The
Arabidopsis Information Resource (TAIR) [27]. These
databases play an important role in providing integrated
access to the data available about these organisms.
169
Protein Sequence Classification
Grouping of related objects is one of the best ways to make
sense of very large data sets, and protein sequences are no
different. The classification of proteins into families helps us
to reduce the overall problem space, as well as to predict
protein function. Proteins with similar sequences should have
similar functions, so if related sequences are grouped and one
or more of these sequences has a known function, this function
can be inferred on the uncharacterized sequences in the group.
There are various methods of classification of protein
sequences that have developed, but arguably the best methods
used protein signatures, which mathematically describe the
common properties of related sequences.
Protein Signature Methods
To create a protein signature, a number of related protein
sequences are required. It is not possible to identify com-
mon features of a protein family or domain from one
sequence, however, an alignment of related sequences can
be used to create a consensus for the protein family, or
identify conserved domains or residues. The highly con-
served areas are likely to be involved in the common
function of the related sequences, for example, highly
conserved residue triads may indicate an active site or
binding site. These conserved areas diagnostic of a protein
family, domain or functional site can be used to develop
protein signatures using several different methods. These
are then used to search a query protein sequence against to
identify which family the protein belongs to.
The simplest protein signature method uses regular
expressions to show patterns of conserved amino acid
residues. The regular expression specifies which amino
acid(s) may or may not occur at each position. This core
pattern is tested against a set of annotated sequences and
optimized until it only hits the correct sequences in the test
set [28]. Regular expressions are useful for identifying
highly conserved active sites and binding sites, but are
limited in their ability to find more distantly related
sequences due to their lack of flexibility in recognizing
single residue variations.
Another widely used protein signature method is a
profile. Profiles are built from multiple sequence align-
ments, and are tables of position-specific amino acid
weights and gap costs, or matrices describing the proba-
bility of finding an amino acid at a given position in the
sequence [29]. The numbers in the table (scores) are used
to calculate similarity scores between a profile and a
sequence for a given alignment. For each set of sequences a
threshold score is calculated to determine whether the
query sequence is related to the original set of sequences in
170
the alignment. An additional method derived from profiles
is a Hidden Markov Model (HMM), which essentially is a
statistical profile based on probabilities rather than scores
[30, 31]. HMMs are the most commonly derived method
from the HMMER package, written by Sean Eddy [32],
which allows a user to create an HMM from a sequence
alignment and to search a database of sequences against the
HMM without the requirement of understanding how the
HMMs work. Profiles and HMMs are powerful protein
signature methods and compensate for the limitations of
regular expressions in that they generally cover larger areas
of the sequence, and are capable of identifying more
divergent family members.
Protein Signature Databases
There are a number of protein signature databases in the
public domain which use the methods described above to
produce diagnostic signatures for protein families and
domains, including the sequence-based PROSITE [33],
PRINTS [34], Pfam [35], SMART [36], TIGRFAMs [37],
PIRSF [16] and PANTHER [38], and the structure-based
SUPERFAMILY [39] and Gene3D [40]. There are also
databases that use sequence clustering and alignment
methods, for example ProDom [41], who cluster all pro-
teins in the database into families.
PROSITE is a database of both regular expressions and
profiles and has a primary focus on signatures for the anno-
tation of UniProtKB/Swiss-Prot proteins. PRINTS uses a
variation on profiles to produce fingerprints, which are a
collection of motifs along the conserved regions of a protein
sequence. Fingerprints are particularly useful for diagnosing
receptors and ion channels and for their high granularity,
showing different levels in a protein family hierarchy.
Pfam, SMART, TIGRFAMs, PIRSF, PANTHER,
SUPERFAMILY and Gene3D all use HMMs but in different
ways. For example, each database may have different criteria
for assembling their multiple sequence alignments, different
means of calculating their thresholds, or different methods
for post-processing their results. Pfam is driven by coverage
and aims to have HMMs to represent all sequence space.
Pfam includes both families and domains in their sets and
where possible they base their HMMs on protein structural
information. There are two parts to Pfam, the curated Pfam-
A and the automatically generated Pfam-B, which covers
those families not represented in Pfam-A. SMART,
SUPERFAMILY and Gene3D tend to create HMMs for
protein domains rather than families. SMART focuses pre-
dominantly on signalling, extracellular and chromatin-
associated proteins, while SUPERFAMILY and Gene3D
base their domains on structural domains in the SCOP [42]
and CATH [43] databases respectively.
Mol Biotechnol (2008) 38:165–177
TIGRFAMs, PIRSF and PANTHER are protein family
orientated, although TIGRFAMs do contain some domain
HMMs. TIGRFAMs are also used for annotation, particu-
larly of microbial genomes, and as a result, their HMMs hit
mostly bacterial proteins. They focus on creating HMMs
based on actual function rather than just sequence simi-
larity, so all proteins belonging to a TIGRFAMs family
must be ‘‘equivalogs’’, i.e. have the same function. They
are therefore quite specific. PIRSF, on the other hand, only
produces HMMs representing protein families, mostly at
the superfamily level. The HMMs cover the full-length of
the protein sequences and are derived for those proteins
containing the same domain composition with very similar
sequence lengths. Multifunctional proteins are represented
by different HMMs to those representing each part of the
multifunctional protein, and no protein should match more
than one unique PIRSF HMM. PANTHER creates HMMs
from sequences derived from higher eukaryotic organisms
and thus has few bacterial hits.
Integrating the Signatures in InterPro
While all the databases described above have significant
overlaps in the protein families and domains they predict,
they arrive at these overlaps by different means. Using just
one of the databases to analyze a query sequence could
result in no hits if the sequence is outside their range of
coverage, and also makes one vulnerable to any limitations
the chosen database may have. Conversely, however, try-
ing to use all of them at the same time but from the separate
sites may lead to confusion in trying to rationalize the
different results obtained at each. This problem was
resolved by the InterPro resource, which integrates all the
protein signature databases into one. Signatures from
PROSITE, PRINTS, Pfam, ProDom, SMART, TIGRF-
AMs, PIRSF, SUPERFAMILY, Gene3D and PANTHER
that describe the same domain, family, repeat, active site,
binding site or post-translational modification, are grouped
into single InterPro entries with unique accession numbers.
InterPro entries that match a subset of proteins matched by
other entries are related to each other as parent/child
(family and subfamily) or contains/found in (domain
composition) relationships.
InterPro Annotation
Each InterPro entry contains high-quality manual annota-
tion providing useful information on the protein family,
domain etc. in question. This is in the form of a name
(short description), abstract and cross-references. Literature
references cited in the abstract are stored in a reference
Mol Biotechnol (2008) 38:165–177
field in each entry. Mapping of InterPro entries to GO
terms [19], where possible, provides additional functional
annotation. The GO project is an effort to provide a uni-
versal ontology for describing gene products across all
species, and has developed a set of terms in a directed
acyclic graph under the three ontologies: molecular func-
tion, biological process and cellular component. Where all
proteins matching an InterPro entry have the same function
then the entry can be mapped to the appropriate GO term
describing that function. InterPro entries are manually
mapped to GO terms taking into account information in the
abstract of the entries and annotation of proteins in the
match lists. The associated GO terms should apply to all
proteins with true hits to all signatures in the InterPro entry.
The mappings provide an automatic means of large-scale
GO characterization of proteins.
Additional annotation is provided in InterPro entries
through a number of different fields, including the ‘‘Taxon-
omy’’, ‘‘Interaction’’, ‘‘Structural links’’ and the ‘‘Database
links’’ fields. The ‘‘Taxonomy’’ field aims to provide an, ‘at a
glance’, view of the taxonomic range of the sequences
associated with each InterPro entry. The lineages were
carefully selected to provide a view of the major groups of
organisms. The circular display has the taxonomy-tree root
as its centre with the model organisms populating the outer
most circle and nodes of the taxonomy-tree placed on the
inner circles. The nodes themselves are either true taxonomy
nodes and have an NCBI taxonomy ID or are artificial nodes
created for this display; of which there are three: ‘Unclas-
sified’, ‘Other Eukaryota (Non-Metazoa)’ and the ‘Plastid
Group’. The number of sequences associated with each
lineage is displayed, with links to the InterPro matches for
these proteins or the option to retrieve the FASTA sequences.
The ‘‘Interaction’’ field provides links to example proteins
that are known to interact with the InterPro domain through
protein-protein interactions.
The ‘‘Structural links’’ field provides information on
curated structure links based on the correspondence
between the proteins matching the InterPro entry and those
proteins of known structure and belonging to SCOP or
CATH superfamilies. The links are only included when the
structural domains overlap considerably with one or more
of the InterPro signatures on the protein sequence. Finally,
the ‘‘Database links’’ field provides links to a number of
external databases such as PANDIT, MEROPS, CAZy,
Enzyme Commission numbers, etc.
InterProScan
In addition to annotation, each InterPro entry contains a list
of precomputed matches to UniProtKB proteins. Protein
matches are calculated using the InterProScan [44]
171
software package, which combines different protein sig-
nature recognition methods from each of the InterPro
member databases into one package. The software is used
to compute all matches for each entry, and is also available
for user query sequence searches. For a query sequence run
through InterProScan via a web interface, the software
outputs the resulting InterPro matches in a graphical for-
mat. These can also be viewed as a table, which lists the
protein signature matches, positions of the matches, rela-
tionships for InterPro entries hit and GO terms where
available from InterPro2GO mappings. For bulk sequences,
InterProScan can also be downloaded and run locally, or
programmatic access is provided via web services.
In InterPro entries, the protein matches may be viewed
in tabular and different graphical formats. The table lists
the protein accession numbers and the positions in the
amino acid sequence where each signature from that In-
terPro entry hits. The match list may be displayed in a
detailed graphical view, in which the sequence is split into
several lines, one for each hit by a unique signature (the
bars are colored coded according to the member database).
The graphical overview splits the protein sequence into
different lines for each InterPro entry matched and displays
the consensus domain boundaries of all signatures within
each entry. The graphical views include hits to all signa-
tures from the same and other InterPro entries, thus for
each sequence, the domain and/or motif organization can
be seen at a glance. Where structures are available for
proteins, there is a link from the graphical view to the
corresponding PDB [12] structures and a separate line in
the display showing the SCOP and/or CATH curated
matches on the sequence as white striped bars. Some pro-
teins have modelled structures from SWISS-MODEL [45]
or MODBASE [46], which are shown separately in match
views, again as white striped bars.
Through its matches to protein sequences, InterPro
provides a powerful tool for protein sequence classification
and function prediction. It has been used in many genome
annotation projects, as well as by UniProt curators for
individual protein sequence annotation.
From Genes and Proteins to Genomes and Proteomes
The availability of large quantities of protein sequence data
changes not just the quantity of analysis that can be per-
formed, but also the types of analysis it is possible to
perform. As detailed experimental investigation cannot be
performed on all predicted proteins, functional annotation
of these proteins is often inferred from automatically
detectable patterns in sequence, meaning that confirmation
of the putative protein’s actual expression and information
about any novel or exceptional functionality (or non-
172
functionality) it may posses, will not be available. How-
ever, the possession of a complete set of proteins for a
species allows us, to the extent that the predictions are
correct, to draw inferences from absence, to identify likely
orthologues between species, and to describe the nature of
a species in terms of the composition of its genome.
Annotated genome sequence thus serves as the central
platform for the evolving field of systems biology of whole
organisms, used in both experimental design and
interpretation.
There are a number of difficulties that a user can
encounter in attempting to obtain complete, non-redundant
proteome sets from protein sequence databases. First, some
databases may contain redundant sequences by design
(there are many senses in which two sequences may be
redundant) or ‘‘accident’’, that is, the volume of redundant
data may be too large to reliably identify and update. For
example, the nucleotide sequence database maintained
jointly by the EMBL, Genbank, and the DDBJ is non-
redundant as a representation of submissions from indi-
vidual experimentalists, i.e. a given submitter should
update, not supplement, sequences they now believe to be
incorrect, but it is redundant for protein sequences. Uni-
ProtKB is non-redundant for protein sequences at the
species level, but identification of variants, splice isoforms,
fragmentary sequence or errors has to be done manually
and thus the database retains a certain element of redun-
dancy in the wider sense. Another problem that may affect
users is the incompleteness of a given data source, if, for
example, different data sets have been submitted to dif-
ferent resources. A further problem is that some, although
not all, of the proteins in a proteome may have been pre-
viously studied, providing information that needs to be
integrated, not replaced by, new prediction data made from
the complete genome sequence. The UniProt non-redun-
dant Complete Proteome sets, and IPI, are two data sets
which have been produced in response to this problem.
UniProt Non-redundant Complete Protein Sets
As shown earlier, the UniProtKB consists of two sections, a
manually curated part, UniProtKB/Swiss-Prot, and an
automatically produced supplement, UniProtKB/TrEMBL.
In UniProtKB/Swiss-Prot, data is subject to manual review,
‘‘redundant’’ sequences are merged according to the defi-
nition that there should only be one Swiss-Prot entry per
gene, and experimental information obtained from pub-
lished reports is available. UniProtKB/TrEMBL contains
mainly data submitted by users to the EMBL/Genbank/
DDBJ databases, although the annotations provided by
submitters are supplemented or replaced by automatic
Mol Biotechnol (2008) 38:165–177
predictions derived through sequence classification based
on InterPro and inferences from manual annotation in
similarly classified UniProtKB/Swiss-Prot entries.
In addition, special procedures are in place to handle
data from species with completely deciphered genomes.
The merging of extant, manually curated entries with
newly submitted data associated with a genome sequencing
project is a priority for UniProt curators; this allows for the
production of non-redundant data sets that still contain all
pertinent experimental information. Data that remains in
UniProtKB/TrEMBL is quickly triaged, errors in refer-
ences are corrected, missing gene names are inserted,
extant gene names are classified by type, taxonomic clas-
sification is standardized, and cross-references to resources
maintained by the organization responsible for the original
generation of the sequence and/or annotation are added. A
keyword ‘Complete proteome’ is also added, indicating
that the entry can be considered as part of a set that col-
lectively describes the complete proteome of an organism.
Entries from bacterial genomes are then subject to anno-
tation by a specialist pipeline, HAMAP [14], designed to
identify families of bacterial proteins and apply appropriate
annotation. Following manual review, entries identified as
family members are then promoted to UniProtKB/Swiss-
Prot.
In general, the level of redundant information is greater
for proteomes of higher species; and the relationship
between biologically real variants more complex. For a
number of species, specialist model organism databases, as
described above, may maintain information about the
various representations of the products of a single gene that
exist in other databases. Data is taken from both these
databases and used to ensure that a single entry per gene is
chosen for inclusion in the UniProt proteome sets.
Another problem for a number of well-studied organ-
isms is that revisions to the genome sequence, and/or
revisions to the protein predictions made from it, have not
necessarily always been submitted back into the standard
public repositories. However, specialist model organism
databases usually maintain this information. UniProt
monitors some of these resources (including the Saccha-
romyces Genome Database and The Arabidopsis
Information Resource) and builds new entries corre-
sponding to any data that appears to be missing, to ensure
that the UniProt proteome sets are complete. Recently this
approach was extended, to include predictions made from
the human genome sequence by the Ensembl protein
annotation pipeline. It is planned to further include
‘‘missing’’ (i.e. new) Ensembl predictions for all species
well annotated by this resource.
UniProt non-redundant complete proteome sets can be
downloaded from ftp://ftp.ebi.ac.uk/pub/databases/integr8.
Mol Biotechnol (2008) 38:165–177
IPI
For many of the most studied species, the level of redun-
dancy in gene and transcript predictions is very high. For
example, as of August 2007, the UniProt Knowledgebase
contains 71,479 human entries, all representing distinct
sequences (and with information about approximately 9000
additional splice isoforms too). By contrast, the latest
Ensembl build predicts the existence of just 43,570 protein-
coding transcripts (some of which may encode the same
protein as each other) from 43,570 genes. While some of
the other sequences in UniProtKB may contain information
of genuine biological interest, others may be fragments or
erroneous predictions; others may contain information that
is interesting only to certain communities (for example,
variants derived from SNPs). Additionally, the Ensembl
database is not the only database containing predictions
made from the complete human genome sequence. The
NCBI’s RefSeq project maintains a set of human protein
sequences corresponding to their prediction of the com-
plete human genome (this contains 29,569 entries) and
numerous other groups make their own predictions, over-
lapping to a greater or lesser extent. The CCDS project
(http://www.ncbi.nlm.nih.gov/CCDS/) is an attempt by
Ensembl, RefSeq and the University of Santa Cruz to agree
on a common set of coding sequences. The current CCDS
set contains a total of 17,700 sequences, but unfortunately
there is a wide divergence between predictions outside this
core set.
To deal with this problem, IPI (the International Protein
Index) [47] was conceived. IPI (http://www.ebi.ac.uk/IPI)
is a database built from a number of distinct sources in
which attempts are made to remove redundancy such as to
include one entry per distinct gene product; each entry is
cross-referenced to the source databases. While IPI aims at
non-redundancy, the method used for building the database
places a higher importance on completeness. Non-identical
sequences can be merged into a single IPI entry if there is a
reciprocal best match between a protein in one database
and a protein in another, but IPI does not generally assert
that a given protein prediction is wrong. It therefore
comprises a maximal subset of all predictions but with
some sequence and annotation-derived filtering for redun-
dancy. It has been used as a database for protein
identification from mass spectrometry data, where users
may be keen not to miss a potential hit, but also wish to
derive statistically significant results, which can be impe-
ded by the use of data sets inflated by redundancy. IPI is
also of use as a supplement to UniProtKB for a few species
where the latest predictions have not yet been incorporated
in that resource. The database is currently produced for a
total of seven species in all, including six vertebrates and
Arabidopsis thaliana. For most lower species, UniProtKB
173
itself is complete and up-to-date and there is no need to
supplement it with information from additional resources.
Genome Reviews
The Genbank/EMBL/DDBJ nucleotide sequence databases
are repositories of stored information. However, this
information may become out-of-date over time, because it
is inferred using the contents of this and other databases at
the time of annotation. Additionally, different submitters
may have different interests, in terms of what they choose
to annotate, quality standards and preferences for how to
express themselves within the possibilities allowable in the
rich data structures these databases support. This can cause
problems for people wishing to use this data for compar-
ative genomics: information may be incorrect or out of
date, or represented in different ways in different entries.
Genome Reviews [48] (http://www.ebi.ac.uk/Genome
Reviews) is a database of information about organisms
with completely deciphered genomes. In Genome Reviews,
sequences and annotations are taken from a variety of pri-
mary sources, and improved by importing the latest manual
and automatic information derivable from UniProtKB, and
additionally supplemented by information calculated by
sequence analysis. All imported data is tagged to indicate the
primary data source from which it was imported, allowing
users to select their annotation according to source. The data
is then distributed in a number of formats, including EMBL-
format files, and an (Ensembl-like) MySQL database. An
Ensembl-like Genome Browser is also available for inter-
active access. The EMBL format files not only contain
additional information calculated since the original sub-
mission, but also represent all information in a standardized
way, making Genome Reviews an ideal database for use in
comparative genomic analysis.
Data is incorporated into Genome Reviews in three main
ways. First, annotation is imported through the use of pre-
existing cross-references between databases that describe
the same entities. This allows the import of data from
references that are more intensely curated, such as Uni-
ProtKB. Second, certain predictions are made directly from
the DNA sequence, for example, predictions of non-protein
coding genes are made using tRNAScan-SE and RFAM
where no annotations are present in the original submis-
sion. Third, where information in the curated protein
sequences is in conflict with the original sequence, the
protein and DNA sequences are compared using the utili-
ties BLAST [49] and ALIGN0 [50] to identify and describe
the conflicts such that they can be annotated in the Genome
Reviews flat file.
Gene sets are also available for all species in Genome
Reviews, providing DNA sequences (complete with
174
annotated features) in EMBL and FASTA formats for each
gene. These are cross-referenced to the UniProt entries that
define the corresponding proteome sets. The current scope
of Genome Reviews is bacteria, archaea, bacteriophage and
a small number of lower eukaryotes, but it is aimed to
expand the database to cover all non-vertebrate genomes.
Integr8
The Integr8 web portal [4] (http://www.ebi.ac.uk/integr8)
provides a single point of entry to information about
complete genomes. Available data includes DNA sequen-
ces from databases including the EMBL Nucleotide
Sequence Database, Genome Reviews and Ensembl, pro-
tein sequences from databases including the UniProtKB
and IPI, statistical genome and proteome analysis per-
formed using InterPro, CluSTr [51], and GOA, and
information about orthology, paralogy and synteny. Integr8
provides tools to enable this data to be searched, browsed,
compared and contextualized, and easy access to files
where the data representing complete genomes and their
constituent components can be downloaded and used for
further, large scale analysis.
Integr8 currently covers all cellular organisms with
completely sequenced genomes, a total of 583 species
(including 490 bacteria, 42 archaea and 53 eukaryotes) as
of August 2007. This represents an increase of over 200%
from just 193 species in July 2004.
Gene, Species and Sequence Search
A simple search form available on every page of the In-
tegr8 web application provides access to summary
information relevant to each species. When a species is
selected, it is used as the default species for any subsequent
gene or sequence search. Additionally, it is possible to
select any portfolio of species to search against. A graph-
ical query interface allows users to browse species
alphabetically or according to their position in the taxo-
nomic hierarchy, and allows species to be added to the
portfolio individually or in groups. For example, one can
add/remove all species below a single taxonomic node to/
from the portfolio with a single mouse click. One can
search for a gene or sequence within all species, the cur-
rently selected species portfolio or a new selection. Gene
searching is simply carried out with any text string that
might have been associated with one’s gene or protein, e.g.
name, description, EC number. Additionally, if one types
in text that comprises the first part of a term from GO, a
menu of potential auto-completes will appear, allowing
users to search for genes annotated with a given term
Mol Biotechnol (2008) 38:165–177
without having prior knowledge of exactly what term is
used to describe a particular function, process or cellular
component. Another advantage of searching with GO terms
is that the search is recursive, if one searches with a general
GO term, all genes that are annotated with a more specific
term will also be retrieved. For all retrieved genes, Integr8
provides direct links to a number of useful views, including
the Integr8or (described below), and an Ensembl-like
scalable genome browser for visualizing genes in genomes
covered by Genome Reviews. Sets of UniProtKB entries
describing the gene products can also be downloaded.
Integr8 also offers a sequence search facility, the
Inquisitor. The Inquisitor analyses submitted sequences
using the protein classification tool InterProScan in com-
bination with a FASTA search specific to the species
against which the user has chosen to search. A single
summary page provides an overview of the results of the
analysis.
Species-centric Information
For each species, Integr8 provides a description, a list of
recent publications, a list of the components of its genome,
and information about the composition of these compo-
nents (such as their length, average GC content, and the
length and codon usage in the CDSs they contain), repre-
sented textually or graphically as appropriate. Additionally,
all genomes are assigned a Genome Annotation Score,
based on the apparent completeness of the annotation and
the degree of experimental evidence that exists to support
it. This allows the change in the degree of annotation of a
genome over time to be measured.
Integr8 also provides information about the composition
of each species’ proteome. Individual proteins are classi-
fied according to InterPro, GO and CluSTr, and an
overview of the composition of each proteome constructed
on these criteria is available. For example, for each pro-
teome, users can identify the most common protein
families and domains, proteins without close relatives, or
clusters of related proteins unclassified by InterPro. GO
classifications for each species are summarized using a
reduced set of high-level terms (GO Slim), presenting an
overview of proteome function even in species where more
specific annotations might not be available. A major
advance in the past year has been the doubling in the
coverage of CluSTr, a database that categorizes proteins
into a hierarchy of clusters based on overall sequence
similarity. Individual hierarchies of clusters have now been
prepared for each of 109 proteomes, enabling the rela-
tionship of all paralogous proteomes to be analyzed in
these species. Additional structural data is also available
based on information derived from the PDB and HSSP
Mol Biotechnol (2008) 38:165–177
[52]. Integr8 also offers comparative analysis, whereby the
composition of multiple proteomes can be compared. A
total of 160 comparative analyses, each featuring between
two and four related species, have been pre-compiled.
Additional comparisons can be specified interactively.
Gene Centric Information
A key feature of Integr8 is a single page that summarizes
what is known about a gene. Emphasis is placed on
showing clearly the relationship between the different
products of a gene, and what information (sequence,
experimental evidence etc.) is known about each; also,
information at the gene, transcript and protein level is
clearly separated and displayed, and the gene can be
visualized in its genomic context. Potential orthologues and
paralogues of each protein, identified using information
derived from the CluSTr database, can also be displayed. A
number of these can then be selected, and either aligned, or
the potentially syntenic regions around each homologue
can be viewed in a stylized comparative view showing the
relationship of protein structure as determined by InterPro
analysis.
Download and Web services
The following data is available for download from Integr8:
(i) Genome Reviews files (ii) UniProt complete proteome
sets (iii) IPI data sets (iv) Files of InterPro matches for each
proteome set (v) GOA files, files of GO annotations for
each proteome set (vi) ‘‘Chromosome tables’’, summary
files with information about each complete genome in an
easy-to-parse, tab-delineated format (vii) Orthologue files,
identifying putative orthologues between each pair of
species in Integr8 (ix) an XML dump of the complete
database. Most of the function of the Integr8 web portal is
mirrored by a set of web service methods to allow pro-
grammatic access to the database. Using standard
technologies, this makes Integr8 available to programs
written in most modern programmatic languages; clients
for Java and Perl are provided, but clients for other lan-
guages can easily be generated from the published service
description.
Discussion
With the advent of whole genome sequencing as a rela-
tively low-cost and common scientific method, huge
quantities of data have been and continue to be generated.
The tools and resources mentioned in this review are on
175
target to, and already have relieved some of the burden of
the influx of raw sequencing data. Sequence repositories
and curated databases are vital for providing the public
with access to all known sequences, and adding value to
that data in the case of the latter. Protein sequence dat-
abases, such as UniProtKB act as integrated resources of
protein information, and derived databases like UniRef
provide smaller subsets of the data that are more man-
ageable for searching. However, some researchers are
interested only in smaller, more complete, and yet more
specific subsets of the data, and for this they can go to
resources like IPI. For non-redundant protein sets users can
access data from RefSeq or Integr8 (for completely
sequenced genomes).
As well as just providing protein sequences, it is nec-
essary for these resources to provide some form of function
associated with them. In UniProtKB/Swiss-Prot annotation
is derived from the literature, but here, and for UniProtKB/
TrEMBL, protein function is also predicted using tools like
InterPro. Through the protein signatures it integrates, In-
terPro acts as a protein sequence classification system,
which facilitates automatic function prediction. It has been
used for annotation of numerous completely sequenced
genomes, and in the analysis and comparison of complete
proteomes as provided in Integr8. In addition to these
protein family and domain analyses, Integr8 provides a
useful source of whole genome analysis and comparisons
where users can get a genome-, gene- or protein-centric
view of a complete genome. As the name suggests, it
integrates data for each organism from a variety of sources,
including GO, CluSTr, UniProtKB, InterPro etc., and
facilitates searching, downloading or simply viewing of
precomputed and user-chosen data.
There is a shift in current research from the single gene
to whole genome focus, and hypothesis-driven bottom-up
approaches are being replaced by exploratory top-down
investigations. Proteins are being studied within their
contexts in a cell to determine what molecules they interact
with and which biological systems they play a role in. This
‘‘systems biology’’ approach is far more relevant to real life
and likely to provide a more in-depth understanding of the
molecular biology of organisms than the single gene focus.
To achieve this, researchers need access to large and
complete data sets with as much functional information as
possible. It is hoped that a combination of tools such as
those described here will help biologists shed some light on
the biological function of newly discovered proteins and
systems. Only once this is done it is possible to use the data
to its full potential in medical or commercial applications.
Acknowledgements UniProt is mainly supported by the National
Institutes of Health (NIH) grant 1 U01 HG02712-01. Additional
support comes from the European Commission’s grant 021902RII3,
176
from the NIH grants 1R01HGO2273-01, HHSN266200400 061C,
NCI-caBIGICR-10-10-01 and ITR-0205470, and the Swiss Federal
Government. InterPro was funded by the award of grant number
QLRI-CT-2000–00517 and in part by grant number QLRI-CT-
2001000015 from the European Union under the RTD programme
‘‘Quality of Life and Management of Living Resources’’. InterPro is a
member database of the MRC-funded eFamily project. Genome
Reviews and Integr8 have been funded or are funded, respectively, by
the European Commission’s grants QLRICT-2001000015, and
021902RII3.
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