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What is Bioinformatics?
A Proposed Definition and Overview of the Field
N. M. Luscombe, D. Greenbaum, M. Gerstein
Department of Molecular Biophysics and Biochemistry
Yale University, New Haven, USA
Table 1 Sources of data used in bioinformatics, the quantity of each type of data that is currently (April 2001) available, quences. While more biological informa-
and bioinformatics subject areas that utilize this data. tion can be derived from a single structure
than a protein sequence, the lack of depth
in the latter is compensated by analysing
larger quantities of data.
ed from a common ancestral gene, and straightforward to access and cross- producing multiple sequence alignments
paralogues, proteins that are related by reference these sources of information be- [48], and searching for functional domains
gene duplication within a genome [35]. cause of differences in nomenclature and from conserved sequence motifs in such
Normally, orthologues retain the same file formats. alignments. Investigations of structural
function while paralogues evolve distinct, At a basic level, this problem is fre- data include prediction of secondary and
but related functions [36]. quently addressed by providing external tertiary protein structures, producing
An important concept that arises from links to other databases. For example in methods for 3D structural alignments [49,
these observations is that of a finite “parts PDBsum, web-pages for individual struc- 50], examining protein geometries using
list” for different organisms [37-39]: an tures direct the user towards corresponding distance and angular measurements, calcu-
inventory of proteins contained within an entries in the PDB, NDB, CATH, SCOP lations of surface and volume shapes and
organism, arranged according to different and SWISS-PROT databases. At a more analysis of protein interactions with other
properties such as gene sequence, protein advanced level, there have been efforts to subunits, DNA, RNA and smaller mole-
fold or function. Taking protein folds as an integrate access across several data sources. cules. These studies have lead to molecular
example, we mentioned that with a few One is the Sequence Retrieval System, SRS simulation topics in which structural data
exceptions, the tertiary structures of pro- [41], which allows flat-file databases to be are used to calculate the energetics in-
teins adopt one of a limited repertoire indexed to each other; this allows the user volved in stabilising macromolecular struc-
of folds. As the number of different fold to retrieve, link and access entries from tures, simulating movements within macro-
families is considerably smaller than the nucleic acid, protein sequence, protein molecules, and computing the energies
number of genes, categorising the proteins motif, protein structure and bibliographic involved in molecular docking. The increa-
by fold provides a substantial simplification databases. Another is the Entrez facility sing availability of annotated genomic
of the contents of a genome. Similar sim- [42], which provides similar gateways to sequences has resulted in the introduction
plifications can be provided by other attri- DNA and protein sequences, genome of computational genomics and proteomics
butes such as protein function. As such, we mapping data, 3D macromolecular structu- – large-scale analyses of complete genomes
expect this notion of a finite parts list to res and the PubMed bibliographic database and the proteins that they encode. Re-
become increasingly common in future [43].A search for a particular gene in either search includes characterisation of protein
genomic analyses. database will allow smooth transitions to content and metabolic pathways between
Clearly, an essential aspect of managing the genome it comes from, the protein different genomes, identification of interac-
this large volume of data lies in developing sequence it encodes, its structure, biblio- ting proteins, assignment and prediction of
methods for assessing similarities between graphic reference and equivalent entries for gene products, and large-scale analyses of
different biomolecules and identifying all related genes. In our own group, we have gene expression levels. Some of these re-
those that are related. There are well-docu- developed the SPINE [44] and PartsList search topics will be demonstrated in our
mented classifications for all of the main [39] web resources; these databases inte- example analysis of transcription regula-
types of data we described earlier. Al- grate many types of experimental data and tory systems.
though detailed descriptions of these clas- organise them using the concept of the Other subject areas we have included in
sification systems are beyond the scope of finite “parts list” we described above. Table 1 are: development of digital libraries
the current review, they are of great impor- for automated bibliographical searches,
tance as they ease comparisons between knowledge bases of biological information
genomes and their products. Links to the from the literature, DNA analysis methods
major databases are available from our
supplementary website.
4. “…UNDERSTAND and in forensics, prediction of nucleic acid struc-
tures, metabolic pathway simulations, and
Organise the Information…” linkage analysis – linking specific genes to
different disease traits.
Having examined the data, we can discuss In addition to finding relationships be-
3.2 Data Integration the types of analyses that are conducted.As tween different proteins, much of bioin-
The most profitable research in bioinfor- shown in Table 1, the broad subject areas in formatics involves the analysis of one type
matics often results from integrating mul- bioinformatics can be separated according of data to infer and understand the obser-
tiple sources of data [40]. For instance, the to the type of information that is used. For vations for another type of data. An exam-
3D coordinates of a protein are more useful raw DNA sequences, investigations involve ple is the use of sequence and structural
if combined with data about the protein’s separating coding and non-coding regions, data to predict the secondary and tertiary
function, occurrence in different genomes, and identification of introns, exons and structures of new protein sequences [51].
and interactions with other molecules. In promoter regions for annotating genomic These methods, especially the former, are
this way, individual pieces of information DNA [45, 46]. For protein sequences, ana- often based on statistical rules derived
are put in context with respect to other lyses include developing algorithms for from structures, such as the propensity for
data. Unfortunately, it is not always sequence comparisons [47], methods for certain amino acid sequences to produce
Paradigm shifts during the past couple of decades have taken much of biology away from the
laboratory bench and have allowed the integration of other scientific disciplines, specifically
computing. The result is an expansion of biological research in breadth and depth. The vertical axis
demonstrates how bioinformatics can aid rational drug design with minimal work in the wet lab.
Starting with a single gene sequence, we can determine with strong certainty, the protein
sequence. From there, we can determine the structure using structure prediction techniques. With
geometry calculations, we can further resolve the protein’s surface and through molecular
simulation determine the force fields surrounding the molecule. Finally docking algorithms can
provide predictions of the ligands that will bind on the protein surface, thus paving the way for the
design of a drug specific to that molecule. The horizontal axis shows how the influx of biological
data and advances in computer technology have broadened the scope of biology. Initially with a pair
of proteins, we can make comparisons between the between sequences and structures of
evolutionary related proteins. With more data, algorithms for multiple alignments of several
proteins become necessary. Using multiple sequences, we can also create phylogenetic trees to
trace the evolutionary development of the proteins in question. Finally, with the deluge of data we
currently face, we need to construct large databases to store, view and deconstruct the
information. Alignments now become more complex, requiring sophisticated scoring schemes and
there is enough data to compile a genome census – a genomic equivalent of a population census –
providing comprehensive statistical accounting of protein features in genomes.
different secondary structural elements. transferred between homologous proteins analysis in two dimension, depth and
Another example is the use of structural [55]. breadth. The first is represented by the
data to understand a protein’s function; vertical axis in the figure and outlines a
here studies have investigated the rela- possible approach to the rational drug
tionship different protein folds and their design process. The aim is to take a single
functions [52, 53] and analysed similarities
4.1 The Bioinformatics Spectrum gene and follow through an analysis that
between different binding sites in the ab- Fig. 2 summarises the main points we maximises our understanding of the
sence of homology [54]. Combined with raised in our discussions of organising protein it encodes. Starting with a gene
similarity measurements, these studies pro- and understanding biological data – the sequence, we can determine the protein
vide us with an understanding of how much development of bioinformatics techniques sequence with strong certainty. From there,
biological information can be accurately has allowed an expansion of biological prediction algorithms can be used to calcu-
-helix and major groove, there is enough interactions of arginine or lysine with 6.2 Genomic Studies
flexibility to allow both the protein and guanine, asparagine or glutamine with
DNA to adopt distinct conformations. adenine and threonine with thymine. Such Due to the wealth of biochemical data that
However, several studies that analysed the preferences were explained through exami- are available, genomic studies in bioin-
binding geometries of -helices demon- nation of the stereochemistry of the amino formatics have concentrated on model
strated that most adopt fairly uniform con- acid side chains and base edges. Also organisms, and the analysis of regulatory
formations regardless of protein family. highlighted were more complex types of systems has been no exception. Identification
They are commonly inserted in the major interactions where single amino acids of transcription factors in genomes invari-
groove sideways, with their lengthwise axis contact more than one base-step simulta- ably depends on similarity search strate-
roughly parallel to the slope outlined by neously, thus recognising a short DNA gies, which assume a functional and evolu-
the DNA backbone. Most start with the sequence. These results suggested that tionary relationship between homologous
N-terminus in the groove and extend out, universal specificity, one that is observed proteins. In E. coli, studies have so far
completing two to three turns within across all protein-DNA complexes, indeed estimated a total of 300 to 500 transcription
contacting distance of the nucleic acid [59, exists. However, many interactions that are regulators [71] and PEDANT [72], a data-
60]. normally considered to be non-specific, base of automatically assigned gene funct-
Given the similar binding orientations, it such as those with the DNA backbone, can ions, shows that typically 2-3% of pro-
is surprising to find that the interactions also provide specificity depending on the karyotic and 6-7% of eukaryotic genomes
between each amino acid position along context in which they are made. comprise DNA-binding proteins.As assign-
the -helices and nucleotides on the DNA Armed with an understanding of ments were only complete for 40-60% of
vary considerably between different pro- protein structure, DNA-binding motifs and genomes as of August 2000, these figures
tein families. However, by classifying the side chain stereochemistry, a major applica- most likely underestimate the actual num-
amino acids according to the sizes of their tion has been the prediction of binding ber. Nonetheless, they already represent a
side chains, we are able to rationalise the either by proteins known to contain a parti- large quantity of proteins and it is clear that
different interactions patterns. The rules of cular motif, or those with structures solved there are more transcription regulators
interactions are based on the simple pre- in the uncomplexed form. Most common in eukaryotes than other species. This is
mise that for a given residue position on are predictions for -helix-major groove unsurprising, considering the organisms
-helices in similar conformations, small interactions – given the amino acid se- have developed a relatively sophisticated
amino acids interact with nucleotides that quence, what DNA sequence would it transcription mechanism.
are close in distance and large amino acids recognise [61, 67]. In a different approach, From the conclusions of the structural
with those that are further [60, 61]. Equi-va- molecular simulation techniques have been studies, the best strategy for characterising
lent studies for binding by other structural used to dock whole proteins and DNAs on DNA-binding of the putative transcription
motifs, like -hairpins, have also been con- the basis of force-field calculations around factors in each genome is to group them
ducted [62]. When considering these the two molecules [68, 69]. by homology and to analyse the individual
interactions, it is important to remember The reason that both methods have families. Such classifications are provided
that different regions of the protein surface been met with limited success is because in the secondary sequence databases
also provide interfaces with the DNA. even for apparently simple cases like - described earlier and also those that
This brings us to look at the atomic level helix-binding, there are many other factors specialise in regulatory proteins such as
interactions between individual amino that must be considered. Comparisons RegulonDB [73] and TRANSFAC [74].
acid-base pairs. Such analyses are based on between bound and unbound nucleic acid Of even greater use is the provision of
the premise that a significant proportion of structures show that DNA-bending is a structural assignments to the proteins;
specific DNA-binding could be rationalised common feature of complexes formed with given a transcription factor, it is helpful to
by a universal code of recognition between transcription factors [58, 70].This and other know the structural motif that it uses for
amino acids and bases, ie whether certain factors such as electrostatic and cation- binding, therefore providing us with a
protein residues preferably interact with mediated interactions assist indirect better understanding of how it recognises
particular nucleotides regardless of the recognition of the nucleotide sequence, the target sequence. Structural genomics
type of protein-DNA complex [63]. Studies although they are not well understood yet. through bioinformatics assigns structures
have considered hydrogen bonds, van der Therefore, it is now clear that detailed rules to the protein products of genomes by
Waals contacts and water-mediated bonds for specific DNA-binding will be family demonstrating similarity to proteins of
[64-66]. Results showed that about 2/3 of all specific, but with underlying trends such as known structure [75]. These studies have
interactions are with the DNA back- the arginine-guanine interactions. shown that prokaryotic transcription fac-
bone and that their main role is one of tors most frequently contain helix-turn-
sequence-independent stabilisation. In helix motifs [71, 76] and eukaryotic factors
contrast, interactions with bases display contain homeodomain type helix-turn-
some strong preferences, including the helix, zinc finger or leucine zipper motifs.
From the protein classifications in each specific but different members bind distinct approach, it was found that at least 27% of
genome, it is clear that different types of base sequences. Here protein residues known E. coli DNA-regulatory motifs are
regulatory proteins differ in abundance and undergo frequent mutations, and family conserved in one or more distantly related
families significantly differ in size. A study members can be divided into subfamilies bacteria [84].
by Huynen and van Nimwegen [77] has according to the amino acid sequences The detection of regulatory sites in
shown that members of a single family have at base-contacting positions; those in the eukaryotes poses a more difficult problem
similar functions, but as the requirements same subfamily are predicted to bind the because consensus sequences tend to be
of this function vary over time, so does same DNA sequence and those of different much shorter, variable, and dispersed over
the presence of each gene family in the subfamilies to bind distinct sequences. On very large distances. However, initial stud-
genome. the whole, the subfamilies corresponded ies in S. cerevisiae provided an interesting
Most recently, using a combination of well with the proteins’ functions and mem- observation for the GATA protein in nitro-
sequence and structural data, we examined bers of the same subfamilies were found to gen metabolism regulation. While the 5
the conservation of amino acid sequences regulate similar transcription pathways. base-pair GATA consensus sequence is
between related DNA-binding proteins, The combined analysis of sequence and found almost everywhere in the genome, a
and the effect that mutations have on structural data described by this study pro- single isolated binding site is insufficient to
DNA sequence recognition. The structural vided an insight into how homologous exert the regulatory function [85]. There-
families described above were expanded to DNA-binding scaffolds achieve different fore specificity of GATA activity comes
include proteins that are related by sequence specificities by altering their amino acid from the repetition of the consensus se-
similarity, but whose structures remain sequences. In doing so, proteins evolved quence within the upstream regions of con-
unsolved. Again, members of the same distinct functions, therefore allowing trolled genes in multiple copies. An initial
family are homologous, and probably derive structurally related transcription factors to study has used this observation to predict
from a common ancestor. regulate expression of different genes. new regulatory sites by searching for over-
Amino acid conservations were calculat- Therefore, the relative abundance of tran- represented oligonucleotides in non-coding
ed for the multiple sequence alignments scription regulatory families in a genome regions of yeast and worm genomes [86,
of each family [78]. Generally, alignment depends, not only on the importance of a 87].
positions that interact with the DNA are particular protein function, but also in the Having detected the regulatory binding
better conserved than the rest of the pro- adaptability of the DNA-binding motifs to sites, there is the problem of defining the
tein surface, although the detailed patterns recognise distinct nucleotide sequences. genes that are actually regulated, commonly
of conservation are quite complex. Residues This, in turn, appears to be best accommo- termed regulons. Generally, binding sites
that contact the DNA backbone are highly dated by simple binding motifs, such as the are assumed to be located directly upstream
conserved in all protein families, providing zinc fingers. of the regulons; however there are different
a set of stabilising interactions that are Given the knowledge of the transcription problems associated with this assumption
common to all homologous proteins. The regulators that are contained in each depending on the organism. For prokary-
conservation of alignment positions that organism, and an understanding of how otes, it is complicated by the presence of
contact bases, and recognise the DNA they recognise DNA sequences, it is of operons; it is difficult to locate the regulat-
sequence, are more complex and could be interest to search for their potential bind- ed gene within an operon since it can lie
rationalised by defining a three-class model ing sites within genome sequences [79]. several genes downstream of the regulatory
for DNA-binding. First, protein families For prokaryotes, most analyses have in- sequence. It is often difficult to predict the
that bind non-specifically usually contain volved compiling data on experimentally organisation of operons [88], especially to
several conserved base-contacting residues; known binding sites for particular proteins define the gene that is found at the head,
without exception, interactions are made in and building a consensus sequence that in- and there is often a lack of long-range con-
the minor groove where there is little corporates any variations in nucleotides. servation in gene order between related
discrimination between base types. The Additional sites are found by conducting organisms [89]. The problem in eukaryotes
contacts are commonly used to stabilise word-matching searches over the entire is even more severe; regulatory sites often
deformations in the nucleic acid structure, genome and scoring candidate sites by act in both directions, binding sites are
particularly in widening the DNA minor similarity [80-83]. Unsurprisingly, most of usually distant from regulons because of
groove. The second class comprise families the predicted sites are found in non-coding large intergenic regions, and transcription
whose members all target the same regions of the DNA [80] and the results of regulation is usually a result of combined
nucleotide sequence; here, base-contacting the studies are often presented in databases action by multiple transcription factors in a
positions are absolutely or highly conser- such as RegulonDB [73]. The consensus combinatorial manner.
ved allowing related proteins to target the search approach is often complemented by Despite these problems, these studies
same sequence. comparative genomic studies searching have succeeded in confirming the transcrip-
The third, and most interesting, class upstream regions of orthologous genes in tion regulatory pathways of well-character-
comprises families in which binding is also closely related organisms. Through such an ized systems such as the heat shock response
system [83]. In addition, it is feasible to relates to the functions associated with the distinction between different forms of
experimentally verify any predictions, most these folds; the former are commonly in- cancer – for example subclasses of acute
notably using gene expression data. volved in metabolic pathways and the latter leukaemia – has been well established, it is
in signalling or transport processes [98]. still not possible to establish a clinical diag-
This is also reflected in the relationship nosis on the basis of a single test. In a
with subcellular localisations of proteins, recent study, acute myeloid leukaemia and
6.3 Gene Expression Studies where expression of cytoplasmic proteins is acute lymphoblastic leukaemia were suc-
Many expression studies have so far high, but nuclear and membrane proteins cessfully distinguished based on the ex-
focused on devising methods to cluster tend to be low [99, 100]. pression profiles of these cells [26]. As the
genes by similarities in expression profiles. More complex relationships have also approach does not require prior biological
This is in order to determine the proteins been assessed. Conventional wisdom is that knowledge of the diseases, it may provide a
that are expressed together under different gene products that interact with each other generic strategy for classifying all types of
cellular conditions. Briefly, the most com- are more likely to have similar expression cancer.
mon methods are hierarchical clustering, profiles than if they do not [101, 102]. How- Clearly, an essential aspect of under-
self-organising maps, and K-means cluster- ever, a recent study showed that this rela- standing expression data lies in understand-
ing. Hierarchical methods originally de- tionship is not so simple [103]. While ex- ing the basis of transcription regulation.
rived from algorithms to construct phyloge- pression profiles are similar for gene pro- However, analysis in this area is still limited
netic trees, and group genes in a “bottom- ducts that are permanently associated, for to preliminary analyses of expression levels
up” fashion; genes with the most similar example in the large ribosomal subunit, in yeast mutants lacking key components of
expression profiles are clustered first, and profiles differ significantly for products the transcription initiation complex [19,
those with more diverse profiles are that are only associated transiently, includ- 107].
included iteratively [90-92]. In contrast, the ing those belonging to the same metabolic
self-organising map [93, 94] and K-means pathway.
methods [95, 96] employ a “top-down”
approach in which the user pre-defines the
As described below, one of the main
driving forces behind expression analysis
7 “… many PRACTICAL
number of clusters for the dataset. The has been to analyse cancerous cell lines APPLICATIONS…”
clusters are initially assigned randomly, and [104]. In general, it has been shown that dif-
the genes are regrouped iteratively until ferent cell lines (eg epithelial and ovarian Here, we describe some of the major uses
they are optimally clustered. cells) can be distinguished on the basis of of bioinformatics.
Given these methods, it is of interest to their expression profiles, and that these
relate the expression data to other attri- profiles are maintained when cells are 7.1 Finding Homologues
butes such as structure, function and sub- transferred from an in vivo to an in vitro
cellular localisation of each gene product. environment [105]. The basis for their phy- As described earlier, one of the driving
Mapping these properties provides an siological differences were apparent in the forces behind bioinformatics is the search
insight into the characteristics of proteins expression of specific genes; for example, for similarities between different biomole-
that are expressed together, and also sug- expression levels of gene products neces- cules. Apart from enabling systematic orga-
gest some interesting conclusions about the sary for progression through the cell cycle, nisation of data, identification of protein
overall biochemistry of the cell. In yeast, especially ribosomal genes, correlated well homologues has some direct practical uses.
shorter proteins tend to be more highly with variations in cell proliferation rate. The most obvious is transferring informa-
expressed than longer proteins, probably Comparative analysis can be extended to tion between related proteins. For example,
because of the relative ease with which they tumour cells, in which the underlying given a poorly characterised protein, it is
are produced [97]. Looking at the amino causes of cancer can be uncovered by possible to search for homologues that are
acid content, highly expressed genes are pinpointing areas of biological variations better understood and with caution, apply
generally enriched in alanine and glycine, compared to normal cells. For example in some of the knowledge of the latter to the
and depleted in asparagine; these are breast cancer, genes related to cell prolif- former. Specifically with structural data,
thought to reflect the requirements of eration and the IFN-regulated signal trans- theoretical models of proteins are usually
amino acid usage in the organism, where duction pathway were found to be upregu- based on experimentally solved structures
synthesis of alanine and glycine are energe- lated [25, 106]. One of the difficulties in of close homologues [108]. Similar tech-
tically less expensive than asparagine.Turn- cancer treatment has been to target specific niques are used in fold recognition in which
ing to protein structure, expression levels of therapies to pathogenetically distinct tu- tertiary structure predictions depend on
the TIM barrel and NTP hydrolase folds mour types, in order to maximise efficacy finding structures of remote homologues
are highest, while those for the leucine and minimise toxicity. Thus, improvements and checking whether the prediction is
zipper, zinc finger and transmembrane in cancer classifications have been central energetically viable [109]. Where biochem-
helix-containing folds are lowest. This to advances in cancer treatment. Although ical or structural data are lacking, studies
Fig. 3 Above is a schematic outlining how scientists can use bioinformatics to aid rational drug discovery. MLH1 is a human gene encoding a mismatch repair protein (mmr) situated on the
short arm of chromosome 3. Through linkage analysis and its similarity to mmr genes in mice, the gene has been implicated in nonpolyposis colorectal cancer. Given the nucleotide sequence,
the probable amino acid sequence of the encoded protein can be determined using translation software. Sequence search techniques can be used to find homologues in model organisms, and
based on sequence similarity, it is possible to model the structure of the human protein on experimentally characterised structures. Finally, docking algorithms could design molecules that
could bind the model structure, leading the way for biochemical assays to test their biological activity on the actual protein.
could be made in low-level organisms like simplifies the problem of understanding missing in humans. On a smaller scale,
yeast and the results applied to homo- complex genomes by analysing simple structural differences between similar pro-
logues in higher-level organisms such as organisms first and then applying the teins may be harnessed to design drug
humans, where experiments are more same principles to more complicated ones – molecules that specifically bind to one
demanding. this is one reason why early structural structure but not another.
An equivalent approach is also employed genomics projects focused on Mycoplasma
in genomics. Homologue-finding is exten- genitalium [75].
sively used to confirm coding regions in Ironically, the same idea can be applied
newly sequenced genomes and functional in reverse. Potential drug targets are quickly
7.2 Rational Drug Design
data is frequently transferred to annotate discovered by checking whether homo- One of the earliest medical applications of
individual genes. On a larger scale, it also logues of essential microbial proteins are bioinformatics has been in aiding rational
drug design. Fig. 3 outlines the commonly protein folds are often related to specific this way, we are able to examine individual
cited approach, taking the MLH1 gene pro- biochemical functions [52, 53], these find- systems in detail and also compare them
duct as an example drug target. MLH1 is a ings highlight the diversity of metabolic with those that are related in order to un-
human gene encoding a mismatch repair pathways in different organisms [36, 89]. cover common principles that apply across
protein (mmr) situated on the short arm of As we discussed earlier, one of the most many systems and highlight unusual fea-
chromosome 3 [110]. Through linkage ana- exciting new sources of genomic information tures that are unique to some.
lysis and its similarity to mmr genes in mice, is the expression data. Combining expression
the gene has been implicated in nonpoly- information with structural and functional Acknowledgments
We thank Patrick McGarvey for comments on
posis colorectal cancer [111]. Given the classifications of proteins we can ask the manuscript.
nucleotide sequence, the probable amino whether the high occurrence of a protein
acid sequence of the encoded protein can fold in a genome is indicative of high ex-
be determined using translation software. pression levels [97]. Further genomic scale
Sequence search techniques can then be data that we can consider in large-scale sur-
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