Xenopus Tropicalis

Kinesin of XENOPUS TROPICALIS


I tried three different organism-specific BLAST sites (TIGR, Ensembl, and JGI) for the frog species, xenopus tropicalis. The non-default TIGR parameters I used were a tblastn search using P17210 as the query, a cutoff of 80, and an E value cutoff of less than or equal 0.00001. However, there was no option allowing a search for the first 350 aa (the head domain of KHC). Therefore, I selected the first 350 aa from the FASTA format of the Drosophilia KHC pasted it into TIGR in FASTA format. My results showed 36 hits with all significantly small E values (TIGR results). All hits showed statistically significant E values and bit scores suggesting homology. I checked my results by clicking on the genbank accession number, clicked "links" (NCBI website), and then "unigene (NCBI website)."

The JGI website (genome.jgi-psf.org) has an organism specific database for the species, Xenopus tropicalis that has been recently updated to version 4.1. Results yielded hits with significantly low E values (SEE JGI Results). By clicking on the genbank link and then the "more info" link, JGI's description of the protein is revealed. In this case, THE #1 HIT, has a molecular aspect of motor activity, a function aspect of ATP binding, and the cellular component is a micotubule associated complex. The protein is classified as part of the kinesin SMY1 subfamily and this 350 aa region is the kinesin protein's motor region.

The Ensembl Multi View search yielded significantly more hits, 413 (Ensembl Results). However, many of these hits could be of redundancy because I used the entire 961 aa of the query (P17210). The top hit had a score of 317 and an E value of 1.9 e^-21. In addition, the results had a nice karyotype diagram.

All in all, these three organism specific BLAST sites were much more useful because of their specificity rather then just using the NCBI website with the nr database.

Modification of Search for All Vertebrate Kinesin: psiBLAST

This week in lab we performed a position-specific iterated BLAST or psiBLAST for all Vertebrate kinesin using the Drosophila KHC as our query. This particular BLAST search is designed for finding distantly related proteins. psiBLAST uses a position-specific scoring matrix (PSSM) and therefore is more sensitive then a regular BLAST search.
Our initial results yielded 995 hits with the top hit being: KIF5C protein [Homo sapiens] (Score 520 and an E value of 9e-147) (psiBLAST Results). Our hits continued to increase after every iteration.

The Taxonomy Report

After 5 iterations, psiBLAST had yet to report convergence (no new results found). We ran into the problem of having too many hits. Therefore we modified our search parameters by typing in "refseq" in the limit by Entrez query box. All though this most likely eliminated redundant database matches, it over-rided our parameter of just Vertebrate kinesin. In the end we only got 17 hits, in which more then half were of non-vertebrate.
In conclusion, we had more success with the BLASTp search. Although we ran into the problems of both too many and too few hits with psiBLAST, we were able to recognize the potentiality of this specific BLAST. More time needs to be spent working with the psiBLAST parameters in order to get the kind of results we are looking for.