COMPARATIVE SEQUENCE ANALYSIS OF LIPOXYGENASE OF SPECIES OF ASPERGILLUS AND FUSARIUM USING WEB-BASED BIOINFORMATICS TOOLS
Bioinformatics is an interdisciplinary field that develops methods and software tools for understanding biological data. As an interdisciplinary field of science, bioinformatics combines computer science, statistics, mathematics, and engineering to analyze and interpret biological data. Bioinformatics is both an umbrella term for the body of biological studies that use computer programming as part of their methodology, as well as a reference to specific analysis “pipelines” that are repeatedly used, particularly in the fields of genetics and genomics. Common uses of bioinformatics include the identification of candidate genes and nucleotides (SNPs). Often, such identification is made with the aim of improved understanding of the genetic basis of diseases, unique adaptations, desirable properties (especially in agricultural species), or differences between populations. In a less formal way, bioinformatics also tries to understand the organizational principles within nucleic acid and protein sequences. Bioinformatics tools aid in the comparison of genetic and genomic data and more generally in the understanding of evolutionary aspects of molecular biology. At a more integrative level, it helps analyze and catalogue the biological pathways and networks that are an important part of systems biology. In structural biology, it aids in the simulation and modeling of DNA, RNA, and protein structures as well as molecular interactions. Computers became essential in molecular biology when protein sequences became available after Frederick Sanger determined the sequence of insulin in the early 1950s (Attwood et al, 2011).
The primary goal of bioinformatics is to increase the understanding of biological processes. What sets it apart from other approaches, however, is its focus on developing and applying computationally intensive techniques to achieve this goal. Examples include: pattern recognition, data mining, machine learning algorithms, and visualization. Major research efforts in the field include sequence alignment, gene finding, genome assembly, drug design, drug discovery, protein structure alignment, protein structure prediction, prediction of gene expression and protein-protein interaction, genome wide association studies, and the modeling of evolution.
1.2 Sequence alignment
In bioinformatics, a sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences (Mount, 2004). Aligned sequences of nucleotide or amino acid residues are typically represented as rows within a matrix. Gaps are inserted between the residues so that identical or similar characters are aligned in successive columns. Sequence alignments are also used for non-biological sequences, such as those present in natural language or in financial data.
If two sequences in an alignment share a common ancestor, mismatches can be interpreted as point mutations and gaps as indels (that is, insertion or deletion mutations) introduced in one or both lineages in the time since they diverged from one another. In sequence alignments of proteins, the degree of similarity between amino acids occupying a particular position in the sequence can be interpreted as a rough measure of how conserved a particular region or sequence motif is among lineages. The absence of substitutions, or the presence of only very conservative substitutions (that is, the substitution of amino acids whose side chains have similar biochemical properties) in a particular region of the sequence, suggest that this region has structural or functional importance (Henikoff, 2001). Although DNA and RNA nucleotide bases are more similar to each other than are amino acids, the conservation of base pairs can indicate a similar functional or structural role.
1.2.1 Sequence alignment methods
Very short or very similar sequences can be aligned by hand. However, most interesting problems require the alignment of lengthy, highly variable or extremely numerous sequences that cannot be aligned solely by human effort. Instead, human knowledge is applied in constructing algorithms to produce high-quality sequence alignments, and occasionally in adjusting the final results to reflect patterns that are difficult to represent algorithmically (especially in the case of nucleotide sequences). Computational approaches to sequence alignment generally fall into two categories: global alignments and local alignments. Calculating a global alignment is a form of global optimization that “forces” the alignment to span the entire length of all query sequences.
By contrast, local alignments identify regions of similarity within long sequences that are often widely divergent overall. Local alignments are often preferable, but can be more difficult to calculate because of the additional challenge of identifying the regions of similarity (Polyanovsky et al., 2011). A variety of computational algorithms have been applied to the sequence alignment problem. These include slow but formally correct methods like dynamic programming. These also include efficient, heuristic algorithms or probabilistic methods designed for large-scale database search, that do not guarantee to find best matches.
1.2.2 Software used in Sequence Alignment
The software tools commonly used for general sequence alignment tasks include ClustalW2 and T-coffee for alignment, and BLAST and FASTA3x for database searching. Commercial tools such as Geneious and PatternHunter are also available. Alignment algorithms and software can be directly compared to one another using a standardized set of benchmark reference multiple sequence alignments known as BAliBASE (Thompson et al., 1999). The data set consists of structural alignments, which can be considered a standard against which purely sequence-based methods are compared. The relative performance of many common alignment methods on frequently encountered alignment problems has been tabulated and selected results published online at BAliBASE (Thompson et al., 1999). A comprehensive list of BAliBASE scores for many (currently 12) different alignment tools can be computed within the protein workbench STRAP.
1.2.3 Basic Alignment Search Tool (BLAST) analysis
The comparison of nucleotide or protein sequences from the same or different organisms is a very powerful tool in molecular biology. By finding similarities between sequences, scientists can infer the function of newly sequenced genes, predict new members of gene families, and explore evolutionary relationships. Now that whole genomes are being sequenced, sequence similarity searching can be used to predict the location and function of protein-coding and transcription regulation regions in genomic DNA. Basic Local Alignment Search Tool is the tool most frequently used for calculating sequence similarity (Altschul et al., 1990). BLAST comes in variations for use with different query sequences against different databases. All BLAST applications, as well as information on which BLAST program to use and other help documentation, are listed on the BLAST homepage. This chapter will first describe the BLAST architecture—how it works at the NCBI site—and then go on to describe the various BLAST outputs. The way most people use BLAST is to input a nucleotide or protein sequence as a query against all (or a subset of) the public sequence databases, pasting the sequence into the textbox on one of the BLAST Web pages. This sends the query over the Internet, the search is performed on the NCBI databases and servers, and the results are posted back to the person’s browser in the chosen display format. However, many biotech companies, genome scientists, and bioinformatics personnel may want to use “stand-alone” BLAST to query their own, local databases or want to customize BLAST in some way to make it better suit their needs. Standalone BLAST comes in two forms: the executables that can be run from the command line; or the Standalone WWW BLAST Server, which allows users to set up their own in-house versions of the BLAST Web pages. There are many different variations of BLAST available to use for different sequence comparisons, e.g., a DNA query to a DNA database, a protein query to a protein database, and a DNA query, translated in all six reading frames, to a protein sequence database. Other adaptations of BLAST, such as PSI-BLAST (for iterative protein sequence similarity searches using a position-specific score matrix) and RPS-BLAST (for searching for protein domains in the Conserved Domains Database, Chapter 3) perform comparisons against sequence profiles. The best known of these outputs is the default display from BLAST Web pages, the so-called “traditional report”. As well as obtaining BLAST results in the traditional report, results can also be delivered in structured output, such as a hit table, XML, or ASN.1. The optimal choice of output format depends upon the application. The final part discusses stand-alone BLAST and describes possibilities for customization. There are many interfaces to BLAST that are often not exploited by users but can lead to more efficient and robust applications. Once BLAST has found a similar sequence to the query in the database, it is helpful to have some idea of whether the alignment is “good” and whether it portrays a possible biological relationship, or whether the similarity observed is attributable to chance alone. BLAST uses statistical theory to produce a bit score and expect value (E-value) for each alignment pair (query to hit).
1.3 Protein structure
Proteins are polymers specifically formed from sequences of monomer amino acids. By convention, a chain under 40 amino acids is often identified as a peptide, rather than a protein (Stephen Stoker, 2015). To be able to perform their biological function, proteins fold into one or more specific spatial conformations driven by a number of non-covalent interactions such as hydrogen bonding, ionic interactions, Van der Waals forces, and hydrophobic packing. To understand the functions of proteins at a molecular level, it is often necessary to determine their three-dimensional structure. This is the topic of the scientific field of structural biology, which employs techniques such as X-ray crystallography, NMR spectroscopy, and dual polarisation interferometry to determine the structure of proteins.
Protein structures range in size from tens to several thousand amino acids (Brocchieri et al., 2005). By physical size, proteins are classified as nanoparticles, between 1–100 nm. Very large aggregates can be formed from protein subunits. For example, many thousands of actin molecules assemble into a microfilament.
A protein may undergo reversible structural changes in performing its biological function. The alternative structures of the same protein are referred to as different conformations, and transitions between them are called conformational changes.
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