ASIAN J. EXP. BIOL. SCI. VOL 2(2) 2011: 201-206
© Society of Applied Sciences
ORIGINAL ARTICLE
COBALT: Multiple Protein Sequences Alignment Tool In Analysis of
Cytochrome b Based Protein Diversity of osteichthyes.
a
K. S. Dangi*, Geeta and S.N. Mishra
a
Department of Biosciences, Maharshi Dayanand University, Rohtak, 124001 Haryana (INDIA).
Centre for Biotechnology, Maharshi Dayanand University, Rohtak, 124001 Haryana (INDIA).
ABSTRACT
A tool that simultaneously aligns multiple protein sequences, automatically utilizes information about protein domains and
has a good compromise between speed and accuracy will have practical advantages over current tools. While, study of
Osteichythyes is vast and cytochrome b enzyme of mitochondria was analysed for evolutionary relationship by using
COBALT. In most of classification system Osteichthyes (bony fishes) are paraphyletic with land vertebrates includes
tetrapods amongst its decedents. The majority rule 75% consensus tree from twenty-five fresh water bony fish using BLASTp
shows only few species in most primitive category provide lineage for other species evolution. COBALT provided the
correlation and phylogenetic relationship of cytochrome b protein sequences that Ompok species, Siluriformes species,
Krytopterus apogon, Silurus glanis, Wallga attu species with high parsimony than the other species. Results show that
COBALT has reasonable runtime performance and alignment accuracy which can be a tool for a broad range of problems of
phylogenetic analysis.
KEYWORDS: Osteichythyes, Phylogenetic analysis, Cytochrome b, COBALT
INTRODUCTION
The simultaneous alignment of multiple sequences (multiple alignments) serves as a building block in several fields of
computational biology such as phylogenetic studies [1], detection of conserved motifs [2], prediction of functional
residues and secondary structure, prediction of correlations and even quality assessment of protein sequences [3]. The
development of algorithms that can automatically produce biologically plausible multiple alignments is a subject of
very active research [4].Unfortunately, finding a multiple alignment that rigorously optimizes the commonly used
'sum-of-pairs' scoring measure is computationally hard and not practical when more than a few sequences are
involved [5]. COBALT has a general framework that uses progressive multiple alignment to combine pairwise
constraints from different sources into a multiple alignment. COBALT does not attempt to use all available constraints
but uses only a high scoring consistent subset that can change as the alignment progresses, where a set of constraints is
called consistent if all of the constraints in the set can be simultaneously satisfied by a multiple alignment.
Furthermore, CDD also contains auxiliary information that allows COBALT to create partial profiles for input
sequences before progressive alignment begins, and this avoids computationally expensive procedures for building
profiles.
In India, fresh water bony fishes are distributed in large amount about 111 species in Krishna River. Indian fresh water
Osteichthyes species are Labeo rohita (Rohu), Labeo calbasu (Calbasu), Catla catla (Catla) and cyprinus carpio
(carp). Labeo rohita include about 29 species, Labeo calbasu include about 28 species, Catla catla include about 32
species and Cyprinus carpio include about 21 species in Krishna river of Karnataka. Some of them are edible species
such as, Labeo rohila, Labeo calbasu, Catla catla and Cyprinus carpio are common fresh water edible species. Bony
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COBALT: Multiple Protein Sequences Alignment Tool In Analysis Of Cytochrome B Based Protein Diversity Of Osteichthyes....... K. S. Dangi et al.
fishes are classified based on comparative anatomy, embryology, genetics, molecular biology and the fossil record.
Cytochrome b is a highly conserved protein across the spectrum of species, found in plants, animals, and many
unicellular organisms. The cytochrome b molecule has been studied for the glimpse it gives into evolutionary biology
[6, 7]. Therefore, genetic variation and specificity of cytochrome b which varied with climatic condition conferred the
genetic complexity which can be taken as a suitable marker enzyme for phylogenetic analysis.
MATERIALSAND METHODS
Collection of DNAsequences
The sequence of cytochrome b were taken from the following source: Amblyceps mangois, gi:11o293492,
Amplypharyngodon mola, gi:299832307, Bagarius yarrelli, gi:110293496, Barilius barna, gi:167887443, Barilius
bendelisis, gi:300136784, Catla catla, gi:194173358, Chanda nama, gi:203287172, Channa punctata,
gi:167887437, C. striata, gi:254055103, Devario aequipinnatus, gi:299832341, D. malbaricus, gi:289546477,
Danio rerio, gi:261337155, Esomus danricus, gi:299832369, E.longinaus, gi:299832371, Wallago attu,
gi:203287200 etc. were used for phylogenetic analysis by selecting the gene of respective species of bony fishes. Each
protein sequence was changed to Pearson format for further analysis by COBALT software.
Sequences were retrieved from the protein database by a single-pass BLASTp search (e-value <0.1) using the aligned
as a result, 555 sequence hits passed the cutoff e-value. The like sequences identical by 75% or more were clustered
using the NCBI BLAST CLUST program (http://www.ncbi.nlm.nih.gov/blast/docs/blastclust.html).
BLAST
Basic Local Alignment Search Tool, or BLAST, is an algorithm for comparing primary biological sequence
information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences [8]. The
BLAST algorithm is a heuristic program and performs local alignments. It is available to the users trough web service
BLAST provided by NCBI. There are several types of algorithm optimized for particular types of queries: BLAST p
for searching a protein database using a peptide query. BLAST works by first making a look-up table of all the “words”
(short subsequences, which for proteins the default is three letters) and “neighboring words” i.e. similar words in the
query sequence. The sequence database is then scanned for these “hot spots”. When a match is identified, it is used to
initiate gap-free and gapped extensions of the “word”[9].
Scoring a multiple alignment
The traditional method of scoring an alignment between a pair of sequences is to use a score matrix based on log-odds
scores, such as the PAM or BLOSUM series of matrices. When there are more than a few sequences, the information
content in a multiple alignment can be measured by its entropy. A small number of sequences, or correlations between
residues, can have a harmful effect on an entropy-based scoring measure. Partitioning residues into a smaller number
of residue classes can sometimes dampen this effect. The intuition used by Tharakaraman et al. [10] is to combine
ideas behind log-odds and entropy-based scoring.
Algorithm
COBALT is a flexible tool for simultaneously aligning a given set of protein sequences, where users can directly
specify pairwise constraints and/or ask COBALT to generate the constraints using sequence similarity, (optional)
CDD searches and (optional) PROSITE pattern searches. COBALT will optionally create partial profiles for input
sequences based on any CDD search results. Aside from these features, the COBALT algorithm is similar to that of
other progressive multiple alignment tools:
Step 1: Find alignments for generating constraints.
Step 2: Find partial profiles and a pairwise consistent set of constraints.
Step 3: Generate a guide tree.
Step 4: Create a multiple alignment using the current set of constraints and guide tree.
Step 5: Create bipartitions and realign.
Computing the multiple sequence alignment
The progressive multiple alignment does a depth-first traversal of the tree generated in Step 3. At each node of the tree,
profiles for both subtrees and align these profiles to produce a multiple alignment for all of the sequences was
generated.
A variant of ordinary NeedlemanWunsch dynamic programming computes a global alignment of two profiles. The
alignment process uses well-known techniques to reduce memory consumption [4] and includes two variations that
are specific to profile alignments. The first variation is the choice of profileprofile score function [11].
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Refinement by finding new constraints
The second refinement phase begins by finding conserved columns in the output from Step 5. A column in a multiple
alignment is considered to be high scoring if its score exceeds a cutoff (set to 0.67 by default), and groups of at least
two adjacent high-scoring columns are considered conserved. Iteration continues as long as the number of conserved
columns increases. Before iterating, the set of constraints found in Step 2 is replaced by constraints that encompass
alignment decisions based on conserved columns, pattern matches and user specified pairs.
COBALT uses an all-against-all collection of pairwise constraints to represent each group of conserved columns.
Conserved columns may contain gaps, but sequences that contain gaps in a conserved column do not participate in
pairwise constraints for that column. This exception allows conserved columns to be used for most profileprofile
alignments, while generating pressure on slightly misaligned sequences to shift position.
RESULTS
BLASTp analysis based on score matrix present a close association between O.bimaculatus, O.pabda, wallago attu
but seems to be widely apart from other fresh water bony fishes (fig.1). Present endeavor therefore may aid
interspecific hybridization in bony fishes and variations released through climate change may enrich genetic diversity
in bony fishes but may be attributed to limitation of BLASTp algorithms used. Although relationship varied among
species depending on the criteria used, it seems that for most of the traits of Ompok species and walago attu were very
closely associated. Phylogenetic tree constructed from cluster analysis using software COBALT of have a better
precision (fig.2).
Fig. 1 BLASTp protein sequence score table, output is a summary of sequences producing significant alignments,
along with both normalized scores and E values (see text for further details; only the few highest-scoring hits are
shown)
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Fig. 2 Neighbor-joining phylogenetic tree for progressive alignment using COBALT. Here we show the same set of twenty
five sequences aligned according to a neighbor-joining tree
Therefore, unrooted tree was prepared by taking one discrete genetic variable and for twenty-five species of bony fish.
Findings revealed that the few species (Ompok pabda, Ompok bimaculatus,P. anguloides, Wallago attu etc) are more
primitive than other (Amplyceps mangois, Bagarius yarrelli, Catla catla, Labeo rohita, Danrio rerio etc). Among
these Ompok species and Wallago attu were found most primitive phylogenetic relationship among the bony fishes
suggested a complex evolutionary trend (fig.3).
Fig. 3 Pairwise sequence alignment of bony fish, mitochondrial Cytochrome b protein sequence analysis by BLASTp.
Phylogenetic tree shown here indicating genetic diversity of bony fishes
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The minimum and maximum p distance indicate that cytochrome b enzyme highly conserved in case of Ompok
bimarculatus, Ompok pabda, Wallago attu and other are quite divergent such as Amplyceps mangois,
Amblypharyngodon mola, Bagarius yarrelli, Catla catla, Labeo rohita, Channa striata etc. The majority rule 75%
consensus tree from twenty-five fresh water bony fish shows phyllogram (fig. 2). Twenty-five species with
cytochrome b enzyme is correlated to each other to phylogenetic relationship, it shows that Ompok species,
Siluriformes species, Krytopterus apogon, Silurus glanis, Wallga attu species have high parsimony than other
species.
DISCUSSION
Results shows that COBALT has a better chance of producing a biologically meaningful multiple alignment
compared to tools that do not utilize this information. The applicability of additional information such as secondary
structure alignments computed with recent algorithms [12] and the detection of short highly conserved motifs found
with de novo methods [13]. Progressive multiple alignment algorithms all have difficulty with highly divergent
sequence inputs, and so COBALT may also benefit from incorporating alignment algorithms that explicitly process
more than two sequences or sequence collections at a time [14].
Figure2 shows that the sequences from the twenty out of twenty five fresh water bony fishes evolved considerably
slower than five fresh water bony fish species. These twenty species formed one cluster outside the other sequences
and other five sequences evolved nearly at the same rate.According to this, cytochrome b enzyme varies their function
species to species may be due to change in climate show change may enrich genetic diversity in bony fish species. The
vast majority of Cytochrome b disorders are linked to mutations in nuclear-encoded proteins referred to as assembly
factors, or assembly proteins. These assembly factors contribute to cytochrome b structure and functionality, and are
involved in several essential processes, including transcription and translation of mitochondrion-encoded subunits,
processing of pre proteins and membrane insertion, and cofactor biosynthesis and incorporation [15]. Mutations in
these proteins can result in altered functionality of sub-complex assembly, copper transport, or translational
regulation. Each gene mutation is associated with the functional changes in species and ultimately leads to species
diversity.
CONCLUSION
Results, shows that when the rate of evolution varies extensively from branch to branch, many methods may fail to
recover the true topology. Cytochrome b enzyme phylogenetic analysis between twenty-five fresh water bony fishes
by using software COBALT align all protein sequences in a line and phyllogram show evolutionary relationship
between all species by comparing them and concluded that five species are more primitive than other twenty fresh
water bony fish species.
ACKNOWLEDGEMENT
We are grateful to University Grant Commission New Delhi, India for providing Meritorious Research Fellowship to
K.S. Dangi.
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Correspondence to Author : K. S. Dangi, Department of Biosciences, Maharshi Dayanand University, Rohtak, 124001
Haryana (INDIA).Email : [email protected]
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