EST database

When the Arabidopsis Expressed Sequence Tag (EST) Database was searched with the tomato fmit Psubunit protein sequence two related cDNAs were identified (Figure 11). cDNA 2 is near full length and has been completely sequenced, cDNA 1 has also been sequenced but currently lacks approximately 100 amino acids of coding region. The two Arabidopsis cDNAs are 81% identical at the protein level and have lower identity to the protein encoded by tomato gene 1, 64 and 63% for cDNA 1 and cDNA 2, respectively. However, both cDNAs encode... 

Neubauer, G., King, A., Rappsilber, J., Calvio, C., Watson, M., Ajuh, P., Sleeman, J., Lamond, A., and Mann, M. (1998). Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat. Genet. 20, 46-50. 

Ewing RM et al. EST databases as multiconditional gene expression datasets. Pac Symp Biocomput 2000 5 430-432. 

PMF is generally used to identify proteins that have been previously separated by 2-D GE so that additional information including the molecular weights and isoelectric points can be used to supplement PMF identification. PMF is not well suited for searching expressed sequence tag (EST) databases that contain incomplete gene coding information for particular ESTs and it is not adequate for the analysis of complex protein mixtures in solution. 

Mining for SNPs in EST databases requires only computer resources and does not incur experimental cost (as do the various techniques of large-scale DNA chip analysis). 

Despite these problems EST databases are a valuable source of large-scale analysis of human variation. They will become even more valuable as the data continue to grow at the present rate. An algorithm for computer-aided SNP mining should contain filters to eliminate the potential sequence errors. Such filters can be based on the probabilistic analysis of sequence features. It can also take into account that multiple occurrences of a variant are more trustworthy, and it may furthermore focus on improving the quality of base-calling if the fluorescent traces are available for closer srcutiny. 

A nucleotide or peptide sequence is searched against the EST database using the BLAST (see BLAST server Karlin and Altschul, 1990 and 1993 Altschul et al., 1997) searching programs. 

D. A., and Boyce-Jacino, M. (1999). Mining SNPs from EST databases. Genome Res. 9, 167-174. 

The 4-coumarate CoA ligase (4CL EC 6.2.1.12) enzyme activates 4-coumaric acid, caffeic acid, ferrulic acid, and (in some cases) sinapic acid by the formation of CoA esters that serve as branch-point metabolites between the phenylpropanoid pathway and the synthesis of secondary metabolites [46, 47]. The reaction has an absolute requirement for Mg " and ATP as cofactors. Multiple isozymes are present in all plants where it has been studied, some of which have variable substrate specificities consistent with a potential role in controlling accumulation of secondary metabolite end-products. Examination of a navel orange EST database (CitEST) for flavonoid biosynthetic genes resulted in the identification of 10 tentative consensus sequences that potentially represent a multi-enzyme family [29]. Eurther biochemical characterization will be necessary to establish whether these genes have 4CL activity and, if so, whether preferential substrate usage is observed. 

Off-target mRNAs with homology to target-mRNA Sequence comparison Comparison of siRNA sequences to EST databases (Is the EST expressed in the cell-line of interest )... 

The second approach is the tandem mass spectrometric method (Wilm et ah, 1996 Link et ah, 1999 Yates, 2000). This method relies on fragmentation of individual peptides in the tryptic peptide mixture to gain sequence information. Its main advantage is that sequence information derived from several peptides is much more specific for the identification of a protein than a list of peptide masses. The sequence data can be used to search not only protein sequence databases but also nucleotide databases such as expressed sequence tag (EST) databases and, more recently, even... 

The advent of rapid DNA cloiung and sequencing techniques, and the pubhc availability of nucleotide sequence information from the tikes of ttie tiuman genome sequencing project, various expressed sequence tag (EST) databases, and various... 

EST databases contain sequence data on cDNA sequences, from a number of organisms, made from single random passes. There are millions of ESTs within these databases and although, commonly, they only cover a small section of organisms, genes and proteins, they are still valuable in the identification of proteins (Andersen and Mann, 2000). However, more recent EST projects are believed to cover a greater proportion of an organism s transcriptome. 

If PMF and sequence homology searches fail using available protein and EST databases, mass spectrum data can, in principle, be used to search genome databases (GenBank http //www.ncbi.nlm.nih.gov/). However, only a small percentage of large genomic sequences code for proteins such that bioinformatics still needs to accurately define exon-intron structures (Andersen and Mann, 2000). 

Further support for the occurrence and activity of neuropeptides in flatworms stems from the presence of enzymes necessary for the production of the mature peptides. For example, the first step in the generation of a mature neuropeptide is often the excision of a propeptide from a longer pre-propeptide. This is achieved by the activity of prohormone convertases, and a number of putative platyhelminth convertase-encoding cDNAs are represented within the EST databases. 

Among the EST database of ragi sequences, there are two groups of bifunctional proteinase inhibitor trypsin a-amylase from seeds of ragi sequences. The upper clade was further subdivided (Fig. 6.10). Wang et al. (2008) concluded that there was great diversity in the sequence of different Bowman-Birk inhibitors in emmer wheat both within and between populations. 

Sorensen, M. B., Rasmussen, S. K. (2005). Construction of an annotated EST resource for developing finger millet (Eleusine coracana L.) seeds. Unpublished. NCBI EST Database. 

Searching EST databases ESTs are generally short and therefore do not usually provide sufficient numbers of matched peptides for a significant score. 

Bera TK, Lee S, Salvatore G, Lee B, Pastan I. MRP8, a new member of ABC transporter superfamily, identified by EST database mining and gene prediction program, is higly expressed in breast cancer. Mol Med 2001 7 509-516. 

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