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Using at least three methods of multiple alignments of mitochondrial genes (Cox1, Cox2, and Cox3) for comparison purpose in the subterranean termites from Genus Reticulitermes (Isoptera: Rhinotermitidae)
Termites are social insects that live in colonies, which, in turn, function because the complementary roles played by the different caste. In United States, subterranean termites, Coptotermes spp. and Reticulitermes spp. are the most destructive and cause substantial economic damage to buildings/structures (Su & Scheffrahn, 1990). Currently, mitochondrial genes were used for taxonomy purpose in termites (Cameron & Whitting, 2007). Thus, the whole mitochondrial genome can be used to predict useful regions for further investigation in the multiple alignments methods application
Multiple alignments are the key starting point for prediction of protein secondary structure, residue accessibility, and the identification of residues important for specificity. Multiple alignments can provide the basis for the most sensitive sequence searching algorithms (Barton & Sternberg, 1990). Furthermore, methods in multiple alignments affective analysis of a well-constructed multiple alignments can provide important clues about which residues in the protein are important for stabilizing the second and tertiary structure of the protein (Altschul et al. 1997)
ClustalW will calculates the best match for the selected sequences, and lines them up so that the identities, similarities and differences can be seen and will provide a better quality for sequences (Thomson et al. 1994). Meanwhile, T-Cofee application is better to overcome with some of the problems that standard hierarchical method s have in aligning sequences of very different length or that share only local region similarity (Notredame et al. 2000). The program works by building a library of pairwise alignments for the sequence interest. Furthermore, T coffee has been adapted to include structural alignment and alignments from threading algorithms since it can work from pairwise alignments that originate from any source (Notredame et al. 2000). In addition, the program PSI-BLAST searches database with a single sequence, any high-scoring sequences that are found are built into a multiple alignment, and this multiple alignment is then used to derive a search "profile" for subsequent search of the database. This process is repeated until no new sequences are added to profile, or a specific number of iteration have been performed (Jones 1999).
Genes alignment (Multiple Alignments)
Selected list of gene (Cox1, Cox2, and Cox3) in termites in R. santonesis & R.hageni & R. flavipes for analysis will be obtain from the NCBI entrez database. Fasta file format sequence will be saved in the separate file. Three set of mitochondrial genes Cox1, Cox2, and Cox3 genes from three different species will be carried out using T-Cofee, PSI-BLAST, Clustal-W. Only one mitochondrial gene will be run from the three different termite species at the same time. All the three methods will be run on the website directly. Furthermore, all the three website has been test on the computer it is perfectly working as long the server is ok
• T-Cofee (http://www.ebi.ac.uk/Tools/clustalw2 )
In the T-cofee alignment methods, the fasta sequence format file of (Cox1, Cox2, and Cox3) from three termites will be paste in the box directly to T coffee website (http://www.ebi.ac.uk/Tools/clustalw2). A T-cofee method will generate a library of all the pairwise alignments between the sequences and gives positional information concerning which residues are homologous to which other residues. This can be used to guide progressive alignments
• PSI-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi)
Fasta sequence format file of (Cox1, Cox2, and Cox3) from three termites will be paste in the box directly to PSI-BLAST website http://blast.ncbi.nlm.nih.gov/Blast.cgi. In PSI-BLAST multiple alignment method is always is the length of the query sequence used in the search. Furthermore, if alignment of the query (or query profile) to a database sequence requires an insertion in the query, then the inserted region from the database sequence simply is discarded. In addition to that, the results alignment will highlights the amino acids that may be aligned to each position in the query. Compare to the other two methods, this can be most informative type of alignment when one is focused on understanding a single sequence set. According to Jones (1999), PSI-Blast multiple alignments and their associated frequency tables and profile have proved very effective as input for programs that predict protein secondary structure.
• Clustal-W (http://www.ebi.ac.uk/Tools/clustalw2/index.html)
This method is a good approach for doing progressive alignment compare to the other two methods. However, depend on the internal node in the tree, the alignments. We may want to align a sequence with a sequence, a sequence with a profile (partial alignment) and a profile with a profile (Thomson et al. 1994). None the less we can use dynamic programming for the profile cases using SP score.
Comparative analysis plan
Three data sets will be uses (Cox1, Cox2, and Cox3) from three termite species, R. santonesis & R.hageni & R. flavipes. All the data set will be gain from ENTREZ search at NCBI web page as a fasta file format sequence (*explanation under the methods). Throughout the three methods of multiple alignments, we will be looking at the list below for comparative work.
§ Secondary and tertiary structure predictions
§ Identification of active sites, domains or other conserved regions
§ Looking at the motif finding
Furthermore, from these three methods, we can look which methods are better in "progressive methods" especially between T-Cofee and ClustalW. In addition to that we can look if iterative methods use in PSI-Blast will give better results compare to progressive methods in T-Cofee and ClustalW.
JalView and Web LOGO Berkeley, (http://weblogo.berkeley.edu/logo.cgi ) will be use for editing and analysis process. Jalview is an interactive alignment editor. This will allow an existing alignment to be read into the program and individual residues or blocks of residues to be moved around. Meanwhile, WebLogo Berkeley application is to design to make the generation of sequence logos as easy and painless as possible. Therefore we can look at the conserved region much more clearly.
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z.Miller, E., and Lipman, D.J. (1997). Gapped blast and psi-blast: a new generation of protein database search programs. Nucl. Acids Res. 28:225-227
Barton, G.J., and Sternberg, M.J.E. (1987). A strategy for the rapid multiple alignment of protein sequences: confident levels from tertiary structure comparisons. J.Mol.Biol. 198:327-337
Cameron, S.L & Whiting, M.F. 2007. Mitochondrial genomic comparisons of the subterranean termites from the Genus Reticulitermes (Insecta: Isoptera: Rhinotermitidae).Genome 50:188-202
Jones, D.J., (1999). Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 17:195-202
Notredame, C., Higgins, D.G., and Heringa, J. (2000). T-cofee:a novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302:205-217
Su, N.-Y., and R. H. Scheffrahn. (1990). Comparison of eleven soil termiticides against the Formosan and eastern subterranean termites (Isoptera: Rhinotermitidae). J. Econ. Entomol. 83: 1918-1924
Thomson,J. D., Higgins, D.G., and Gibson, T.J. (1994). Clustal w: improving the sensitivity of pregressive multiple sequence alignment through sequence weighting, position-specific gap penalties abd weight matrix choice. Nucl. Acids Res. 22: 4673-4680