Vertebrate Kinesins

Created by: Jason Close, Matt Morale and Lisa Warner

blastp

A BLAST search revealed 1033 predicted vertebrate kinesins. Vertebrate Kinesin BLAST Results

When broken down by organism we see that the human has 248 predicted kinesins. Vertebrate Kinesin Tax BLAST Report. Some of these 248 kinesins are repeats of one another and others are not kinesin at all. According to the Kinesin Homepage there are 41 kinesin proteins currently known in humans.

The animals that the 1033 predicted kinesins come from are:

• Bos taurus 30 hits
• Canis familiaris 144 hits
• Coturnis coturnix 3 hits
• Danio rerio 60 hits
• Gallus gallus 47 hits
• Homo sapiens 248 hits
• Macaca fascicularis 15 hits
• Morone saxatilis 6 hits
• Mus musculus 178 hits
• Oryzias latipes 12 hits
• Pan troglodytes 32 hits
• Pongo pygmaeus 13 hits
• Rattus norvegicus 70 hits
• Tetraodon nigroviridis 39 hits
• Xenopus laevis 63 hits
• Xenopus tropicalis 5 hits

Our methodology for searching for all vertebrate kinesins was to use a BLASTp search. We used the Drosophilia KHC accession number (P17210) as our query sequence. Next we set our subsequence limits from 1 to 350 amino acids as the first 350 aa are a conserved head domain of most known kinesin. We used the default nr database as the subject because it includes all of the other databases and therefore it is the broadest. In the options for advanced blasting section, we limited our search to Vertebrata [ORGN] proteins only. We kept the default matrix (BLOSUM 62) as well as the gap costs- existence to extension ratio (11:1). Lastly, we increased descriptions and alignments to 1000. An important part of in BLAST searching is deciding whether an alignment is significant. Our cutoff for evaluating whether or not each protein was homologous to our query was an E value of less than or equal to 0.01. In addition, we were able to expand and organize our results using the taxonomy tool.

blastp v. 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.