EST sequences

Since 1988, the methods that we use to isolate cDNAs of alkaloid biosynthesis have become ever more facile and sensitive, allowing for more efficient cDNA identification. We do not, however, yet understand enough about the cellular localization of alkaloid formation or about the nature of the catalysts to move completely away from enzymology and biochemistry and to use only molecular genetic techniques to dissect these biosynthetic pathways. Even our most recently successful cDNA isolations and identifications involved classical protein purification. We are beginning now to use proteomics and EST sequencing to identify natural product biosynthetic cDNAs, but these approaches are more feasible when a specialized cell/tissue type in which secondary metabolite biosynthetic pathways are active, can be isolated and used as a protein or RNA source. 

Expressed Sequence Tags and In Silico Methods Expressed sequence tags (ESTs) are short nucleotide sequences of complementary DNA with about 200-500 base pairs. They are parts of the DNA that code for the expression of particular proteins. EST sequencing provides a rapid method to scan for all the protein coding genes and to provide a tag for each gene on the genome. 

A database search reveals that YPClp and YDClp are not homologous to any proteins with known functions, but are homologous to putative proteins from Arabidoposis, C. elegans, peptides deduced from EST sequences of human, mouse, pig, zebra fish, and human genomic sequences. A human homologue has been identified and its cDNA has been cloned. Preliminary results show that this human homologue is also an alkaline ceramidase that selectively hydrolyzes phytoCer. 

Kiister H etal.. Development of bioinfo rmatic tools to support EST-sequencing, in silico- and microarray-based transcriptome profiling in mycorrhizal symbioses, Phytochemistry 68 19—32, 2007. 

Fig. 6.1. Summary data for the large-scale expressed sequence tag (EST) sequencing projects for Schistosoma mansoni and S. japonicum. Numbers refer to ESTs obtained for each of the six stages of the parasite s life cycle.

Note-. Unpublished sequences (C,E) are referenced by accession number. EST sequences may have been listed as examples of multiple ESTs in the database. 

EST Sequence databases Bioinfofmatics Unique gene clusters... 

Of 43 337 pairs of donor and acceptor splice sites (splice pairs) 22 489 were supported by EST sequences. 98.71% of those contain canonical dinucleotides GT and AG for donor and acceptor sites. 0.56% hold non-canonical GC-AG splice site pairs. The reminder 0.73% occurs in a lot of small groups (with maximum size of 0.05%). 53.6% of canonical and just 27.3% of non-canonical splice pairs were supported by ESTs. Based on these figures it was supposed that at least half of annotated non-canonical sites presents annotation errors, as was shown in some previous works [8, 17]. 

Kodzius, R., Matsumura, Y., Kasukawa, T., et al. (2004) Absolute expression values for mouse transcripts re-annotation of the READ expression database by the use of CAGE and EST sequence tags. FEBS Lett. 559, 22-26. 

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